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WO2011056658A1 - Microscopie multiphotonique par l'intermédiaire d'une lentille d'objectif à interface hertzienne - Google Patents

Microscopie multiphotonique par l'intermédiaire d'une lentille d'objectif à interface hertzienne Download PDF

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Publication number
WO2011056658A1
WO2011056658A1 PCT/US2010/054303 US2010054303W WO2011056658A1 WO 2011056658 A1 WO2011056658 A1 WO 2011056658A1 US 2010054303 W US2010054303 W US 2010054303W WO 2011056658 A1 WO2011056658 A1 WO 2011056658A1
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Prior art keywords
specimen
objective lens
glass
microscope
photon
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Chay Titus Kuo
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Duke University
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Duke University
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence

Definitions

  • the present disclosure relates to an improved microscope for the examination of objects, such as biological tissues.
  • the present disclosure further relates to a tissue culture vessel that provides easy access to cells, tissue and variability of growth substrate for continuous microscopic examinations at high magnification.
  • the present disclosure further relates to an assay for long term live imaging of tissue using multi-photon microscopy.
  • Confocal microscopy involves scanning a specimen to produce microscopic images of a slice or section of specimen. Such microscopic imaged sections may be made in vivo and can image at cellular resolutions (Milind Rajadhyaksha et al, (1995) J. of Invest. Derm., 104(6): 1-7; Milind Rajadhyaksha et al, (1997) Laser Focus World, Feb., pp 119-127). These systems have confocal optics which direct light to tissue and image the returned reflected light.
  • Such confocal microscope systems can focus and resolve a narrow width of tissue as an imaged section, such that tissue structures can be viewed at particular depths within the tissue, thereby avoiding invasive biopsy procedures for pathological examination of the tissue, or allow pathological examination of unprepared excised tissue.
  • NA numerical aperture
  • the various devices of the prior art are of two types of construction.
  • the first type allows for growth and easy access to the culture sample, but the materials used do not permit high magnification microscopy.
  • These include such growth containers as glass and plastic bottles, flasks and petri dishes that have thick and/or irregular surfaces which distort the image when viewed at high magnifications.
  • stress lines, bubbles and scratches interfere with the visual morphological assessments.
  • phase and interference microscopy are far from optimal because of the thickness of the growth surface.
  • These devices also fail to offer any variability in the nature of the growth substrate.
  • the second type incorporates thin glass or plastic coverslips as the growth substrate sandwiched between metal plates.
  • the coverslips are separated by a ring-like gasket or are permanently cemented to one of the plates, and holes in the plates have a circumference substantially matched to the ring-like gasket.
  • Access to this type of chamber is either with a syringe needle or by a tube projecting radially from the enclosed area. Maintenance of a sterile environment during disassembly is difficult if not impossible.
  • Such devices permit the exchange of liquid or media contained within, but do not provide access to the cellular material located on the growth surface.
  • the present disclosure provides an optical system for depth-resolved imaging, e.g., multiphoton microscopy, of living tissue in vitro, methods of using said optical system, as well as methods of visualizing neural stem cells in mouse brain slices using the optical system of the present disclosure.
  • the present disclosure further provides an assay for viewing and manipulating neural stem cells in living brain slices in vitro, which can be used for screening smaller molecules added to culture media that can change neural stem cell behavior in intact brain slices in vitro.
  • One aspect of the present disclosure provides a microscope comprising, a multi- photon excitation light source, the multi-photon excitation light source comprising a laser generating device capable of emitting laser light energy having a wavelength of about 690 to 1050 nm; a plurality of optical elements, the plurality of optical elements comprising a primary dichroic mirror capable of passing wavelengths of 1020 nm and 488 nm simultaneously; an objective lens which comprises an NA of less than 1.0; a multi-photon detector for signal detection that provides for the detection of red and green light; and a specimen incubator.
  • the multi-photon light source further comprises an optical element selected from the group consisting of a half-wave plate, a beam dump, a beam splitter an electro-optic modulator, a beam expanding lens, and combinations thereof.
  • the laser generating device comprises a Ti-Sapphire laser.
  • the plurality of optical elements comprises at least one optical element selected from the group consisting of at least one scan head, a scan lens, a tube lens and a long pass dichroic mirror, and combinations thereof.
  • the objective lens comprises a numerical aperture (NA) of about 0.4 to 1.0 and a working distance (WD) of about 2.5 and 9.0 mm.
