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WO2024253915A1 - Systèmes et procédés de cartographie de température à l'aide d'agents thermosensibles - Google Patents

Systèmes et procédés de cartographie de température à l'aide d'agents thermosensibles Download PDF

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Publication number
WO2024253915A1
WO2024253915A1 PCT/US2024/031427 US2024031427W WO2024253915A1 WO 2024253915 A1 WO2024253915 A1 WO 2024253915A1 US 2024031427 W US2024031427 W US 2024031427W WO 2024253915 A1 WO2024253915 A1 WO 2024253915A1
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photoluminescent
temperature
environment
thermosensitive
time
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Baohong Yuan
Tingfeng YAO
Liqin REN
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/48—Diagnostic techniques
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/01—Measuring temperature of body parts ; Diagnostic temperature sensing, e.g. for malignant or inflamed tissue
    • A61B5/015—By temperature mapping of body part
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/08—Clinical applications
    • A61B8/0833—Clinical applications involving detecting or locating foreign bodies or organic structures
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/52—Devices using data or image processing specially adapted for diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/5207—Devices using data or image processing specially adapted for diagnosis using ultrasonic, sonic or infrasonic waves involving processing of raw data to produce diagnostic data, e.g. for generating an image
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
    • G01K11/00—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00
    • G01K11/20—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00 using thermoluminescent materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/08—Clinical applications
    • A61B8/0825—Clinical applications for diagnosis of the breast, e.g. mammography
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/48—Diagnostic techniques
    • A61B8/481—Diagnostic techniques involving the use of contrast agents, e.g. microbubbles introduced into the bloodstream

Definitions

  • the present invention generally relates to systems and methods for imaging and/or mapping temperature in an environment, and more particularly, to noninvasive imaging and/or mapping temperature in deep tissue using temperature-sensitive agents.
  • BACKGROUND [0004] Tissue temperature is largely dependent on cellular metabolism and localized blood flow. For example, most solid tumors generate more heat than surrounding healthy tissues because of increased vascularization and metabolic activity. The temperature of lung, bladder, and breast tumors often have a temperature that is 1-2 °C higher than the surrounding healthy tissues. Fast- growing tumors were found to be hotter than benign or slowly growing tumors and normal tissues ( ⁇ 1-3 °C or higher).
  • tissue thermography methods that observe heat near the surface of the skin were shown to be unviable for the diagnosis of cancer, particularly for breast cancer.
  • Conventional tissue thermography methods rely on infrared photon detection within a wavelength spectrum of approximately 3-10 microns for imaging temperature. In cases where these infrared photons generated from deep breast tissues are completely absorbed by water molecules present in the tissue, a complete loss of information beneath the skin can occur.
  • the absolute value of the surface temperature of the skin is not specific to the tumor’s activeness and can be affected by many other factors, and thermography diagnosis based on skin surface temperature is neither specific nor sensitive to tumor activeness for diagnosis purposes. Accordingly, the technology described herein overcomes issues in conventional tissue thermography, particularly in imaging and/or mapping deep tissue environments.
  • a method described herein comprises disposing a population of photoluminescent thermosensitive agents in the environment, the population having a first switching temperature threshold and a second switching temperature threshold that is higher than the first switching temperature threshold.
  • the method further comprises exposing the environment to an activation source at an initial time to create an activation region within the environment having a temperature greater than or equal to the first switching temperature threshold at a first time, and exposing the environment to a beam of electromagnetic radiation to excite the population of photoluminescent thermosensitive agents at the first time.
  • the method further comprises detecting a photoluminescent signal emitted by the excited population of photoluminescent thermosensitive agents at the first time. In some cases, the method further comprises exposing the environment to the activation source at a second time to increase the temperature of the activation region to a temperature greater than or equal to the second switching temperature threshold, and exposing the environment to a beam of electromagnetic radiation to excite the population of photoluminescent thermosensitive agents at the second time. In some embodiments, the method further comprises detecting a photoluminescent signal emitted by the population of photoluminescent thermosensitive agents at the second time, and determining the initial temperature of the environment based on detecting the photoluminescent signal at the first time and the second time.
  • the population of photoluminescent thermosensitive agents comprises a first photoluminescent thermosensitive agent having the first switching temperature threshold and a second photoluminescent thermosensitive agent having the second switching temperature threshold.
  • a single photoluminescent thermosensitive agent may have a first switching temperature threshold and a second switching temperature threshold.
  • the photoluminescent thermosensitive agents comprise ultrasound-switchable fluorophores.
  • exposing the environment to a beam of electromagnetic radiation comprises exposing the environment to a first excitation beam and a second excitation beam, and the first excitation beam and the second excitation beam have differing wavelengths.
  • the photoluminescent signal at the first time is detected with a first detector or detection channel
  • the photoluminescent signal at the second time is detected with a second detector or detection channel.
  • the photoluminescent signal at the first time and/or the photoluminescent signal at the second time comprises visible light.
  • determining the initial temperature of the environment comprises determining a function corresponding to a photoluminescence signal-temperature profile of the population of photoluminescent thermosensitive agents.
  • methods of measuring an initial temperature in an environment further comprise determining a linear heating speed of the environment (Vh).
  • the activation source comprises at least one of an ultrasound beam, electromagnetic radiation, a magnetic field, an electron or particle beam, and a chemical stimulus.
  • the activation source is an ultrasound beam.
  • the environment comprises biological tissue.
  • the environment comprises a tumor.
  • the system comprises a population of photoluminescent thermosensitive agents, the population having a first switching temperature threshold and a second switching temperature threshold, at least one activation source, at least one excitation beam, and at least one photoluminescence detector. Additionally, in some embodiments, the activation source is an ultrasound beam.
  • the photoluminescent thermosensitive agents comprise ultrasound switchable fluorophores (USF).
  • the population of photoluminescent thermosensitive agents comprises a first photoluminescent thermosensitive agent having the first switching temperature threshold and a second photoluminescent thermosensitive agent having the second switching temperature threshold.
  • first photoluminescent thermosensitive agent can have a first emission profile and the second photoluminescent thermosensitive agent can have a second emission profile differing from the first emission profile.
  • Figure 2 illustrates a plot of normalized fluorescent intensity and the corresponding normalized differences versus temperature (°C) for ICG-liposomes with a higher LCST (H), ICG-liposomes with a lower LCST (L), and a mixture of H and L liposomes according to one embodiment described herein.
  • Figure 3A illustrates a plot of the temporal normalized USF intensity and the normalized differential curve versus time (sec) according to one embodiment described herein.
  • Figure 3B illustrates a plot of the normalized USF signal over time (sec) according to one embodiment described herein.
  • Figure 3C illustrates an acquired image of the USF signal from Figure 3A.
  • Figure 3D illustrates an acquired image of the USF signal from Figure 3A.
  • Figure 3E illustrates an acquired image of the USF signal from Figure 3A.
  • Figure 3F illustrates an acquired image of the USF signal from Figure 3A.
  • Figure 3G illustrates an acquired image of the USF signal from Figure 3A.
  • Figure 4A illustrates a plot of the temperature (°C) calculated using USF thermometry versus the temperature (°C) determined with an IR camera (IR reading) with a reference line of a slope of 1 according to one embodiment described herein.
  • Figure 4B illustrates a plot of the time for the first switching peak versus the background temperature as an IR reading (°C) according to one embodiment described herein.
  • Figure 4C illustrates a plot of the time for the second switching peak versus the background temperature as an IR reading (°C) according to one embodiment described herein.
  • Figure 5A illustrates a plot of the temporal profiles of the normalized differences of USF signal when switching the thermosensitive USF agents with different ultrasound parameters according to one embodiment described herein.
  • Figure 5B illustrates a plot of the temporal profiles of the normalized differences of the USF signal when switching the thermosensitive USF agents with different ultrasound parameters according to one embodiment described herein.
  • Figure 5C illustrates a plot of the normalized USF signals and signal differences versus time at different background temperatures according to one embodiment described herein.
  • Figure 6 schematically illustrates a USF thermometry system according to one embodiment described herein.
  • Figure 7A illustrates a plot of the UV-vis-NIR spectra of 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC)-1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-ADP_bot, ADP_bot, DPPC- ⁇ /CD-ICG, ⁇ /CD-ICG, and free ICG according to one embodiment described herein.
  • DPPC 1,2-dipalmitoyl-sn-glycero-3- phosphocholine
  • DSPC disistearoyl-sn-glycero-3-phosphocholine
  • ADP_bot DPPC- ⁇ /CD-ICG
  • ⁇ /CD-ICG free ICG according to one embodiment described herein.
  • Figure 7B illustrates a plot of the emission spectra of DPPC-DSPC-ADP_bot and DPPC- ⁇ /CD-ICG according to one embodiment described herein.
  • Figure 8A is a plot of the normalized fluorescent intensity versus temperature (°C) of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-DSPC-ADP_bot, DPPC- ⁇ /CD-ICG, their difference curve according to one embodiment described herein.
  • Figure 8B illustrates a plot of the derivative curve of the difference curve shown in Figure 8A.
  • Figure 9A illustrates a plot of the normalized intensity of the USF signal of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-ADP_bot and DPPC- ⁇ /CD-ICG versus time (sec) at various water temperatures according to one embodiment described herein.
  • Figure 9B illustrates a plot of the maximum temperature (°C) recorded by the IR camera at the ultrasound focal zone versus time (sec) during ultrasound exposure and the corresponding linear fitted curve according to one embodiment described herein.
  • Figure 9C illustrates a plot of the derivative curves of the difference between the USF signals and the characteristic temperature points at water temperatures of 36.90°C and 38.62°C according to one embodiment described herein.
  • Figure 9D illustrates USF images acquired by the 857 nm detection channel of the USF thermometry system according to one embodiment described herein.
  • Figure 9E illustrates USF images acquired by the 730 nm detection channel of the USF thermometry system according to one embodiment described herein.
  • Figure 9F illustrates a plot of temperature (°C) versus time (sec) for the characteristic points and a regression line based on the points wherein the water temperature was 36.90°C with the arrow indicating the initiation of focused ultrasound exposure according to one embodiment described herein.
  • Figure 9G illustrates a plot of temperature (°C) versus time (sec) for the characteristic points and a regression line based on the points wherein the water temperature was 38.62°C with the arrow indicating the initiation of focused ultrasound exposure according to one embodiment described herein.
  • Figure 10 illustrates a plot of the temperature of the water, the temperature of the water as measured by USF, and the temperature of the water as measured by an IR camera according to one embodiment described herein.
  • a stated range of “1.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9.
  • All ranges disclosed herein are also to be considered to include the end points of the range, unless expressly stated otherwise.
  • a range of “between 5 and 10” or “5 to 10” or “5-10” should generally be considered to include the end points 5 and 10.
  • the phrase “up to” is used in connection with an amount or quantity, it is to be understood that the amount is at least a detectable amount or quantity (that is, the amount is a non-zero amount).
  • a material present in an amount “up to” a specified amount can be present from a detectable (or non-zero) amount and up to and including the specified amount.
  • the terms “substantially,” “approximately,” and “about” may be substituted with “within [a percentage] of” what is specified, where the percentage includes 0.1, 1, 5, and 10 percent.
  • IR Infrared
  • MRI magnetic resonance imaging
  • ultrasound ultrasound
  • photoacoustic techniques are generally used to image or map temperature change induced by externally applied energy, such as high intensity focused ultrasound.