  • NA numerical aperture
  • WD working distance
  • the objective lens comprises a NA of about 0.6 to 0.45 and a WD of about 3.6 to 8.2 mm.
  • the microscope further comprises a second laser generating device, wherein the second laser generating device is capable of emitting light energy having wavelengths of about 475 to 650 nm.
  • the microscope further comprises a third laser generating device that is capable of emitting light energy having wavelengths of about 400 to 500 nm.
  • the microscope further comprises a confocal detector comprising at least one photomultiplier tube, at least one dichroic mirror, and a pinhole aperture.
  • the specimen incubator comprises a first housing having an internal chamber comprising a top panel, wherein the top panel comprises an aperture in the geometric center of the top panel and wherein the aperture comprises a material that is suitable for optical viewing such as glass; a second housing positioned within the internal chamber of the first housing comprising a bottom section, a removable top section, wherein the removable top section and the bottom section each comprise an aperture positioned at the geometric center of the top panel and bottom section, the aperture comprising a material that is suitable for optical viewing such as glass; and a specimen stand comprising of at least two legs and a platform supported by the at least two legs; and a growth medium located within the second housing.
  • the top portion of the second housing is removable.
  • the top portion of the first housing is removable.
  • the specimen incubator further comprises a growth membrane positioned on said platform.
  • the growth membrane is gas permeable.
  • the growth membrane is microporous.
  • the apertures comprise a material having a refractive index of about 1.0 to 2.0 n D at 20°C.
  • tissue specimen is a brain section.
  • the brain section is from a transgenic mouse, wherein the transgenic mouse is engineered to have neural stem cells whose progeny are fluorescently labeled.
  • Another aspect of the present disclosure provides a method of screening the effect of a compound on neural stem cell behavior using the optical system according to the present disclosure comprising placing a brain section on the platform of the specimen incubator, wherein the brain section is from a transgenic mouse engineered to have neural stem cells fluorescently labeled; adding a compound of interest to the medium; and imaging the brain slice.
  • the method further comprises comparing the behavior of neural stem cells in the brain section cultured in the compound of interest with neural stem cells from a brain section cultured in medium without the compound of interest.
  • FIG 1 is a block diagram illustrating a configuration of a multi-photon microscope according to the present disclosure.
  • FIG 2 is a block diagram showing a multi-photon microscope in accordance with the present disclosure.
  • FIGS 3A and 3B are cross sections depicting specimen incubators.
  • FIG 3A is a cross section of a specimen incubator according to one embodiment of the present disclosure
  • FIG 3B is a specimen incubator for viewing from the bottom.
  • FIGS 4A and 4B are images generated of neural tissue using embodiments of the microscope and specimen incubator of the present disclosure.
  • FIG 4A is an image resulting from viewing the specimen from the bottom.
  • FIG 4B is an image resulting from viewing the specimen form the top.
  • FIGs 5A and 5B are schematic and image results demonstrating the two-photon Jablonski Energy diagram of single vs. multi-photon excitation.
  • FIG 8A is a schematic demonstrating the two-photon Jablonski energy concept.
  • FIG 8B are image results comparing the single-photon and multi-photon excitation.
  • FIG 6A and 6B are images results of neural tissue generated using one embodiment of a microscope of the present disclosure.
  • FIG 7A - 7B are images generated using one embodiment of a microscope of the present disclosure pre- and post deconvolution, respectively, using an objective lens having a 0.45 NA.
  • FIG 8 is an image of neural tissue demonstrating the depth of vision using an optical system according to the present disclosure.
  • FIG 9A and 9B are schematic diagrams showing the differences between embryonic stem cells and adult stem cells.
  • FIGS 10A and 10B are schematic diagrams showing location of neural stem cells within the brain.
  • FIG IOC shows the neurogenesis of the sub ventricular zone (SVZ) region and imaging the of the SVZ region using an embodiment of the the optical system of the present disclosure.
  • SVZ sub ventricular zone
  • FIG 1 1A and 1 IB are block diagrams showing gene constructs used to generate transgenic mice having labeled stem cells for imaging with embodiments of the optical system of the present disclosure.
  • FIG 12 is a two-color, three-dimensional 2-photon image obtained from transgenic mice created via the constructs shown in FIG 11A and 1 1B using the optical system of the present disclosure.
  • Articles "a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article.
  • an element means at least one element and can include more than one element.
  • the present disclosure provides an optical system for depth-resolved imaging, e.g., multiphoton microscopy, of living tissue in vitro.
  • the optical system 100 of the present disclosure comprises a microscope 1.