  • non-invasive imaging, measuring, and/or mapping of absolute temperature in deep tissue e.g., tissue background temperature
  • measuring the subtle, small, or incremental temperature changes of an object e.g., a nanoparticle or tumor
  • a surrounding environment e.g., surrounding tissue
  • aspects of the present technology are directed towards improved measuring, mapping, and/or imaging technology to quantify the local background temperature in deep tissue and/or to measure small, subtle, and/or incremental temperature change, such as temperature changes caused by disease (e.g., a tumor) or other reasons.
  • temperature determinations can be made for an object within a deep tissue environment.
  • the determined temperature or temperature change may be the temperature of an object with respect to an environment and/or a temperature change caused in the environment by an object.
  • temperature sensitive agents, high resolution imaging systems and methods, ultra-sound switchable fluorescence, and temperature reconstruction methods can in some cases be leveraged to image and/or map the background temperature of an environment and/or an object within the environment with high resolution and accuracy.
  • systems and methods provided herein can, in some embodiments, measure, map, and/or image temperature in centimeter-deep environments, for example, in tissue and with respect to an object in such tissue (e.g., a tumor or nanoparticle). I.
  • methods of mapping and/or imaging temperature in an environment are provided. For instance, in some embodiments, such methods may be used for measuring an initial temperature in an environment. Such methods can comprise disposing a population of photoluminescent thermosensitive agents in an environment, the population having a first switching temperature threshold and a second switching temperature threshold that is higher than the first switching temperature threshold. In some implementations, the environment can subsequently be exposed to an activation source at an initial time to create an activation region within the environment having a temperature that is greater than or equal to the first switching temperature threshold at a first time.
  • the environment can be exposed to a beam of electromagnetic radiation to excite the population of photoluminescent thermosensitive agents at the first time.
  • the method can further comprise detecting a photoluminescent signal (or plurality of photoluminescent signals) emitted by the excited population of photoluminescent thermosensitive agents at the first time.
  • the method can further include exposing the environment to the activation source (or another activation source) at a second time to increase the temperature of the activation region to a temperature that is greater than or equal to the second switching temperature threshold, exposing the environment to a beam of electromagnetic radiation to excite the population of photoluminescent thermosensitive agents at the second time, and detecting a photoluminescent signal (or plurality of photoluminescent signals) emitted by the population of photoluminescent thermosensitive agents at the second time.
  • the method can include determining the initial temperature of the environment based on detecting the photoluminescent signal at the first time and detecting the photoluminescent signal at the second time.
  • a biological environment comprises an in vivo environment, such as a biological tissue, organs, blood vessels, or other portion of a living organism.
  • the biological environment comprises a tumor or tumor vasculature.
  • the tumor or tumor vasculature can be located in any tissue or organ in a living organism, such as the breast, prostate, head, neck, throat, mouth, thyroid, skin, colon, cervix, or uterus.
  • a biological environment comprises an in vitro environment, such as a tissue culture or biological samples.
  • the biological environment of a method described herein can also comprise or be replaced by a biological phantom material or tissue-mimicking phantom material, such as an agar, silicone, polyvinyl alcohol (PVA) gel, polyacrylamide (PAA) gel, or a dispersion of an oil in gelatin. Other phantom materials may also be used.
  • a biological environment comprises deep tissue.
  • “Deep” tissue for reference purposes herein, comprises tissue (or, in the case of a phantom material, an interior region of the phantom material) that is located at least about 1 cm below the exterior or outer surface of an organism, tissue culture, or other larger structure associated with the biological environment (such as, in the case of a phantom material, the outer surface of the phantom material).
  • tissue or, in the case of a phantom material, an interior region of the phantom material that is located at least about 1 cm below the exterior or outer surface of an organism, tissue culture, or other larger structure associated with the biological environment (such as, in the case of a phantom material, the outer surface of the phantom material).
  • deep tissue is located between about 1 cm and about 10 cm, between about 1 cm and about 6 cm, or between about 1 cm and about 5 cm below an outer surface.
  • deep tissue is located more than 10 cm below an outer surface.
  • an outer surface in some embodiments, comprises the surface of the skin of an organism.
  • the environment can be a non-biological material.
  • the non-biological material may comprise a polymeric material or an inorganic material.
  • the inorganic material comprises a nanoparticle.
  • many combinations of these materials are also contemplated.
  • a variety of photoluminescent thermosensitive agents can be used in a system and/or method described herein.
  • a photoluminescent thermosensitive agent has a photoluminescence that changes in a temperature-dependent manner.
  • the photoluminescence of a photoluminescent thermosensitive agent described herein is or comprises a fluorescence signal and/or a phosphorescence signal.
  • the temperature-dependent change in the photoluminescence of a photoluminescent thermosensitive agent described herein may be a discontinuous or binary change (e.g., in which the photoluminescence change is from an “on” state to an “off” state (or vice versa), or from a “bright” state to a “dark” state (or vice versa) in which the luminescence of the agent in the “bright” state is at least two times, at least 5 times, at least 10 times, at least 50 times, or at least 100 times the luminescence of the agent in the “dark” state).
  • a discontinuous or binary change e.g., in which the photoluminescence change is from an “on” state to an “off” state (or vice versa), or from a “bright” state to a “dark” state (or vice versa) in which the luminescence of the agent in the “bright” state is at least two times, at least 5 times, at least 10 times, at least 50 times, or at least 100 times the
  • a photoluminescent thermosensitive agent described herein has a switching temperature threshold between an “on” or “bright” state and an “off” or “dark” state.
  • the switching temperature threshold of the photoluminescent thermosensitive agent corresponds to or is a temperature at which the photoluminescence of the photoluminescent thermosensitive agent exhibits a maximum rate of change (more specifically, a maximum increase), when the photoluminescence is observed as a function of temperature (e.g., ranging from 15°C to 55°C or 20°C to 40°C) during exposure to the relevant activation source.
  • the maximum rate of increase in photoluminescence may be a local maximum or an absolute maximum for that agent, when determined as described above.
  • a photoluminescent thermosensitive agent described herein can have more than one switching temperature threshold.
  • each of the multiple switching temperature thresholds can be defined as described above.
  • each of the switching temperature thresholds (of the single agent) is a local or global maximum in the rate of increase of photoluminescne as a function of temperature, as described above.
  • a population of photosensitive thermoresponsive agents can comprise a single photoluminescent thermosensitive agent or species that can have both a first switching temperature threshold and a second switching temperature threshold.
  • the population of photoluminescent thermosensitive agents can comprise a first photoluminescent thermosensitive agent (or plurality of first photoluminescent thermosensitive agents) having the first switching temperature threshold and a second photoluminescent thermosensitive agent (or plurality of second photoluminescent thermosensitive agents) having the second switching temperature threshold.
  • Concerning populations of more than one photoluminescent thermosensitive agents, in some other instances, photoluminescent thermosensitive agents can be mixed in any proportion.
  • the ratio of a first photoluminescent thermosensitive agent to a second photoluminescent thermosensitive agent may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.
  • the photoluminescent thermosensitive agents can further be mixed with other agents or prepared and/or combined in a solution.
  • a photoluminescent thermosensitive agent described herein can have any photoluminescent emission profile not inconsistent with the objectives of the current invention.
  • a photoluminescent thermosensitive agent may exhibit an emission profile including visible light or centered in the visible region of the electromagnetic spectrum, such as between 450 nm and 750 nm, 500 nm and 700 nm, or 550 nm and 650 nm.
  • a photoluminescent thermosensitive agent exhibits an emission profile including infrared (IR) light or centered in the IR region of the electromagnetic spectrum.
  • a photoluminescent thermosensitive agent described herein exhibits an emission profile centered in the near-IR (NIR, 750 nm-1.5 ⁇ m), short-wavelength IR (SWIR, 1.4-3 ⁇ m), mid-wavelength IR (MWIR, 3-8 ⁇ m), or long-wavelength IR (LWIR, 8-15 ⁇ m).
  • a photoluminescent thermosensitive agent described herein has an emission profile overlapping with a wavelength at which water and/or biological tissue has an absorption minimum, such as a wavelength between about 700 nm and about 800 nm or between about 1.25 ⁇ m and about 1.35 ⁇ m.
  • different populations of photoluminescent thermosensitive agents described herein comprise photoluminescent thermosensitive agents having the same emission profiles.
  • a population of photoluminescent thermosensitive agents described herein comprises different populations of photoluminescent thermosensitive agents having differing emission profiles.
  • a first photoluminescent thermosensitive agent can emit in the NIR and a second photoluminescent thermosensitive agent can emit in the visible region of the electromagnetic spectrum.
  • a first photoluminescent thermosensitive agent has an emission spectrum in one portion of the NIR
  • a second photoluminescent thermosensitive agent has an emission spectrum in a different portion of the NIR.
  • the photoluminescent thermosensitive agents may be encapsulated.
  • the photoluminescent thermosensitive agents may be encapsulated in liposomes or vesicles.
  • the liposomes comprise phospholipids.
  • Non- limiting examples of phospholipids include 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine.
  • DPPC 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine
  • the liposome may also be modified to have other functionality, such as surface functionality.
  • the liposome or vesicle may comprise a PEGylated surface.
  • Such a PEGylated surface can include one or more polyethylene glycol (PEG) chains conjugated or associated with the surface of the liposome or vesicle.
  • the liposome or vesicle further comprises a therapeutic species. Any therapeutic species not inconsistent with the objectives of the present disclosure may be used.
  • the therapeutic species can be a drug such as an anti-tumor drug.
  • the photoluminescent thermosensitive agents may be encapsulated in a phantom material. [0062]
  • the photoluminescent thermosensitive agent can comprise an ultrasound switchable fluorophore.
  • An “ultrasound switchable” fluorophore for reference purposes herein, comprises a fluorophore operable to switch between an “on” state and an “off” state in response to exposure to an ultrasound beam.
  • the ultrasound beam can be either directly or indirectly responsible for the switching response of the fluorophore. For example, in some cases, the ultrasound beam interacts directly with the fluorophore, resulting in a switch between fluorescence states of the fluorophore.
  • the ultrasound beam interacts directly with the immediate environment or microenvironment of the fluorophore and changes at least one property of the fluorophore’s microenvironment.
  • the fluorophore can switch between on and off fluorescence states in response to the environmental change induced by the ultrasound beam.
  • a non-limiting example of an environmental change would be a change in temperature.
  • the fluorophore can be indirectly switchable in response to exposure to an ultrasound beam.
  • the fluorophore can be indirectly switchable in response to exposure to an ultrasound beam.
  • the “on” state of a fluorophore comprises either (1) a state at which the fluorescence intensity of the fluorophore is relatively high compared to the “off” state of the fluorophore, at which the fluorescence intensity is relatively low (assuming the fluorophore is similarly excited in both the “on” state and the “off” state); or (2) a state at which the fluorescence lifetime of the fluorophore is relatively long compared to the “off” state of the fluorophore, at which the fluorescence lifetime is relatively short (again assuming the fluorophore is similarly excited).
  • the “on” and “off” states substantially define a step function in the fluorescence intensity or lifetime profile when plotted as a function of a critical switching parameter such as temperature.
  • a fluorophore having a longer lifetime in an “on” state than an “off” state can be particularly suitable for use in methods described herein using time-gated or time-delayed detection of emitted photons from fluorophores, such as time- gated detection in which only those photons received after a relatively long delay following excitation are counted by the detector as part of the USF signal.