  • a microscope comprises at least one multi-photon excitation light source 2, a plurality of optical elements 3, and an objective lens 26 and a signal detector 108.
  • the optical system 100 of the present disclosure further provides a specimen incubator 5, in which the specimen to be examined is maintained.
  • the present disclosure further provides methods of using the microscope and optical system, as well as methods of labeling and viewing neural stem cells using the microscope and optical system of the present disclosure.
  • the multi-photon excitation light source 1 generally comprises a laser generating device 6, a half- wave plate 7, a beam dump 8, a beam splitter 9, a beam expanding lens 10, an electro-optic modulator 11 and a mirror 12.
  • the laser generating device 6 is preferably a tunable semiconductor laser source which emits a laser beam Q having a wavelength of between 690 to 1050 nm.
  • the laser beam Q has a wavelength of 1020 nm.
  • An exemplary laser source as used herein is a Ti-Sapphire laser, such as the Spectra-Physics® Mai Tar One Box Ti Laser.
  • a laser Q is emitted from the laser generating device 6 and passed through a half-wave plate 7.
  • the half-wave plate 7 refers to any optical device that alters the polarization state of a light wave travelling through it.
  • the half-wave plate is a birefringement crystal with a predetermined orientation and thickness, both of which can be readily determined by those skilled in the art.
  • the half-wave plate 7 is capable of modulating or tuning the laser beam Q such that when there is no power, red light is visible, and when at full power, unsaturated green light is visible.
  • the beam splitter 9 may be any optical device which can split an incident light beam into two or more beams, which mayor may not have the same optical power.
  • Suitable devices for use with embodiments of the present disclosure include, but are not limited to, dielectric mirrors, beam splitter cubes (e.g., Wollaston prisms, Nomarski prisms, Glan-Thompson prisms, Nicol prisms, etc.), fiber-optic beam splitters, metallic mirrors (e.g., half-silvered mirrors), pellicles, micro-optic beam splitters, waveguide beam splitters, and the like.
  • dielectric mirrors e.g., Wollaston prisms, Nomarski prisms, Glan-Thompson prisms, Nicol prisms, etc.
  • fiber-optic beam splitters e.g., metallic mirrors (e.g., half-silvered mirrors), pellicles, micro-optic beam splitters, waveguide beam splitters
  • the beam dump 8 may be any device which quickly dissipates and absorb unwanted light (i.e. incident light) generated by the laser entering the beam splitter 9.
  • suitable beam dumps are known to those skilled in the art.
  • a suitable beam dump for use with embodiments of the present disclosure would have the following characteristics: (1) being optimized for between 400 and 800 nm wavelengths; (2) having a dimensional tolerance of about ⁇ 0.2 nm; (3) have a reflectivity factor of ⁇ 10 4 , and (4) being comprised of a material such as black anodized aluminum.
  • the split laser beam Q is next passed through a beam expanding lens 10.
  • the beam expanding lens 10 may be any lens or combination of lenses that can increase the diameter of the laser beam. Suitable type of beam expanding lenses for use with embodiments of the present disclosure include YAG laser beam expanders, red laser beam expanders, green laser beam expanders, NIR laser beam expanders, MID and Far IR laser beam expanders, Zinc- Selenide Laser beam expanders, UV laser beam expanders and the like.
  • the beam expanding lens is tuned for a wavelength of between 405 and 1064 nm.
  • the beam expanding lens 10 is a 20x lens.
  • the electro-optic modulator comprises any optical device in which a signal-controlled element displaying an electro-optic effect is used to modulate (i.e. modulate the phase, frequency, amplitude or direction of) a beam of light.
  • the electro-optic modulator provides a delay, whereby the S-wave light of the laser light Q is modulated by the external voltage applied to the modulator to change the direction of its polarization axis.
  • Many electro-optic modulators are readily available and known to those skilled in the art.
  • the electro-optic modulator is similar to a liquid crystal phase retarder or an electro-optic crystal such as LiNb0 3 , ADP (NH4H2PO4), or KPD(KH 2 P04).
  • the electro-optic modulator is a device such as a Mach-Zehnder inferometer.
  • the laser Q passes through a second beam splitter 209 and a portion of the laser beam striking the second beam splitter 209 is directed to a power meter 208.
  • the power meter 208 detects the amount of light energy contained in the laser Q and provides a signal indicative of the measured energy to a computer controlling operation of various components of the microscope.
  • the laser Q then enters a first combining element 12, combines the laser beam Q of the first laser generator device 6 with light Qb from a second laser generating source (i.e. confocal source) 13 to form a common beam path Qa which intersects a mirror 212.
  • the second laser 13 generating source is a diode laser.
  • the light of the confocal source Qb has a shorter wavelength than the laser beam Q.
  • the first combining element 12 combines laser light Q having wavelengths of between 690 and 1050 nm with light from the confocal source Qb having wavelengths between 450 and 650 nm (i.e., in the visible spectrum).
  • the first combining element 12 combines laser light Q having a wavelength of about 1020 nm with light from the confocal source Qb having wavelengths of 488,561 and 633 nm.
  • the reflected light Qa from the mirror 212 next passes through a beam shutter 214 and focusing mirrors 216 before intersecting a primary dichroic mirror 15.
  • the term dichroic mirror refers to any mirror that can accurately filter color by selectively passing light or a small range of colors while reflecting other colors.
  • the dichroic mirror comprises a substrate and a dielectric multilayered film formed on the surface of a substrate.
  • the primary dichroic mirror is capable of allowing light beams having a wavelength of about 488 nm and of about 1020 nm to pass simultaneously with high rate of transmission (i.e., laser light emitted from the first laser generating device 6 comprises a wavelength of about 1020 nm from the Ti-sapphire laser and 488 nm from the second laser generating source 13, and light returning from the specimen comprises a wavelength of between 490 and 530 nm).
  • the light Qa then passes through a first scan head 16 that rasterizes the excitation scans, as well as passes the photons from the specimen 30 to a confocal detector 34 for assembling into the final image.
  • the scan head 16 comprises inputs from the first laser generating device 6 and at least one deflecting mirror 17 to direct the laser beam Qa to the specimen incubator 5 and specimen 30.
  • the scan head 16 comprises a galvanometer-based scanning motor with at least one optical mirror 17 mounted on the shaft and a detector that provides positional feedback to the control board. Moving magnets can be used to maximize the response times and resonant frequency of the optical system.
  • the laser light Qa next exits the scan head 16 and enters a second combining element 18 which combines the laser light Qa with laser light Qc emitted from a third laser source 19, to form the combined laser light Qd.
  • Laser light Qc emitted from the third laser source 19 passes through a second scan head 21, whereby the laser light Qc is then reflected by a second mirror 22 and combined with the laser light Qa within the second combining element 18.
  • the scan head 21 includes a second deflecting mirror 17a for rasterizing the laser light Qc.
  • the laser light Qc comprises wavelengths of between 690 and 1050 nm.
  • the laser light Qc comprises wavelengths of between 400 and 600 nm.
  • the laser light Qc comprises wavelengths of 405 and 473 nm.
  • the third laser light source 19 emits ultraviolet and visible light to cause uncagging or photoactivation within the specimen 30.
  • a scan lens 23 refers to the lens which focuses the line scan generated by the mirrors 17, 17a of the scan heads 16, 21 at a conjunctive image plane of the optical system 100. This raster scans are then relayed by the optics of the system to the conjugate plane within the specimen 50 to excite, for example, a fluorescent protien in the specimen.
  • Such scan lenses are readily available to those skilled in the art and may have, for example, a wavelength range of at least 450 to 1100 nm, a focal length of between 28.6 and 172 nun, and a field size of between 3 and 36 mm.
  • the objective lens 26 allows high ultra-violet transmission, high numerical aperature (NA) and good chromatic correction, and includes a correction collar for viewing through thickness of optical grade glass from 0.17 to 2.5 mm, as well as improved signal-to-noise ratio for short wavelengths.
  • NA numerical aperature
  • suitable objective lenses have an NA of between 0.4 to 1.3, between 0.6 to 1.0, or between 0.7 and 0.95, and a working distance (WD) of between 2.5 and 9.0 mm, between 3.5 and 8.5 mm, or between 3.6 and 8.2 mm.
  • Exemplary objective lenses are the S Plan Fluor EL WD 40x and S plan Fluor EL WD 20x lenses available from Nikon, Inc.
  • the beam splitter 9 is not necessarily accommodated upstream from the scan lens 23, tube lens 24, and long pass dichroic mirror 25, , but the configuration may be such that the beam splitter 9 is located anywhere within the plurality of optical elements.
  • the scan lens 23, tube lens 24, long pass dichroic mirror 25, and objective lens 26 define an optical axis of the optical system 100, and is configured as a focusing arrangement, and may also be configured to provide a desired image magnification.