  • the “on” state of a fluorophore exhibits at least about 70 percent, at least about 80 percent, or at least about 90 percent of the theoretical maximum fluorescence intensity of the fluorophore
  • the “off” state of the fluorophore exhibit no more than about 50 percent, no more than about 30 percent, no more than about 10 percent, or no more than about 5 percent of the theoretical maximum fluorescence intensity of the fluorophore.
  • the fluorescence intensity or fluorescence lifetime of a fluorophore changes dues to a conformational or chemical change of the fluorophore in response to a change in environmental conditions, such as exhibited by some thermoresponsive polymers, pH- sensitive chemical species, or pressure sensitive materials.
  • the fluorescence intensity or fluorescence lifetime of a fluorophore changes in response to internal fluorescence quenching, wherein such quenching can be directly or indirectly induced by the presence of ultrasound.
  • an ultrasound-switchable fluorophore described herein comprises a Förster resonance energy transfer (FRET) donor species and a FRET acceptor species, and the distance between the FRET donor species and the FRET acceptor species is altered by the presence of an ultrasound beam.
  • the FRET donor species can be a first fluorescent species or other chromophore
  • the FRET acceptor species can be a second fluorescent species or other chromophore.
  • FRET energy transfer between the donor species and the acceptor species can result in quenching of the fluorescence of the donor species.
  • the acceptor species can be considered to be a fluorescence quenching species of the fluorophore.
  • an ultrasound switchable fluorophore described herein comprises a microbubble comprising one or more FRET donor species and one or more FRET acceptor species attached to the exterior surface of the microbubble, wherein the microbubble is operable to change in size in response to the presence of an ultrasound beam. The change in size can increase or decrease the distance between the FRET donor species and the FRET acceptor species, thus reducing or increasing the FRET energy transfer efficiency.
  • a microbubble described herein can have any size and be formed of any chemical species not inconsistent with the objectives of this disclosure.
  • a microbubble has a diameter between about 1 ⁇ m and about 10 ⁇ m or between about 1 ⁇ m and about 5 ⁇ m.
  • the diameter of the microbubble is not limited to these sizes, and in some cases, other sizes of microbubbles can also be used.
  • a microbubble described herein comprises a gas core surrounded by a shell formed from a polymeric material, such an organic polymeric material.
  • a microbubble comprises a shell formed from one or more of albumin, galactose, lipid, and sulfur hexafluoride.
  • the gas core of a microbubble described herein can comprise one or more of air, nitrogen, and a perfluorocarbon such as octafluoropropane.
  • a microbubble described herein can be formed from a commercially available microbubble, such as a SonoVue TM , Optison TM , Imagent TM , Definity TM , or Targestar TM microbubble.
  • a FRET donor and/or acceptor species described herein can be attached to the surface of such a microbubble in any manner not inconsistent with the objectives of the current invention.
  • a donor and/or acceptor species is attached to the exterior surface of a commercially available microbubble using one or more of a carbodiimide, maleimide, or biotin-streptavidin coupling scheme.
  • any other coupling scheme not inconsistent with the objectives of the current disclosure can be used to attach a donor and/or acceptor species to a microbubble.
  • gas-filled micro-particles such as the above described microbubbles, generate a short but high temperature pulse in and around the particle surface when the microbubble is irradiated with an ultrasound pulse at diagnostic intensity level.
  • This short temperature pulse spatially decays very fast (only ⁇ 0.2°C left at a distance of 1 micron away from the bubble surface).
  • tissue overheating caused by microbubbles is minimalized from this fast temperature decay.
  • this microscopic heating principle is effective for heating ultrasound switchable fluorophores because ultrasound switchable fluorophores are small nanoparticles that can be attached on the microbubble’s surface.
  • ultrasound switchable fluorophores can be attached to a microbubble via a biotin/streptavidin linkage. Moreover, any other linkage not inconsistent with the objectives of this disclosure can be used to attach ultrasound switchable fluorophores to a microbubble.
  • a highly ultrasound-absorbing polymer such as a biodegradable polyurethane with pendent carboxyl groups (PU-COOH), can alternatively be used instead of the microbubbles. These ultrasound-absorbing polyurethanes can form relatively rigid gas-filled sub- micro-particles ( ⁇ 700 nm in diameter).
  • an ultrasound- absorbing polymer can comprise a Pluronic polymer with pendent carboxyl groups similar in size to the polyurethanes, such as F127, F98, F98-PEG20k, F98-PEG30k, F98-PEG40k, F68 and its PEGylated polymers, which have been functionalized to incorporate pendent carboxyl groups.
  • These ultrasound-absorbing polymers are generally smaller in diameter than microbubbles, reducing their acoustic attenuation compared to microbubbles. However, their relatively rigid structures can sometimes display more resilient bio-stability than microbubbles.
  • an ultrasound switchable fluorophore described herein comprises a thermoresponsive polymer.
  • a “thermoresponsive” polymer for reference purposes herein, comprises a polymer having a physical or chemical property that changes in a temperature- dependent manner, wherein the change is a discontinuous or binary change.
  • the physical conformation or polarity of a thermoresponsive polymer changes in a temperature-dependent manner, and the thermoresponsive polymer exhibits a first conformation below a threshold temperature and a second, substantially different conformation above the threshold temperature.
  • a thermoresponsive polymer exhibits an expanded coil or chain confirmation below a threshold temperature and exhibits a compact or globular conformation above the threshold temperature.
  • this threshold temperature can be referred to as the “lower critical solution temperature” (LCST) of the polymer.
  • LCST lower critical solution temperature
  • Other conformational changes (not limited to this example) can also be characterized similarly as having a LCST.
  • thermoresponsive polymer comprises a poly(N- isopropylacrylamide) or a copolymer of N-isopropylacrylamide with one or more of acrylamide, N-tert-butylacrylamide, acrylic acid, allylamine, or a polyoxypropylene-polyoxyethylene block copolymer.
  • a thermoresponsive polymer comprises a poly(N-vinylcaprolacatam) (PVCL) or a poloxamer such as a Pluronic polymer.
  • PVCL poly(N-vinylcaprolacatam)
  • Pluronic polymer a poloxamer
  • thermoresponsive polymer of a fluorophore described herein comprises one or more fluorescent moieties or is conjugated to one or more fluorescent species, such as one or more fluorescent dye molecules.
  • the fluorescent dye molecules can comprise any fluorescent dyes not inconsistent with the objectives of this disclosure, such as the commercially available ZnPC (Zinc phthalocyanines) family of dyes (e.g., ZnPc, ZnPcTTB, ZnPcHF, ZnPcOB, among others), the ADP(CA) 2 family of dyes, or ICG-based agents (indocyanine greens).
  • ZnPC Zinc phthalocyanines
  • ADP(CA) 2 ADP(CA) 2 family of dyes
  • ICG-based agents indocyanine greens
  • thermoresponsive polymer is coupled to a fluorescent species through one or more covalent bonds such as one or more ester bonds or one or more amide bonds.
  • covalent bonds such as one or more ester bonds or one or more amide bonds.
  • a fluorophore described herein comprises a fluorescent material dispersed in and/or attached to the surface of a thermoresponsive polymer nanoparticle.
  • the fluorescence properties of the fluorescent material can be dependent on a change of the conformation, polarity, or other physical or chemical property of the polymer nanoparticle.
  • the property change can be a temperature-dependent change.
  • a change in temperature of the thermoresponsive polymer nanoparticle can result in a change in fluorescence intensity and/or lifetime of the fluorescent material, including a change between an “on” state of the fluorescent material and an “off” state of the fluorescent material.
  • a thermoresponsive polymer nanoparticle can exhibit a temperature-dependent polarity, and the fluorescent material dispersed in the nanoparticle can exhibit a polarity-dependent fluorescence intensity and/or lifetime.
  • a change in the temperature of the nanoparticle can result in a change in the fluorescence intensity and/or lifetime of the fluorophore.
  • a thermoresponsive polymer nanoparticle can have a hydrophilic interior below a threshold temperature and a hydrophobic interior above the threshold temperature.
  • a nanoparticle can exhibit a temperature-dependent size when dispersed in a polar or non-polar solvent.
  • the nanoparticle when dispersed in water or another polar solvent below the threshold temperature, the nanoparticle can exhibit a larger size due to the presence of water in the hydrophilic interior of the nanoparticle.
  • an ultrasound-switchable fluorophore is formed by incorporating a fluorescent material such as a fluorescent dye within the interior of a polymeric nanoparticle or micelle, such that the polymeric nanoparticle or micelle acts as a nanocapsule for the fluorescent material.
  • the polymeric nanoparticle can be formed from a thermoresponsive polymer, such as a thermoresponsive polymer described hereinabove.
  • a thermoresponsive polymer such as a thermoresponsive polymer described hereinabove.
  • polymers suitable for forming nanocapsules described herein include Pluronic F127, F98, F98- PEG20k, F98-PEG30k, F98-PEG40k, F68 and its PEGylated polymers, poly(N- isopropylacrylamide) or a copolymer of N-isopropylacrylamide with one or more of acrylamide, N-tert-butylacrylamide, acrylic acid, allylamine, or a polyoxypropylene-polyoxyethylene block copolymer, or poly(N-vinylcaprolacatam) (PVCL).
  • a nanoparticle or nanocapsule can be formed by copolymerizing a thermoresponsive polymer described hereinabove with a PEG and/or by conjugating a PEG as a pendant group to a thermoresponsive polymer.
  • a fluorophore in some cases, can have a switching threshold that is controlled at least in part by the inclusion of PEG, as described further in the ‘014 publication.
  • a polymer nanoparticle such as a thermoresponsive polymer nanoparticle or a polymer nanocapsule described herein can have any size or shape not inconsistent with the objectives of the current disclosure.
  • thermoresponsive polymer nanoparticle is substantially spherical and has a diameter between about 10 nm and about 300 nm, between about 50 nm and about 250 nm, between about 50 nm and about 200 nm, or between about 70 nm and about 150 nm.
  • a polymer nanocapsule is substantially spherical and has a diameter of less than about 100 nm or less than about 50 nm.
  • a polymer nanocapsule has a size between about 20 nm and about 90 nm, between about 20 nm and about 80 nm, or between about 20 nm and about 70 nm. Other sizes and shapes are also possible.
  • any fluorescent material not inconsistent with the objectives of the current invention can be dispersed in and/or attached to a thermoresponsive polymer nanoparticle or other polymer nanoparticle to form a fluorophore described herein.
  • the fluorescent material exhibits a polarity-sensitive fluorescence intensity and/or lifetime.
  • the fluorescent material exhibits a temperature-dependent, viscosity-dependent, pH-dependent, and/or an ionic strength-dependent fluorescence intensity and/or lifetime.
  • Non-limiting examples of fluorescent materials suitable for use in some embodiments described herein include organic dyes such as N,N-dimethyl-4-benzofurazansulfonamide (DBD); 4-(N,N-dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DBD-ED); indocyanine green (ICG); a Dylight-700 such as Dylite-700-2B; IR-820; 3,3′- Diethylthiatricarbocyanine iodide (DTTCI); LS-277; LS-288; a cypate; a rhodamine dye such as rhodamine 6G or rhodamine B; or a coumarin.
  • organic dyes such as N,N-dimethyl-4-benzofurazansulfonamide (DBD); 4-(N,N-dimethylaminosulfonyl)-7-(2-amin
  • a fluorescent material comprises an azadipyrromethene.
  • a fluorescent material comprises an inorganic species such as a semiconductor nanocrystal or quantum dot, including a II-VI semiconductor nanocrystal, such as ZnS or CdSe or a III-V semiconductor nanocrystal such as InP or InAs.
  • a fluorescent material comprises a Lanthanide species.
  • fluorescent materials suitable for use in an ultrasound- switchable fluorophore described herein include the fluorescent materials described in Amin et al., “Syntheses, Electrochemistry, and Photodynamics of Ferrocene-Azadipyrromethane Donor- Acceptor Dyads and Triads,” J. Phys. Chem. A 2011, 115, 9810-9819; Bandi et al., “A Broad- Band Capturing and Emitting Molecular Triad: Synthesis and Photochemistry,” Chem.
  • An ultrasound-switchable fluorophore described herein can have any fluorescence emission profile not inconsistent with the objectives of the current disclosure.
  • a fluorophore exhibits an emission profile including visible light or centered in the visible region of the electromagnetic spectrum, such as between 450 nm and 750 nm, 500 nm and 700 nm, or 550 nm and 650 nm.
  • a fluorophore exhibits an emission profile including infrared (IR) light or centered in the IR region of the electromagnetic spectrum.
  • IR infrared
  • a fluorophore described herein exhibits an emission profile centered in the near-IR (NIR, 750 nm-1.5 ⁇ m), short-wavelength IR (SWIR, 1.4-3 ⁇ m), mid- wavelength IR (MWIR, 3-8 ⁇ m), or long-wavelength IR (LWIR, 8-15 ⁇ m).
  • a fluorophore described herein has an emission profile overlapping with a wavelength at which water and/or biological tissue has an absorption minimum, such as a wavelength between about 700 nm and about 800 nm or between about 1.25 ⁇ m and about 1.35 ⁇ m.
  • a population of ultrasound-switchable fluorophores described herein comprise fluorophores having differing emission profiles.
  • a first fluorophore of a population can emit in the NIR and a second fluorophore of the population can emit in the visible region of the electromagnetic spectrum.
  • a fluorophore of the population has an emission spectrum in one portion of the NIR, and the second fluorophore of a population has an emission spectrum in a different portion of the NIR.
  • different populations of ultrasound switchable fluorophores described herein comprise the same fluorophore having the same emission profiles.
  • different populations of ultrasound-switchable fluorophores described herein comprise different fluorophores having different emission profiles.
  • an emission profile of a first population of ultrasound switchable fluorophores can have a first fluorophore between about 680 nm and about 710 nm
  • the emission profile of a second population of ultrasound switchable fluorophores having a second fluorophore can be between about 740 nm and about 770 nm.
  • the first or second ultrasound switchable fluorophores comprise a fluorescent material having a peak emission wavelength between 680 nm and 710 nm, between 740 nm and 770 nm, or > 800 nm.
  • the first ultrasound- switchable fluorophores are configured to emit light having a first average peak wavelength
  • the second ultrasound-switchable fluorophores are configured to emit light having a second average peak wavelength, and wherein the second average peak wavelength is 25-75 nm longer than the first average peak wavelength.
  • a method described herein comprises exposing the environment to an activation source to create an activation region with the environment having a temperature greater than or equal to the first switching temperature threshold and/or second switching temperature threshold.
  • an “activation region” comprises a region of the environment in which the temperature is greater than or equal to the first switching temperature threshold and/or second switching temperature threshold of the population of photoluminescent thermosensitive agents described herein.
  • the size, shape, and/or other properties of the activation region may be determined by the activation source used to form the activation region.
  • the activation region has a lateral dimension (or two lateral dimensions, such as an x-dimension and a y-dimension) and/or an axial dimension (such as a z-dimension) of less than about 2 mm, less than 1.5 mm, or less than about 1 mm. In some embodiments, the activation region has a lateral dimension (or lateral dimensions) and/or an axial dimension of less than about 700 ⁇ m or less than about 500 ⁇ m.
  • the activation region has a lateral dimension (or lateral dimensions) and/or an axial dimension of about 300 ⁇ m to about 2 mm, about 400 ⁇ m to about 1.5 mm, about 400 ⁇ m to about 1 mm, about 400 ⁇ m to about 700 ⁇ m, or about 400 ⁇ m to about 500 ⁇ m.
  • the lateral and axial dimensions e.g., the x-, y-, and z-dimensions
  • the lateral and axial dimensions of the activation region are different, thereby providing a relatively anisotropic activation region.
  • the lateral and axial dimensions are substantially the same, thereby providing a relatively “square” or “cubic” or isotropic activation region.
  • the activation source may vary.
  • the activation source is at least one of an ultrasound beam, a source of electromagnetic radiation, a magnetic field, an electron or particle beam, or a chemical stimulus. Other activation sources may also be used.
  • a single activation source is used. Alternatively, in other instances, a plurality or combination of activations sources is used.
  • the activation source can be applied wide-field, and in some other instances, the activation source can be applied in a focused manner, such as using a focused beam (e.g., by raster scanning).
  • a focused beam e.g., by raster scanning.
  • a single ultrasound transducer such as a high intensity focused ultrasound (HIFU) transducer, is used to provide the ultrasound beam.
  • HIFU high intensity focused ultrasound
  • a plurality of ultrasound beams is provided using a plurality of ultrasound transducers.
  • the ultrasound beam can have any ultrasound frequency not inconsistent with the objectives of the current disclosure.
  • an ultrasound beam comprises an oscillating sound pressure wave with a frequency of greater than about 20 kHz or greater than about 2 MHz.
  • an ultrasound beam described herein has a frequency of up to about 5 GHz or up to about 3 GHz.
  • an ultrasound beam has a frequency between about 20 kHz and about 5 GHz, between about 50 kHz and about 1 GHz, between about 500 kHz and about 4 GHz, between about 1 MHz and about 5 GHz, between about 2 MHz and about 20 MHz, between about 2 MHz and about 10 MHz, between about 5 MHz and about 200 MHz, between about 5 MHz and about 15 MHz, between about 200 MHz and about 1 GHz, between about 500 MHz and about 5 GHz, or between about 1 GHz and about 5 GHz.
  • an ultrasound beam described herein can have any power not inconsistent with the objectives of the current disclosure.
  • an ultrasound beam has a power between about 0.1 W/cm 2 and about 10 W/cm 2 , between about 0.1 W/cm 2 and about 5 W/cm 2 , between about 0.5 W/cm 2 and about 5 W/cm 2 , between about 1 W/cm 2 and about 10 W/cm 2 , or between about 1 W/cm 2 and about 5 W/cm 2 .
  • an ultrasound beam has a power between about 100 W/cm 2 and about 5000 W/cm 2 , or between about 100 W/cm 2 and about 3000 W/cm 2 .
  • the use of an ultrasound beam having a high power, such as a power described herein, can result in the generation of non-linear effects within the activation region.
  • the effective size of the activation region can be reduced in this manner, leading to improved imaging resolution.
  • the activation source may comprise or be a source of electromagnetic radiation. It is to be understood that any source of the electromagnetic radiation not inconsistent with the objectives of the present disclosure may be used. Many suitable sources of electromagnetic radiation will be readily apparent to those of ordinary skill in the art.
  • the source of electromagnetic radiation comprises a source of visible light, NIR light, or IR light.
  • the electromagnetic radiation comprises a source of ultraviolet (UV) light.
  • the electromagnetic radiation comprises a wavelength maximum of approximately 671 nm, 730 nm, 800 nm, or 810 nm.
  • the source of electromagnetic radiation may provide electromagnetic radiation (e.g., a beam of electromagnetic radiation) having a peak or average wavelength between 600 nm and 900 nm, between 650 nm and 850 nm, between 700 nm and 800 nm, between 600 nm and 800 nm, between 600 nm and 700 nm, between 700 nm and 900 nm, between 800 nm and 900 nm, between 900 nm and 1000 nm, between 1000 nm and 1100 nm, between 1100 nm and 1200 nm, between 1200 nm and 1300 nm, between 1400 nm and 1500 nm, or between 1600 nm and 1700 nm.
  • the source of electromagnetic radiation comprises a light-emitting diode.
  • the source of electromagnetic radiation comprises a laser.
  • a “laser” can refer to a single lasing device that produces a single beam of laser light from a single lasing medium.
  • a laser described herein can be a pulsed laser or a continuous wave (CW) laser.
  • CW continuous wave
  • the laser can produce time-modulated pulses of the laser beam.
  • a laser diode may be used.
  • a laser or laser beam described herein can have any power and any peak or average emission wavelength not inconsistent with the objectives of this disclosure.
  • a laser or laser beam of a device described herein has a peak or average emission wavelength in the infrared (IR) region of the electromagnetic spectrum.
  • the laser or laser beam has a peak or average emission wavelength in the range of 1-4 ⁇ m, 1-3 ⁇ m, 2-4 ⁇ m, 2-3 ⁇ m, 8-12 ⁇ m, or 9-11 ⁇ m.
  • the laser or laser beam comprises an erbium-doped yttrium aluminum garnet (Er:YAG) laser or laser beam or a neodymium-doped YAG (Nd:YAG) laser or laser beam having a peak or average emission wavelength of 2940 nm or 1064 nm.
  • the laser or laser beam comprises a carbon dioxide laser or laser beam.
  • a laser beam described herein can also have a peak or average emission wavelength in the visible region of the electromagnetic spectrum.
  • peak or average emission wavelengths suitable for use in some embodiments described herein include 532 nm, 695 nm, 755 nm, 1064 nm, and 1470 nm, or 2940 nm.
  • the laser beam can have an average wavelength between 700 and 1500 nm.
  • the laser beam can have an average wavelength between 900 and 1300 nm.
  • a laser or laser beam of a device described herein has an average power of 1 to 10 W, 10 W to 50 W, or 50 to 200 W.
  • the activation source may comprise a magnetic field.
  • the magnetic field is a static field.
  • the magnetic field is a pulsed magnetic field.
  • the magnetic field is an alternating magnetic field.
  • the magnetic field may induce eddy currents in the environment and/or the activation region.
  • a magnetic field can have any maximum magnetic flux density not inconsistent with the objectives of the current disclosure.
  • a magnetic field described herein can have a maximum magnetic flux density between 0.5 and 5 T, between 0.5 and 4 T, between 0.5 and 3 T, between 0.5 and 2 T, between 0.5 and 1 T, between 1 and 5 T, between 1 and 4 T, between 1 and 3 T, between 1 and 2 T, between 2 and 5 T, between 2 and 4 T, between 2 and 3 T, between 3 and 5 T, between 3 and 4 T, or between 4 and 5 T.
  • the source of a magnetic field may be a ferromagnetic material.
  • Non-limiting examples of a ferromagnetic material include iron, cobalt, and nickel.
  • the source of a magnetic field is an electromagnet.
  • an electromagnet may comprise a magnet wherein the magnetic field of the magnet is produced by an electric current and the magnetic field is absent when the electric current is absent.
  • an electromagnet comprises a wire configured into a coil shape.
  • the source of the magnetic field may comprise a plurality of sources to produce the magnetic field.
  • the activation source may comprise an electron or particle beam. An electron or particle beam described herein can have any energy not inconsistent with the objectives of the current disclosure.