  • the optical system 100 of the present disclosure also provides a signal detector 108, comprising a first and second photomultiplier tube 27, 28, an infrared filter 29, a focusing lens 30, a green/red dichroic mirror 31 and red and green filters, 32, 33, respectively.
  • the signal detector 108 is a multi-photon detector. As shown in FIG 2, the reflected light Qe (dashed line) reflected from the specimen 30 is passed back through the objective lens 26 and long pass dichroic mirror 25, which reflects the portion of the light Qe having a wavelength of about 581 nm to the multi-photon detector 108.
  • the multi-photon detector 108 may be configured to define an array of image pixels (e.g., CCD), may be configured for intensified or non-intensified light direction, as well as for gated or non-gated light detection.
  • the green/red dichroic mirror 31 provides the ability to detect fluorescence from a variety of dyes or proteins, either simultaneously or individually.
  • a combination of dichroic filters and band pass filters can be placed in front of the first and second photomultiplier tubes 27, 28, when the first an second photomultiplier tubes 27, 28 are behind the scan heads 16, 21 with respect to the light path Qe from the specimen 30.
  • the filters may be arranged to reflect short wavelengths and pass longer wavelengths.
  • the red/green dichroic mirror 31 is removable so that light losses are not encountered when wavelength separation is not required. The signals are then sent to a digitizer, amplifier and computer (not shown) for analysis and display.
  • the light Qf comprises a wavelength of between 450 and 650 nm, or between 490 and 530 nm. In one embodiment, the light Qf comprises a wavelength of 507 nm.
  • the light Qf reflected into the confocal detector 34 by the primary dichroic mirror 15 passes through a pin hole 35 and a first and second dichroic mirror 36, 37 whereby the light is split into blue, green and red beams Qblue, Qgreen, Qred and directed into a corresponding photomultiplier tube 38, 39, 40 for collection and processing.
  • the confocal detector 33 comprises variable pinhole apertures 35. These variable apertures may vary in diameter and may be contained on a rotating turret that enables an operator to adjust the pinhole size (and optical section thickness). The pinhole 35 aperture also serves to eliminate much of the stray light passing through the optical system.
  • a microscope according to the present disclosure includes a motorized multi-repeatable position stage (such as Prior Scientific H101A ProScan II stage).
  • the optical device 100 of the present disclosure also provides a specimen incubator 5 which houses a specimen such as a tissue specimen for viewing.
  • the specimen incubator 5 provides a sterile, humidified, temperature, CO 2 as well as O 2 controlled chamber in which a tissue specimen may be cultured for extended periods of time (i.e. kept alive) as well as allow for the viewing of the specimen by the optical system 100 disclosed herein.
  • FIG 3A is a side cross section of the specimen incubator 5 described herein.
  • the specimen incubator 5 comprises a first housing 51 having an internal chamber 52.
  • the first housing 51 depicted in FIG 3 A and throughout this disclosure is rectangular shaped, one skilled in the art would recognize that any housing, or suitable shape or dimensions, would be suitable. Such shapes include cylindrical, spherical, trapezoidal, etc.
  • FIG 3A depicts the rectangular housing 51 enclosed by a bottom panel 53 and a top panel 54. In certain embodiments, the top panel 54 is removable.
  • the housing 51 also includes a first end wall 55 and an opposing second side wall 56.
  • the interior chamber 52 is defined between the bottom panel 53, the top panel 54, the first and second end walls 55, 56.
  • the housing 51 can be manufactured using standard machining techniques or can be specifically injection molded to desired dimensions.
  • the housing 51 is made of polycarbonate; however, any suitable material will be sufficient.
  • the material of the housing 51 is capable of being sterilized (e.g., exposure to ethylene oxide).
  • injection molding with a certain plastics would allow for autoclaving of the housing 51.
  • An aperture 57 is formed in the geometric center of the area of the top panel 54.
  • the aperture 57 comprises a material 58 that is suitable for optical applications, such as glass.
  • the material 58 has a refractive index of between 1.0 to 2.0 n D at 20°C.
  • the material 58 comprises a refractive index of between 1.2 to 1.8 no at 20°C. In another embodiment, the material 58 comprises a refractive index of between 1.4 to 1.7 no at 20°C.
  • the aperture 57 is located within the geometric center of the top panel, thereby allowing for viewing by the optical system from the top of the specimen incubator 5. One skilled in the art will recognize that the aperture 57 may be located other than in the geometric center of the top panel of the housing 51.