  • the energy of the electron or particle beam may be between 5 eV and 100,000 eV, between 5 eV and 50,000 eV, between 5 eV and 10,000 eV, between 5 eV and 1,000 eV, between 5 eV and 500 eV, between 5 eV and 100 eV, between 5 eV and 50 eV, between 50 eV and 100,000 eV, between 50 eV and 50,000 eV, between 50 eV and 10,000 eV, between 50 eV and 1,000 eV, between 50 eV and 500 eV, between 50 eV and 100 eV, between 100 eV and 100,000 eV, between 100 eV and 50,000 eV, between 100 eV and 10,000 eV, between 100 eV and 1,000 eV, between 100 eV and 500 eV, between 500 eV and 100,000 eV, between 500 eV and 100,000 eV, between 500 eV and 100,000 eV, between 500 eV and 100,000 eV
  • an electron or particle beam described herein can have any beam current not inconsistent with the objectives of the current disclosure.
  • the beam current may be between 1 nA and 2,000,000 nA, between 1 nA and 1,000,000 nA, between 1 nA and 500,000 nA, between 1 nA and 250,000 nA, between 1 nA and 100,000 nA, between 1 nA and 50,000 nA, between 1 nA and 25,000 nA, between 1 nA and 10,000 nA, between 1 nA and 5,000 nA, between 1 nA and 1,000 nA, between 1 nA and 100 nA, between 100 nA and 2,000,000 nA, between 100 nA and 1,000,000 nA, between 100 nA and 500,000 nA, between 100 nA and 250,000 nA, between 100 nA and 100,000 nA, between 100 nA and 100,000 nA, between 100 nA and 50,000 nA, between 100 nA and 25,000 nA, between 100
  • the spot size of the electron or particle beam may be between 0.5 mm and 100 mm, between 0.5 mm and 90 mm, between 0.5 mm and 80 mm, between 0.5 mm and 70 mm, between 0.5 mm and 60 mm, between 0.5 mm and 50 mm, between 0.5 mm and 40 mm, between 0.5 mm and 30 mm, between 0.5 mm and 20 mm, between 0.5 mm and 10 mm, between 0.5 mm and 5 mm, between 0.5 mm and 1 mm, between 1 mm and 100 mm, between 1 mm and 90 mm, between 1 mm and 80 mm, between 1 mm and 70 mm, between 1 mm and 60 mm, between 1 mm and 50 mm, between 1 mm and 40 mm, between 1 mm and 30 mm, between 1 mm and 20 mm, between 1 mm and 10 mm, between 1
  • the source of an electron or particle beam may be an electron gun.
  • an electron gun may comprise a thermionic electron gun.
  • the thermionic source of the thermionic electron gun may comprise a tungsten filament source, a lanthanum hexaboride source, or a cerium hexaboride source.
  • the electron gun may comprise a field emission gun (FEG).
  • the FEG may be a Schottky field emission gun (FEG) or a cold FEG.
  • the activation source may comprise a chemical stimulus.
  • such a chemical stimulus may comprise a stimulus that creates or helps to create an activation region within the environment through chemical means.
  • the chemical stimulus may react or interact with the photoluminescent thermosensitive agent directly or indirectly to produce the activation region.
  • the chemical stimulus may react or interact with a component already present in the environment to produce the activation region.
  • the chemical stimulus may comprise multiple chemical stimuli that react or interact to create the activation region.
  • the chemical stimulus may create or help to create an activation region having a temperature greater than or equal to the first and/or second switching temperature threshold through chemical means via a chemical reaction.
  • the chemical stimulus may modulate, alter, or change the pH of the environment to create or help to create the activation region. It is also to be understood that any chemical stimulus not inconsistent with the objectives of the present disclosure may be used. Many suitable sources of chemical stimuli will be readily apparent to those of ordinary skill in the art.
  • the chemical stimulus may be the components of a reaction that when applied to the environment, react in such a way that the reaction is exothermic, heating the environment to create the activation region.
  • Methods described herein also comprise exposing the environment to a beam of electromagnetic radiation to excite the population of photoluminescent thermosensitive agents.
  • the population of photoluminescent thermosensitive agents can be excited with a beam of electromagnetic radiation in any manner not inconsistent with the objectives of the current disclosure.
  • the population of photoluminescent thermosensitive agents is excited using a laser excitation source, such as a diode laser.
  • the population of photoluminescent thermosensitive agents is excited using one or more light emitting diodes (LEDs) or a broadband excitation source.
  • LEDs light emitting diodes
  • an excitation source described herein can provide any wavelength of light not inconsistent with the objectives of the current disclosure.
  • the population of photoluminescent thermosensitive agents described herein is excited with a beam of electromagnetic radiation comprising visible light, NIR light, or IR light. In other cases, the beam of electromagnetic radiation comprises ultraviolet (UV) light.
  • UV ultraviolet
  • a population of photoluminescent thermosensitive agents described herein is excited with a beam of electromagnetic radiation comprising a wavelength maximum of approximately 671 nm, 730 nm, 800 nm, or 810 nm.
  • the population of photoluminescent thermosensitive agents can also be excited with a beam of electromagnetic radiation having a wavelength between 600 nm to 900 nm, 650 nm to 850 nm, 700 nm to 800 nm, 600 nm to 800 nm, 600 nm to 700 nm, 700 nm to 900 nm, 800 nm to 900 nm, 900 nm to 1000 nm, 1000 nm to 1100 nm, 1100 nm to 1200 nm, 1200 nm to 1300 nm, 1400 nm to 1500 nm, or 1600 nm to 1700 nm.
  • the beam of electromagnetic radiation used to excite the population of photoluminescent thermosensitive agents may be continuously applied to the population of photoluminescent thermosensitive agents. That is, the beam of electromagnetic radiation used to excite the population of photoluminescent thermosensitive agents is continuously applied from the first time to the second time without its removal or being turned “off.” Moreover, in some embodiments, a single beam may be used to excite the population of photoluminescent thermosensitive agents at both the first time and the second time. [00100] In contrast, methods described herein may implement multiple sources of the beam of electromagnetic radiation to excite the population of photoluminescent thermosenstive agents.
  • methods described herein may use multiple excitation beams to excite one or more photoluminescent thermosensitive agents. Further, in some embodiments, methods described herein may use multiple excitation beams with the same wavelength or differing wavelengths.
  • exposing the environment to a beam of electromagnetic radiation comprises exposing the environment to a first excitation beam and a second excitation beam where the first excitation beam and the second excitation beam can, in some cases, have differing wavelengths.
  • methods described herein can further comprise detecting a photoluminescent signal (or plurality of signals) emitted by the excited population of photoluminescent thermosensitive agents at a given time, for example, at a first time.
  • a photoluminescent signal emitted by the excited population of photoluminescent thermosensitive agents is a fluorescence signal and/or a phosphorescence signal.
  • a “fluorescence” signal can exhibit luminescence by emitting a photon from a singlet excited state, as opposed to emitting a photon from a triplet excited state or other high spin multiplicity state.
  • a “fluorescent” photoluminescent thermosensitive agent can exhibit relatively rapid emission following excitation (or absorption) due to emission through a quantum mechanically “allowed” energy transition. For example, in some cases, absorption and subsequent fluorescent emission can occur in about 10 nanoseconds or less.
  • a photoluminescent signal emitted by the excited population of photoluminescent thermosensitive agents may be a phosphorescence signal.
  • a “phosphorescence” luminescence occurs by the photoluminescent thermosensitive agents emitting a photon from (or through) a triplet excited state or other higher spin multiplicity state, as opposed to emitting a photon from a singlet excited state.
  • phosphorescent emission can be due to intersystem crossing of a charge carrier (e.g., an electron).
  • a “phosphorescent” population of photoluminescent thermosensitive agents can exhibit relatively slow emission following excitation (or absorption), due to emission through a quantum mechanically “forbidden” energy transition. For instance, in some cases, absorption and phosphorescent emission can occur on a timescale no shorter than 1- 10 milliseconds.
  • the photoluminescent signal emitted by the excited population of photoluminescent thermosensitive agents at the first time and/or the second time may also comprise visible light.
  • visible light comprises the range of wavelengths from 380 nm to 750 nm.
  • the photoluminescent signal at the first time is different than the photoluminescent signal at the second time.
  • the difference between the photoluminescent signal at the first time and the photoluminescent signal at the second time is a difference in the wavelength of visible light emitted (i.e., the color of the visible light).
  • a “color” can refer to color perceived by an average adult, healthy human eye and brain in the visible region of the electromagnetic spectrum, where different visible colors can be assigned to different wavelengths from approximately 380 nm to approximately 750 nm, more specifically from about 400 nm to about 700 nm (e.g., such that a wavelength of 520 nm corresponds to a “green” color, and a wavelength of 470 corresponds to a “blue” color).
  • a perceived color can be based on absorption, reflection, and/or scattering of light such as white light, natural sunlight, or broadband light. It is also possible, in some cases, for perceived color to be based on emission of light.
  • a photoluminescent signal at a first time can be detected with a first detector or detection channel, and the photoluminescent signal at the second time can be detected with a second detector or detection channel.
  • the photoluminescent signals of a method described herein can be detected using any detector configuration not inconsistent with the objectives of the current disclosure.
  • the photoluminescent signal emitted by the population of photoluminescent thermosensitive agents can be detected using a detector comprising a plurality of optical fiber collectors coupled to a camera or photon counter, such as a charge coupled device (CCD) or a photomultiplier tube (PMT), wherein the optical fiber collectors are spatially distributed around an exterior surface of the environment. Further, in some cases, the optical fiber collectors are spatially distributed around the environment or around a detection surface of the environment (such as skin or another exterior surface of the environment). Any desired number of optical fiber collectors can be used. In some embodiments, up to 30, up to 20, or up to 10 optical fiber collectors can be used.
  • optical fiber collectors can be used. Other configurations are also possible. Moreover, resolution or isolation of photoluminescent signals can be achieved or improved, if needed, by using excitation and/or emission filters for one or more excitation beams and/or one or more detectors or detection channels. [00104] Additionally, in some cases, a plurality of photoluminescent signals at a plurality of locations within an environment is detected by raster scanning the environment. Such raster scanning can include raster scanning of one or more of the activation sources across or within the environment, such that the activation source sequentially generates a series of activation regions at different locations within the environment.
  • a method can comprise determining an initial temperature of an environment and can comprise determining a function corresponding to a photoluminescent signal-temperature profile of the population of photoluminescent thermosensitive agents. Moreover, in some instances, determining an initial temperature of the environment can further comprise determining a linear heating speed (Vh) of the environment.
  • Vh linear heating speed
  • T is the initial temperature
  • T a is the first switching temperature threshold
  • T b the second switching temperature threshold
  • t a is the first time
  • t b is the second time
  • t 0 is the initial time (e.g., the time of the initial temperature).
  • a system can comprise a population of photoluminescent thermosensitive agents, the population having a first switching temperature threshold and a second switching temperature threshold, at least one activation source, at least one excitation beam, and at least one photoluminescence detector.
  • the photoluminescent thermosensitive agents, activation source(s), excitation beam(s), and photoluminescence detector(s) described herein with respect to methods for measuring, imaging, and/or mapping temperature(s) within an environment as described in Section I can be used for systems described herein.
  • the population of photoluminescent thermosensitive agents used in systems described herein is created, configured, or adapted to create an activation region within the environment with a temperature greater than or equal to the first switching temperature threshold and/or a second switching temperature threshold of the population of photoluminescent thermosensitive agents.
  • the population of photoluminescent thermosensitive agents can comprise a first photoluminescent thermosensitive agent having the first switching temperature threshold and a second photoluminescent thermosensitive agent having the second switching temperature threshold.
  • a single photoluminescent thermosenstive agent can have a first switching temperature threshold and a second switching temperature threshold.
  • the first photoluminescent thermosensitive agent has a first emission profile
  • the second photoluminescent thermosensitive agent has a second emission profile differing from the first emission profile.
  • the excitation beam used in systems described herein is created, configured, or adapted to excite the population of photoluminescent thermosensitive agents.
  • the excitation beam is a beam of electromagnetic radiation.
  • the beam of electromagnetic radiation can be any beam of electromagnetic radiation not inconsistent with the objectives of the current disclosure.
  • the excitation beam can be produced by a laser excitation source such as a diode laser.
  • the excitation beam is produced using one or more light emitting diodes (LEDs) or a broadband excitation source.
  • the excitation beam described herein can provide any wavelength of light not inconsistent with the objectives of the current disclosure.
  • the excitation beam is a beam of electromagnetic radiation comprising visible light, NIR light, or IR light. In other cases, the beam of electromagnetic radiation comprises ultraviolet (UV) light.
  • the excitation beam is a beam of electromagnetic radiation comprising a wavelength maximum of approximately 671 nm, 730 nm, 800 nm, or 810 nm.
  • the excitation beam can also be a beam of electromagnetic radiation having a wavelength between 600 nm to 900 nm, 650 nm to 850 nm, 700 nm to 800 nm, 600 nm to 800 nm, 600 nm to 700 nm, 700 nm to 900 nm, 800 nm to 900 nm, 900 nm to 1000 nm, 1000 nm to 1100 nm, 1100 nm to 1200 nm, 1200 nm to 1300 nm, 1400 nm to 1500 nm, or 1600 nm to 1700 nm.
  • the photoluminescence detector used in systems described herein is created, configured, or adapted to detect the photoluminescent signal emitted by the population of photoluminescent thermosensitive agents.
  • a plurality of photoluminescence detectors may be used.
  • the photoluminescence detector can be any photoluminescence detector not inconsistent with the objectives of the current disclosure.
  • the photoluminescence detector comprises an image recording device or a camera.
  • the image recording device is controlled by a software trigger, such as from an external or separate software, as further described herein. For instance, in some cases, the image recording device does not use an external hardware trigger and/or does not use a trigger mode integrated into the image recording device.
  • the image recording device is an electron-multiplying CCD (EMCCD).
  • EMCCD electron-multiplying CCD
  • the gain of the image recording device is set to a value of 5 or greater or 9 or greater. In some embodiments, the gain in such a system is between 5 and 9. In some cases, the image recording device is an EMCCD, and the EMCCD is set to a gain greater than 5 or greater than 9. In some cases, the EM gain is between 5 and 9.
  • the image recording device is an EMCCD
  • the EMCCD is set to an electron-multiplying (EM) gain corresponding to a peak signal-to-noise ratio (SNR) at a preselected imaging depth.
  • EM gain settings can permit higher resolution and more effective imaging, including in deep tissue.
  • Such EM gains can also be stable.
  • a maximum change of photoluminescence intensity detected by the image recording device of the system over a 5-second period is 5% or less, 3% or less, or 1% or less, including when measured in a manner described herein.
  • the intensity change is 0.1 to 5%, 0.1 to 3%, or 0.1 to 1%.
  • the internal temperatures of breast cancers were invasively measured in patients. Fast-growing tumors were found to be hotter than benign or slowly growing tumors and normal tissues ( ⁇ 1-3 °C or higher).
  • the temperature distribution in cancerous breast tissue has two apparent changes: (1) The absolute temperature is elevated compared with the surrounding healthy tissue, especially in the region where the tumor is located, and (2) the spatial distribution of the temperature is distorted by the tumor.
  • the temperature gradient (°C/mm) in the healthy breast is spatially smooth.
  • the temperature gradient is significantly distorted in the cancerous breast, especially in the tumor area. Oscillations of the temperature gradients occur around the tumor boundary area.
  • thermography was used as a breast cancer screening tool in the 1960s- 1980s.
  • breast thermography relies on infrared photon detection within a wavelength spectrum of approximately 3-10 microns for imaging temperature.
  • infrared photons generated from deep breast tissues are completely absorbed by water molecules present in the tissue, a complete loss of information beneath the skin can occur.
  • the absolute value of the surface temperature of the skin is not specific to the tumor’s activeness and can be affected by many other factors, and breast thermography diagnosis based on skin surface temperature is neither specific nor sensitive to tumor activeness for diagnosis purposes.
  • breast thermography has several drawbacks, rendering it an unviable option for clinical imaging.
  • thermometry In magnetic resonance imaging (MRI)-based thermometry, temperature sensitivity is ⁇ 2% per °C in the spin-lattice relaxation method, which leads to a limited temperature resolution of ⁇ 1 °C in a clinical scanner and ⁇ 0.3-0.5 °C in a pre-clinical scanner with a strong magnetic field.
  • Ultrasound-based thermometry has even lower temperature sensitivity ( ⁇ 0.05% per °C) and temperature resolution.
  • Photoacoustic-based thermometry has relatively higher sensitivity ( ⁇ 4- 5% per °C), but this sensitivity is still very limited.
  • most technologies aim to image the relative change in tissue temperature induced by externally applied energy, such as high intensity focused ultrasound or other radiation, including MRI-, ultrasound-, and photoacoustic- based thermometry.
  • ICG ultrasound switchable fluorescence
  • NIR near-infrared
  • Liposome-based USFs typically exhibit a sharp fluorescence transition from an “off” to an “on” state when the environmental temperature rises above a threshold, the lower critical solution temperature (LCST). Thus, their fluorescence-vs.-temperature profiles appear as sharp step functions.
  • the LCST is controlled slightly above the background tissue temperature.
  • an ultrasound pulse is applied to increase the tissue temperature in its focal volume above the LCST. These ultrasound-induced fluorescence photons are called the USF signal.
  • the threshold i.e., the LCST
  • the threshold can be controlled by adjusting the lipid compositions of the outer liposomal shell.
  • the second threshold LCST 2
  • Tt tissue background temperature
  • Tb is the temperature where the highest slope of the fluorescence-vs.-temperature profile curve of the second liposome is reached.
  • T a and T b (and LCSTs) can be independently found by measuring the characteristic curves of the fluorescence-vs.-temperature profiles of the two liposomes and their mixture, and the two time points (ta and tb) can be found based on the rate of change of the dynamic USF signal.
  • Ethanol and cholesterol were from Fisher Scientific International, Inc., USA.
  • ICG was obtained from Chem-Impex Int’L Inc., USA.1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti Polar Lipids, Inc., USA.
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • Liposome Preparation [00126] The liposomes were prepared via an ethanol injection method.20 mg phospholipids (DPPC and DSPC) and 1 mg cholesterol were dissolved in 700 ⁇ L ethanol.
  • the resultant organic phase was injected into 9 mL of ICG aqueous phase (0.06 mg/mL) under magnetic stirring at 1000 rpm. After stirring for 15 minutes, the residual ethanol was removed by rotary evaporation under reduced pressure. The liposome suspension was then purified by ultracentrifugation (Avanti J-E, Beckman Coulter, USA) at 16,500 rpm for 25 min.
  • the mass ratio of DPPC and DSPC is 1.5 for the first liposome (denoted by L) with a lower threshold LCST 1 , while the lipids ratio is 0.6 for the second liposome (denoted by H) with a higher threshold LCST 2 .
  • Liposome Characterization [00127] The hydrodynamic size and polydispersity index of the liposomal samples were obtained by a dynamic light scattering instrument (NanoBrook 90PlusPALS, Brookhaven Instruments, USA). All measurements were performed in triplicate at room temperature. An in- house built spectrometer system was used to measure the fluorescence intensity with respect to temperature. The fluorescent sample in a 3 mL quartz cuvette was placed into a temperature- controlled holder (Quantum Northwest, Inc., USA) and excited by an 808 nm laser (MGL-II- 808-2W, Dragon Lasers, China).
  • EMCD electron multiplying charge-coupled device
  • the fluorescence passed through three long-pass filters (104) (BLP01- 830R-50/25, Semrock Inc., USA), and then was captured by an EMCCD camera (105) (ProEM®-HS: 1024BX3, Princeton Instruments, USA; camera lens: AF NIKKOR 50 mm f/1.8D Lens, Nikon, Japan).
  • a function generator (106) (FG, 33500B, Agilent, USA) generated the driven voltage to a 2.5 MHz FU transducer (107) (H-108, Sonic Concepts Inc., USA) after being amplified by a 50 dB-gain radio frequency power amplifier (108) (RF-AMP, A075, E&I, USA) and then passed through the matching network (109) (MNW).
  • an IR camera (110) (A300, Teledyne FLIR, USA) was also synchronized by receiving trigger signals from a pulse delay generator (111) (PDG, P40, Highland Technology, USA) connected to the FG (106).
  • the temperature of the water was controlled by a temperature controller system (112) (PTC10, Stanford Research Systems, USA).
  • a heater (113) was placed in the tank.
  • a temperature probe (114) submerged in the water was placed near the tube to get a close estimation of temperature of the USF agents.
  • a computer (115) was connected to the EMCCD camera (105) and the function generator (106).
  • a magnetic stir bar (116) (11-100-16S, Fisher Scientific, USA) with a magnetic stirrer (not shown) was used to ensure a relatively uniform temperature distribution in the water tank.
  • the purposes of using a relatively thin silicone sample are mainly: (1) combining the relative thinness with the transparency of silicone to IR light, the IR-camera-measured temperature from the sample surface would be approximately equal to the temperature in the sample; and (2) to enable the acquisition of dynamic signals at a high frame rate using the EMCCD camera, a strong USF signal without significant attenuation may be used.
  • Figure 1B shows a time sequence diagram of each temperature measurement.
  • the heating FU pulse with a duration of 1.2 sec was triggered 0.1 sec after the EMCCD camera (105) started acquiring.
  • the EMCCD camera (105) was initiated by the trigger command sent by the computer (Trigger 1), while the FU transducer (107) was externally triggered by the function generator.
  • Both the function generator (106) and EMCCD (105) were synchronized by executing the customized MATLAB script on the computer (115).
  • the frame rate of the EMCCD camera (105) was 100 Hz.
  • the IR reading was performed synchronously with the camera acquisition after FU exposure, and a single IR thermograph was obtained right before the FU heating to evaluate the background temperature at the target zone.
  • the 10 frames obtained from the EMCCD camera (105) before the FU exposure were averaged and considered the background fluorescence image.
  • the time point when the FU (107) initiated and the rising edge of Trigger 1 occurred can be denoted as t0.
  • a continuous set of 500 fluorescence frames was captured immediately at t0 to monitor the real-time change of fluorescence in the focal area.
  • Fluorescence Signal Processing [00131] This section explains the procedures of acquiring and processing the signals and further estimating the local temperature based on the acquired dynamic USF signal.
  • the main procedures include measuring the fluorescence-vs.-temperature based on two characteristic profiles of the two liposomes and their mixture in cuvettes, and the dynamic USF signals in tissue phantoms from the mixture of the two liposomes.
  • the dynamic USF signal of the mixed USFs was acquired from the camera-based USF system. Briefly, a continuous series of fluorescence images were acquired before, during, and after the FU exposure. The images acquired before the exposure were subtracted from those acquired during and after the exposure. The subtracted images represented the USF signals at different time points. These subtracted images were subsequently and spatially filtered based on the correlation between a normalized averaging filter. Then, the USF intensity values of each USF image were averaged within a 0.33 ⁇ 0.66 mm 2 region of interest from the center of the heating spot, where the temperature distribution was relatively uniform. The computation of the temporal variation of USF intensity can be achieved following the steps mentioned above.