  • a second housing 59 Positioned within the first housing 51 is a second housing 59comprising a bottom section 60 and a removable top section 51, first side wall 62 extending upward from the bottom section 60, and a removable top section extending downwardly about an exterior circumference of the first side wall 62.
  • the second housing 59 is therefore defined between the bottom section 60, the removable top section 61, and the first side wall 62 and forms a second interior chamber 52a.
  • the second housing 59 can be any suitable shape or dimension, such as cylindrical, spherical, trapezoidal, and the like, such that is can be positioned within the interior chamber of the first housing 52. Such dimensions can be readily determined by one skilled in the art.
  • the second incubation chamber 59 can be manufactured using standard machining techniques or can be specifically injection molded to desired dimensions.
  • the second incubation chamber 59 is made of polycarbonate; however, any suitable material will be sufficient.
  • the material of the second incubation chamber 59 is capable of being sterilized (e.g., exposure to ethylene oxide). Injection molding with certain plastics would allow for autoclaving of the second incubation chamber 59.
  • the second incubation chamber 59 may be a sterile, disposable container, such as a petri dish, in which growth medium 64 is contained.
  • a second aperture 65 is formed in the geometric center of the area of the removable top 61 of the second incubation chamber 59.
  • a third aperture 66 is positioned in the geometric center of the bottom section 60 such that it is in line with the second aperture 65.
  • the second and third apertures 65, 66 comprise a material 67, 68 that is suitable for optical applications, such as glass.
  • the material 67, 68 comprises a refractive index of between 1.0 to 2.0 no at 20°C.
  • the material 67, 68 comprises a refractive index of between 1.2 to 1.8 no at 20°C.
  • the material 67, 68 comprises a refractive index of between 1.4 to 1.7 n D at 20°C.
  • a specimen stand 69 Positioned within the second housing 59 is a specimen stand 69, comprising at least two legs 70, 71 which supports a platform 72, upon which the specimen 30 is positioned.
  • the platform comprises polycarbonate or a similar material, such that the material for the passage of light through the specimen 50.
  • the platform 72 further comprises a growth substrate 73.
  • growth substrates may include glass or plastic coverslips, a sheet of gas-permeable membrane or any other non-toxic material that will support the growth of cells, tissue, etc.
  • the growth substrate comprises a microporous membrane.
  • microporous membrane Any type of microporous membrane that is commonly used for tissue culture purposes may be used, such as hydrophilic PTFE membranes, mixed cellulose ester membranes, polycarbonate membranes, polyethylene terephthalate membranes, and the like.
  • exemplary microporous membranes for use in the present disclosure are those available from Millipore® Corp. (Billerica, MA).
  • the interior chambers 52, 52a comprises an atmosphere conducive for the maintenance of a tissue sample. Specifically, proper amounts of carbon dioxide (CO 2 ), oxygen (O 2 ), pH, humidity, and temperature improve maintenance of tissue samples.
  • CO 2 carbon dioxide
  • O 2 oxygen
  • pH pH
  • humidity temperature
  • mammalian culture media contain buffering systems that require a CO 2 atmosphere. Severe pH shifts may occur if these CO 2 levels are not maintained.
  • low humidity levels can lead to the evaporation of the water from the culture medium resulting in concentrated salt levels and conditions that cause cell lysis. If the pH of the medium is too acidic or too basic conditions will result in poor cell growth and/or permanent cell damage.
  • the atmospheric conditions within the interior chamber 52 are about 95% humidity, 37°C, and 5% C0 2 .
  • the tissue samples to be imaged comprise brain tissue.
  • mediums for culturing the brain tissue are provided below in Tables I and II.
  • Insulin lmg/ml solution 0.5 ml 0.25ml
  • a section of tissue i.e. specimen 30
  • a biological membrane 73 within the second housing 59.
  • Growth medium 64 is added such that the tissue specimen 30 is in contact with the medium 64, but not completely submerged.
  • the second housing 59 is then placed within the first housing 51, which is environmentally-controlled to have a humidity of about 95%, a temperature of about 37°C, and a CO 2 concentration of about 5%.
  • the specimen incubator 5 is then placed within the optical system 100, such that the objective lens 26 is able to view through the first and second apertures 57 and 65 allowing viewing of the specimen 30.
  • Example 1 Viewing of neuronal tissue using the optical system and specimen incubator of the present disclosure.
  • the optical system 100 of the present disclosure was tested to determine if optimal viewing occurred from the top or bottom of the specimen incubator.
  • FIGS 3A and 3B two specimen incubators were constructed.
  • the first specimen incubator comprised an aperture 57 within the geometric center of the bottom panel of the first housing (see FIG. 3B).
  • the second specimen incubator 5 comprised an aperture 57 within the geometric center of the top panel (see FIG. 3A).
  • the objective lens 26 of the optical system 100 was then placed adjacent to each of the apertures, and the specimen viewed.
  • FIG. 4A shows the specimen 30 as imaged from the bottom through the membrane 73
  • FIG. 4B sows the specimen 30 as imaged form the top of the first housing 51.
  • imaging of the specimen was far superior when viewed through an aperture 57 positioned in the top panel 54 of the first housing 51.
  • Example 2 Benefits of using multi-photon excitation. Absorption of energy by fluorochromes occurs between the closely spaced vibrational and rotational energy levels of the excited states in different molecular orbitals.
  • the various energy levels involved in the absorption and emission of light by a fluorophore are typically represented in a Jablonski diagram, such as that found in FIG 5A.
  • the singlet ground state (SO), and the first excited singlet state (S I) are shown in FIG. 5A as a stack of horizontal lines. The thicker lines represent electronic energy levels, while the thinner lines denote the various vibrational energy states (rotational energy states are ignored).
  • Transitions between the states are illustrated as straight or wavy arrows, depending upon whether the transition is associated with absorption or emission of a photon (straight arrow) or results from a molecular internal conversion or nonradiative relaxation process (wavy arrows).
  • Vertical upward arrows are utilized to indicate the instantaneous nature of excitation processes, while the wavy arrows are reserved for those events that occur on a much longer timescale.
  • irradiation with a wide spectrum of wavelengths will generate an entire range of allowed transitions that populate the various vibrational energy levels of the excited states. Some of these transitions will have a much higher degree of probability than others, and when combined, will constitute the absorption spectrum of the molecule.
  • Example 3 Determination of optimal objective lens for depth visualization of brain tissue.
  • brain sections were isolated and imaged using the multi-photon excitation approach. Briefly, brains from young (p2 to plO) mice were quickly dissected and placed in ice-cold ACSF (see Table II herein) bubbled with 95%0 2 /5%C0 2 for 1 minute before chopping. Three-hundred micron coronal slices were made on the tissue chopper and then manually separated in warmed (37°C) culture media. Individual slices were placed on culture inserts and excess solution was removed. Culture inserts were placed in small lidded dishes milled to appropriate height and containing 1ml of culture media. Dishes were placed in the incubator for at least 30 minutes prior to transfer to the imaging microscope. Antibiotic was added for long-term imaging conditions. Additionally, some samples contained 40ng/ml EGF and FGF in the culture media.
  • the optimal conditions for viewing neural tissue through the glass apertures 57, 65 of the incubation chamber 5 were tested. Brain tissue was isolated and positioned within the specimen incubator 5 as described above. The brain tissue was then imaged using either oil dipped objective lens having an N.A. of 1.4 at 63x or an objective having an N.A. of 0.65 under air conditions at 40x. As shown in FIGS 6A and 6B, the objective having a lower N.A. with long working distance gave detailed image in the XY plane similarly to 1.4 N.A. 63x oil objective with short working distance.
  • the optimal conditions for imaging brain tissue through the specimen incubator include a long working distance between the glass aperture and objective lens, good image resolution, glass correction, and high IR transmittance.
  • FIG. 7A and 7B show brain tissue imaged with one embodiment of the optical system 100 of the present disclosure using a 0.45 N.A. objective lens 26 having a 7 mm WD under 20x power (air) and corrected using deconvolution software.
  • Example 4 Visualization a/neurological stem cells.
  • ESC embryonic stem cells
  • ASC adult stem cells
  • FIGS 12A and 12B ESCs are largely niche-independent, found in a transient state, and have a single overall goal- to differentiate.
  • ASCs are largely niche-dependent, maintained in homeostasis, and are balanced between differentiation and self-renewal.
  • the postnatal stem cells have been determined to be in a few specialized locations, including the subventricular zone (SVZ).
  • the SVZ niche is found in the lateral wall of the lateral ventricle (FIG 10A). It has been demonstrated that adult SVZ niche stem cells can produce new neurons.
  • FIG 10B shows a cut-away section of the brain illustrating the lateral ventricle and the SVZ.
  • Stem cells B-cells
  • C-cells transiently amplifying cells
  • A-cells neuroblasts
  • Ependymal cells are multi-ciliated and line the brain ventricle wall.