  • the differential profile of the dynamic USF signal from the mixed liposomes was computed similarly by subtracting any two adjacent data points of the USF signal, and then normalized. Two switching peaks were found in the time domain, corresponding to two fast switching temperatures revealed by the characteristic curves of the sample curves measured from the cuvettes. As a result, the information in both the time and temperature domains corresponding to each switching peak were found. As mentioned above, the thermal diffusion in the focal volume was ignored within such a short FU exposure. Thus, the local background temperature of the sample can be calculated based on the methods discussed previously.
  • Figure 2 shows the temperature-dependent fluorescence profiles of the liposomes and their differential profiles.
  • the dashed line with circles is for the liposomes with a high LCST (H, 43.8°C), and the dashed line with triangles shows the profiles of the liposomes with a low LCST (L, 38.9°C).
  • Their differential profiles are represented by the corresponding solid lines, respectively. The differences of fluorescence intensity of two adjacent points were calculated from the corresponding solid line.
  • the fluorescence-vs.-temperature profiles exhibit a step function, which is a typical feature of USF imaging.
  • each differential profile shows a peak, representing the highest speed of the fluorescence rising at a specific temperature.
  • the ICG-liposomes had similar sizes, polydispersity index (PDI) values, and zeta potentials, as shown in Table 1. All liposomes had PDI values below 0.31, demonstrating the homogeneity of the liposomes.
  • the low LCST liposome showed a relatively narrower transition band (3.1°C) relative to the high LCST liposomes (6.8°C).
  • the LCST shift from high (43.8°C) to low (38.9°C) is mainly caused by the increase in the mass ratio between the two main components of DPPC and DSPC from 0.6 to 1.5.
  • the primary components of the liposomal shell were DSPC and DPPC, with each possessing a distinct phase transition temperature of 55°C and 41°C, respectively. Therefore, the LCSTs of liposomal USF agents were shown to be tuned effectively by varying the DPPC:DSPC ratio. Table 1. Characteristics of ICG-Liposomes.
  • the voltage from the function generator was 56 mV, which was amplified 50 dB by the power amplifier (RF-AMP) before being applied to the ultrasound transducer.
  • the transmitted electrical power was estimated to be 0.39 W.
  • the USF signal did not show a linear relationship with time. This can be seen from its differential curve, which is not a constant.
  • the speed of USF signal rose. After that, the speed fell gradually. Not intending to be bound by theory, it is believed that this non-linear relationship is caused by two factors. First, in the rising period from 0 to t 3 , the thermal energy is usually confined in the focal volume (i.e., “thermal confinement”).
  • thermal diffusion can be ignored, and the temperature grows linearly within the FU exposure time (i.e., the heating speed usually remains constant in this period).
  • the non- linearity of the USF signal is mainly caused by the nonlinear features of the step function of the fluorescence-vs.-temperature of the two liposomes, rather than the heating.
  • thermal diffusion may not be ignorable.
  • the non-linearity of the USF signal is caused by both the nonlinear thermal response of the liposomes and thermal diffusion.
  • FIG. 4A displays the estimated background temperature (T t ) from the USF method compared to measurements from the IR camera.
  • the background temperature of the liposome mixture was controlled and varied from 35.27°C to 39.31°C based on the IR camera reading.
  • a solid line is also plotted as a reference in the figure.
  • the USF-estimated background temperature data points are closely aligned with the reference line, indicating a high degree of correlation between the variables being compared (IR readings in this case).
  • the average of the temperature difference between the two methods was 0.64 ⁇ 0.43°C. Measurements were limited to a small range (from 35.27°C to 39.31°C) to match typical physiological temperatures.
  • the measurement range can be modulated by the composition of the lipid shells.
  • DSPC, DPPC, and dimyristoyl phosphatidylcholine (DMPC) are fully saturated lipids with different lengths of hydrocarbon chains.
  • DMPC exhibits a phase transition temperature that is below body temperature, at 24°C, while the other two phospholipids can transition around and above physiological temperature.
  • FIG. 4B-C display how the two peak times (ta and tb) shift with the increase in the background temperature.
  • both t a and t b decrease, which means the two peaks occur earlier.
  • Ta and Tb of the liposomes are mainly dependent upon the properties of the material component of the liposomes, they are stable during the experiments.
  • Increasing the background temperature (Tt) will lead to the reduction of the temperature difference between the background temperature and the Ta (and also Tb).
  • the FU heating speed is considered to be fixed.
  • the duration for raising the temperature of the sample from T t to T a or T b decreases.
  • the USF signals are represented using dashed lines, while their differential results are presented using solid lines. When the background temperature is consistent, these signals are depicted using the same marker.
  • the differential USF curves indicate that the switching peaks occur earlier as the background temperature increases, owing to the relatively constant heating speed, which leads to faster attainment of Ta and Tb. This is also the reason why the corresponding USF signals display higher slopes, particularly at the start of the FU heating. [00143] Not intending to be bound by theory, it is believed that switching temperature points possibly allows for higher accuracy and a broader estimation range.
  • the linear heating assumption and the linear equation to calculate the heating speed may be used with other models, such as thermal diffusion and photon diffusion.
  • the local background temperature can be assessed by analyzing the temperature-dependent fluorescence variations from USF contrast agents during ultrasound heating.
  • the ICG-liposome agents provide a tunable measurement range that can be adjusted by modifying the compositions of the outer lipid shells.
  • USF thermometry was tested with a mixture of ICG-liposomes with two distinct temperature switching-on thresholds, and the dynamic USF signals were recorded using a camera-based USF system. By fitting the results into a customized algorithm, the local background temperature of the sample was calculated over a typical physiological range.
  • EXAMPLE 2 Two-Color Ultrasound-Switchable Fluorescence Thermometry
  • Measuring tissue temperature with high sensitivity and specificity is highly desirable because it can provide valuable insights into the thermal behavior of biological systems, enabling researchers to study thermoregulation, metabolic activity, neuron and cancer cell activity, and other temperature-dependent processes. Therefore, it can have diverse applications across various fields, including drug development, biotechnologies, life sciences, medical and health care, and molecular biology.
  • Traditional temperature measurement methods present several limitations. They can be invasive, potentially disrupting delicate biological systems. Additionally, some methods can only measure the surface temperature of the sample, as is the case with infrared (IR) thermometry.
  • IR infrared
  • Example 1 the single-color method of Example 1 is extended to a two-color approach by encapsulating two types of fluorophores into the two liposomes with distinct transition temperatures. Specifically, ICG was encapsulated in a liposome with a lower threshold. Another NIR aza-BODIPY-base fluorophore was encapsulated into a liposome with a higher threshold.
  • thermosensitive liposomes were prepared by thin-film hydration, followed by extrusion.5.5 mg 1, 2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) and 0.6 mg cholesterol were dissolved in 2 mL chloroform. To form the lipid film, the final mixture was evaporated in a rotor evaporator at 120 rpm and 55°C for 1 hour under reduced pressure (-80 kPa).
  • ADP_bot BF2-chelated [5-(4-hydroxyphenyl)-3-phenyl-1H-pyrrol-2-yl]-[5-(4-hydroxyphenyl)-3- phenylpyrrol-2-ylidene] amine
  • NIR near-infrared
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • the two-color USF thermometry system (200), as shown in Figure 6, utilized two excitation lasers with wavelengths of 808 nm (201) (MGL-II-808-2W, Dragon Lasers, China) and 671 nm (202) (MLL-FN-671-500mW, Dragon Lasers, China) that were driven by a first function generator (203) (FG1, 33500B, Agilent, USA).
  • the excitation filters (204, 205) (FF01-673/11-25 and LL01-808-25, Semrock Inc., USA) were used to filter the light that was produced by the 671 nm and 808 nm lasers (202, 201), respectively.
  • the filtered light was coupled into two fiber optic light guides (206, 207) (1/4” x 48”, Edmund Optics Inc., USA) and illuminated the sample (208) placed in a water tank from different directions.
  • the fluorescence emitted by two distinct USF contrast agents was separated by employing two different combinations of emission filters.
  • a 2-inch 715 nm long-pass filter (209) (FF01-715/LP-50, Semrock Inc., USA), a 2-inch 730 nm band-pass filter (210) (FF01-730/39-50, Semrock Inc., USA), a camera lens (211) (AF NIKKOR 50 mm f/1.8D Lens, Nikon, Japan), and a 1-inch 775 nm short-pass filter (212) (FF01-775/SP-25, Semrock Inc., USA) were used.
  • two 2-inch 830 nm long-pass filters (213, 214) BLP01-830R-50, Semrock Inc., USA
  • a 2-inch 857 nm band-pass filter (215) FF01-857/30-50, Semrock Inc., USA
  • a camera lens (216) AF NIKKOR 50 mm f/1.8D Lens, Nikon, Japan
  • a 1-inch 857 nm band-pass filter (217) FF01- 857/30-25, Semrock Inc., USA
  • the filtered fluorescence signal was collected by an EMCCD camera (218, 219) (ProEM®-HS:1024BX3, Princeton Instruments, USA).
  • the fields of view for the 730-nm channel and 857-nm channel were 4 cm x 4 cm and 3 cm x 3 cm, respectively.
  • the driving signal from the second function generator (220) (FG2, 33500B, Keysight Technologies, USA) was amplified by a 50 dB-gain radio frequency power amplifier (221) (RF-AMP, A075, E&I, USA) and fed to a 2.5 MHz focused ultrasound (FU) transducer (222) through a matching network (223) (H-108, Sonic Concepts Inc., USA).
  • the FU transducer (222) was mounted onto a three-axis motorized translation stage (224) (XSlideTM and VXMTM, Velmex Inc., USA).
  • a temperature-controlling system comprising a temperature controller (225), a heater (226), and a sensor (227) were used to maintain the water temperature at various values, while a long magnetic stir bar (228) with a magnetic stirrer (11-100-16S, Fisher Scientific, USA) (not shown) was utilized to ensure uniform heating.
  • the surface temperature of the sample was monitored by an infrared (IR) camera (229) (FLIR A300, FLIR Systems, USA).
  • IR infrared
  • a pulse delay generator (230) and a computer (231) are also connected to the system.
  • a MATLAB-based program (The MathWorks, Inc., USA) was developed to synchronize the exposure of the ultrasound (222) and EMCCD cameras (218, 219).
  • the master program sent a trigger to FG2 (220) for FU driving signal generation through a multifunctional input/output device (232) (I/O, PCIe-6363, National Instruments, USA) and controlled the EMCCD cameras (218, 219) to acquire sequential images simultaneously. Additionally, the master program sent a trigger to a slave program controlling the EMCCD (218) through the I/O device (232).
  • the EMCCD (218) started capturing images when the USB-oscilloscope (UO, USB-5133, National Instruments, USA) (not shown), controlled by the slave program, received the trigger.
  • the IR camera (229) was synchronized with the ultrasound exposure by detecting the output trigger from FG2 (220) relayed by the pulse delay generator (230) (P400, Highland Technology Inc., USA).
  • Fluorescence-Temperature Characteristic Curves [00153] The details of an in-house built cuvette system used to measure the fluorescence- temperature characteristic curve were described previously (15). A quartz cuvette (Hellma, Germany) of 3.5 mL capacity was filled with 3 mL contrast agent and placed into a temperature- controlled sample compartment (qpod 2e, Quantum Northwest, Inc., USA).
  • a 671-nm laser was employed to excite the sample, and the emitted fluorescence was collected by a modular USB spectrometer (USB2000+, Ocean Inlight, USA) after passing through a 715 nm long-pass filter (FF01-715/LP-25, Semrock Inc., USA). The temperature range measured was from 31.0°C to 55.0°C in steps of 0.1°C. The fluorescence spectra were captured at the preset temperature points. The fluorescence intensity of DPPC-DSPC-ADP_bot and DPPC- ⁇ /CD-ICG were calculated by summing the corresponding spectrum from 715 nm to 750 nm and from 830 nm to 1000 nm, respectively.
  • a silicone tube (ST 60-011-01, Helix Medical, USA) with an outer diameter of 0.64 mm and an inner diameter of 0.30 mm was incorporated in the center of the silicone phantom, which had a thickness of approximately 4 mm.
  • DPPC-DSPC-ADP_bot and the DPPC- ⁇ /CD-ICG were mixed according to a volume ratio of 3:1.
  • the FU focus was fixed on the silicone tube filled with the mixed solution during the experiment.
  • the exposure time of the ultrasound was 1600 msec, and the estimated ultrasound power was 0.24 W.
  • the fluorescence intensity changes of the mixed contrast agents were continuously monitored in two detection channels by the EMCCD cameras.
  • the exposure time for a single frame was 15 msec.
  • the USF signal pattern at each time point was obtained by subtracting the average of the 10 frames captured before ultrasound exposure from the frame captured after exposure.
  • the USF signal intensity value was then calculated by summing the pixel values around the ultrasound focus.
  • the areas used for calculating signal intensity for EMCCD 1 and EMCCD 2 were 0.76 x 1.20 mm 2 and 0.94 x 1.60 mm 2 , respectively.
  • DPPC- ⁇ /CD-ICG had an effective size of 149.91 ⁇ 19.06 nm, and the polydispersity was 0.057.
  • the effective size of DPPC-DSPC-ADP_bot was 316.37 ⁇ 40.05 nm, and the polydispersity was 0.123.
  • the lower critical solution temperature (LCST) defined as the point where the slope of the curve reaches 20% of its maximum value, was determined to be 38.9°C and 41.9°C for DPPC- ⁇ /CD-ICG and DPPC-DSPC-ADP_bot, respectively.
  • LCST The lower critical solution temperature
  • the fluorescence intensity of DPPC- ⁇ /CD-ICG increased by 2.93-fold and 1.17-fold, respectively.
  • the fluorescence intensity of DPPC-DSPC-ADP_bot increased by 1.32-fold and 2.67- fold upon elevating the temperature from 38.9°C to 41.9°C and from 41.9°C to 46.8°C, respectively.
  • Figure 8B shows the slope curve of the difference between the two fluorescence- temperature characteristic curves depicted in Figure 8A.
  • the curve defines four characteristic points: the first zero crossing point (38.6°C), the peak point (39.5°C), the second zero crossing point (42.4°C), and the valley point (44.8°C), which are characteristic temperature points for assessing the background temperature via dynamic USF signals.
  • the USF signal of DPPC- ⁇ /CD-ICG exhibits an earlier onset of significant growth compared to that of DPPC-DSPC-ADP_bot at the same water temperature of 36.90°C (solid line with squares vs. solid line with circles) or 38.62°C (solid line with triangles vs. solid line with bold triangles).
  • a linear regression model may be used to determine the background temperature.
  • the slope curve of the difference between the USF signal curves of DPPC- ⁇ /CD-ICG and DPPC-DSPC-ADP_bot at a higher water temperature of 38.62°C (line with circles), exhibits a significant temporal leftward shift compared to that observed at a water temperature of 36.90°C (bold line with circles).
  • the slope curve derived from the USF signals ( Figure 9C) shares a similar shape with the fluorescence temperature characteristic curves obtained from measurements in the cuvette system ( Figure 9B).
  • the first zero crossing point shall not be utilized for background temperature calculation.
  • the EMCCD camera was programmed to initiate the recording 0.15 sec prior to ultrasound exposure. This means that the background temperature should be estimated at 0.15 sec instead of 0 sec.
  • the background temperature was estimated to be 36.12°C at 0.15 sec when the water temperature was 36.90°C.
  • the background temperature was estimated to be 38.20°C based on three characteristic points with corresponding time points of 0.420 sec, 0.855 sec, and 1.410 sec, respectively, while the water temperature was at 38.62°C.
  • the water temperature represented by squares
  • the background temperature estimated via USF thermometry indicated by circles
  • the background temperature measured with an IR camera denoted by triangles
  • this two-color thermometry estimated the local temperature based on four characteristic temperatures, which allows for finer adjustments to the parameters of the linear fitting model, thus improving the overall accuracy by reducing the impact of outliers and random fluctuations.
  • the turning points on the USF signals induced by FU can be identified.
  • the local background temperature can be measured via the application of a linear regression model.
  • Embodiment 1 A method of measuring an initial temperature in an environment, the method comprising: disposing a population of photoluminescent thermosensitive agents in the environment, the population having a first switching temperature threshold and a second switching temperature threshold that is higher than the first switching temperature threshold; exposing the environment to an activation source at an initial time to create an activation region within the environment having a temperature greater than or equal to the first switching temperature threshold at a first time; exposing the environment to a beam of electromagnetic radiation to excite the population of photoluminescent thermosensitive agents at the first time; detecting a photoluminescent signal emitted by the excited population of photoluminescent thermosensitive agents at the first time; further exposing the environment to the activation source to increase the temperature of the activation region to a temperature greater than or equal to the second switching temperature threshold at a second time; exposing the environment to a beam of electromagnetic radiation to excite the population of photolumin
  • Embodiment 2 The method of Embodiment 1, wherein the population of photoluminescent thermosensitive agents comprises a first photoluminescent thermosensitive agent having the first switching temperature threshold and a second photoluminescent thermosensitive agent having the second switching temperature threshold.
  • Embodiment 3. The method of any of the preceding Embodiments, wherein: exposing the environment to a beam of electromagnetic radiation comprises exposing the environment to a first excitation beam and a second excitation beam; and the first excitation beam and the second excitation beam have differing wavelengths.
  • Embodiment 5 The method of any of the preceding Embodiments, wherein: the photoluminescent signal at the first time is detected with a first detector or detection channel; and the photoluminescent signal at the second time is detected with a second detector or detection channel.
  • Embodiment 5 The method of any of the preceding Embodiments, wherein the photoluminescent signal emitted by the population of photoluminescent thermosensitive agents is detected using a detector comprising a plurality of optical fiber collectors coupled to a camera or photon counter, wherein the optical fiber collectors are spatially distributed around an exterior surface of the environment.
  • Embodiment 6 Embodiment 6.
  • determining the initial temperature of the environment comprises determining a function corresponding to a photoluminescent signal-temperature profile of the population of photoluminescent thermosensitive agents.
  • Embodiment 7 The method of any of the preceding Embodiments, further comprising determining a linear heating speed of the environment (Vh).
  • the activation source comprises at least one of an ultrasound beam, electromagnetic radiation, magnetic field, electron or particle beam, and chemical stimulus.
  • Embodiment 11 The method of any of the preceding Embodiments, wherein the activation source is an ultrasound beam.
  • Embodiment 11 The method of any of the preceding Embodiments, wherein the environment comprises biological tissue.
  • Embodiment 12 The method of Embodiment 11, wherein the environment comprises a tumor.
  • Embodiment 13 The method of any of the preceding Embodiments, wherein the photoluminescent thermosensitive agents comprise ultrasound switchable fluorophores (USF).
  • Embodiment 14 The method of any of the preceding Embodiments, wherein the photoluminescent signal at the first time and the second time comprises visible light.
  • Embodiment 16 A system for measuring an initial temperature in an environment, the system comprising: a population of photoluminescent thermosensitive agents, the population having a first switching temperature threshold and a second switching temperature threshold; at least one activation source; at least one excitation beam; and at least one photoluminescence detector.
  • Embodiment 17 The system of Embodiment 16, wherein the activation source is an ultrasound beam.
  • Embodiment 16 or Embodiment 17 wherein the population of photoluminescent thermosensitive agents comprises a first photoluminescent thermosensitive agent having the first switching temperature threshold and a second photoluminescent thermosensitive agent having the second switching temperature threshold.
  • Embodiment 19 The system of Embodiment 18, wherein the first photoluminescent thermosensitive agent has a first emission profile and the second photoluminescent thermosensitive agent has a second emission profile differing from the first emission profile.
  • Embodiment 20 The system of any of Embodiments 16-19, wherein the photoluminescent thermosensitive agents comprise ultrasound switchable fluorophores (USF).
  • USF ultrasound switchable fluorophores

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Abstract

L'invention concerne des systèmes et des procédés de cartographie de la température ambiante à l'aide d'agents thermosensibles photoluminescents. Dans un mode de réalisation, un procédé comprend : la disposition d'une population d'agents thermosensibles photoluminescents dans l'environnement, la population présentant des premier et deuxième seuils de température de commutation ; l'exposition de l'environnement à une source d'activation pour créer une région d'activation dont la température est supérieure ou égale au premier seuil de température de commutation ; l'exposition de l'environnement à un rayonnement électromagnétique ; et la détection d'un signal photoluminescent émis à un premier instant. Un procédé peut en outre comprendre : l'exposition de l'environnement à une source d'activation pour augmenter la température de la région d'activation à une température supérieure ou égale au second seuil de température de commutation ; l'exposition de l'environnement à un rayonnement électromagnétique ; et la détection d'un signal photoluminescent émis à un second instant.
PCT/US2024/031427 2023-06-09 2024-05-29 Systèmes et procédés de cartographie de température à l'aide d'agents thermosensibles Pending WO2024253915A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6132958A (en) * 1999-05-27 2000-10-17 The Rockefeller University Fluorescent bead for determining the temperature of a cell and methods of use thereof
US20040267137A1 (en) * 2003-06-27 2004-12-30 Michael Peszynski Apparatus and method for IC-based ultrasound transducer temperature sensing
US20080033300A1 (en) * 2006-08-04 2008-02-07 Anh Hoang Systems and methods for monitoring temperature during electrosurgery or laser therapy
US20110098609A1 (en) * 2007-11-12 2011-04-28 Koninklijke Philips Electronics N.V. Tissue temperature indicating element for ultrasound therapy
US20190293498A1 (en) * 2016-06-02 2019-09-26 Board Of Regents, The University Of Texas System Systems and methods for thermometry and theranostic applications
US20210153971A1 (en) * 2019-11-26 2021-05-27 Board Of Regents, The University Of Texas System Systems and methods for surgical guidance in breast cancer surgery and lymph node dissection
US20210299281A1 (en) * 2018-07-17 2021-09-30 Board Of Regents, The University Of Texas System Multiple biomarkers imaging for high specificity

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6132958A (en) * 1999-05-27 2000-10-17 The Rockefeller University Fluorescent bead for determining the temperature of a cell and methods of use thereof
US20040267137A1 (en) * 2003-06-27 2004-12-30 Michael Peszynski Apparatus and method for IC-based ultrasound transducer temperature sensing
US20080033300A1 (en) * 2006-08-04 2008-02-07 Anh Hoang Systems and methods for monitoring temperature during electrosurgery or laser therapy
US20110098609A1 (en) * 2007-11-12 2011-04-28 Koninklijke Philips Electronics N.V. Tissue temperature indicating element for ultrasound therapy
US20190293498A1 (en) * 2016-06-02 2019-09-26 Board Of Regents, The University Of Texas System Systems and methods for thermometry and theranostic applications
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