  • B-cells are mono-ciliated and line the ventricle surface as well as contacting blood vessels that are underneath the ventricle surface.
  • FIG IOC is a diagram showing SVZ neurogenesis.
  • neuroblasts migrate from the lateral ventricle, along the rostral-migratory stream, and integrate into established networks in the olfactory bulb primarily as inhibitory granule cells.
  • the lineage of the stem cells expressing the Nestin-CreER gene with a lac-z reporter (described in Example 5) can be imaged using the optical system of the present disclosure.
  • Example 5 Imaging neural stem cells with the optical system a/the present disclosure.
  • transgenic mice can be generated to express the Nestin-CreER transgene (see FIG 1 1A and 1 IB;) with an enhanced green fluorescent protein (EGFP) or tdTomato reporter transgenic mouse lines. All postnatal and adult neural cells in the double transgenic mice express the tamoxifen- inducible CreER tm enzyme. However, without tamoxifen CreER tm is rendered non-functional by Hsp90 sequestration in the cytoplasm, preventing its translocation into the nucleus.
  • EGFP enhanced green fluorescent protein
  • FIG 12 shows the results of two-color, deep three-dimension, 2-photon live imaging of transgenic mice engineered to express fluorescent proteins in neural stem cells.
  • the ependymal SVZ niche cells are labeled with Foxj l-EGFP transgene and stem cells with Nestin- CreER tm crossed to tdTomato-reporter mice.
  • the Image stacks are rendered with Imaris software.
  • Example 6 Screening the effect of a compound of interest on the behavior of neural stem cells.
  • brain sections obtained from transgenic mice engineered to express the Nestin-CreER transgene are prepared as described in Example 3 and placed in the specimen incubator 5.
  • a compound of interest which may include any compound, such as those that effect the nervous system, is placed in the growth medium 73 of specimen incubator 5. Concentrations of the compound of interest may vary and be dependent on a number of variables, such as potency of the compound of interest, dosages typically administered to humans in the clinical setting, etc. and can be readily determined by one skilled in the art.
  • brain slices from the transgenic mice are cultured in growth medium without the compound of interest.
  • the brain slices are allowed to incubate in the medium for a pre-determined amount of time before viewing. Images are taken of the neural stem cells using an embodiment of the optical system 100 of the present disclosure, and changes in neural stem cell behavior as compared between the experimental and control brain sections can be noted.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

La présente invention a trait à un système optique pour imagerie à résolution en profondeur in vitro d'un échantillon, tel qu'un tissu vivant (par exemple un tissu cérébral). Le système optique comprend un microscope confocal et multiphotonique comportant un objectif à correction de verre réglable et un gaz entre la lentille d'objectif et un échantillon. Ce système optique comporte également une chambre d'incubation munie d'un premier logement ayant une ouverture en verre et d'un second logement entouré par le premier, ledit second logement ayant une ouverture en verre. L'échantillon est placé à l'intérieur du second logement. La présente invention a trait également à des procédés d'utilisation dudit système optique.
PCT/US2010/054303 2009-10-27 2010-10-27 Microscopie multiphotonique par l'intermédiaire d'une lentille d'objectif à interface hertzienne Ceased WO2011056658A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111788471A (zh) * 2017-11-14 2020-10-16 思迪赛特诊断有限公司 用于光学测量的样品载体
WO2022061191A1 (fr) * 2020-09-20 2022-03-24 The Regents Of The University Of California Microscopie à éclairage à feuilles transversales (transim)
US11733150B2 (en) 2016-03-30 2023-08-22 S.D. Sight Diagnostics Ltd. Distinguishing between blood sample components
US11796788B2 (en) 2015-09-17 2023-10-24 S.D. Sight Diagnostics Ltd. Detecting a defect within a bodily sample
US11808758B2 (en) 2016-05-11 2023-11-07 S.D. Sight Diagnostics Ltd. Sample carrier for optical measurements
US12174175B2 (en) 2016-05-11 2024-12-24 S.D. Sight Diagnostics Ltd. Performing measurements on a sample
US12189112B2 (en) 2019-12-12 2025-01-07 S.D. Sight Diagnostics Ltd. Artificial generation of color blood smear image
US12393010B2 (en) 2013-08-26 2025-08-19 S.D. Sight Diagnostics Ltd. Distinguishing between entities in a blood sample
US12436101B2 (en) 2019-12-12 2025-10-07 S.D. Sight Diagnostics Ltd. Microscopy unit
WO2025235411A1 (fr) * 2024-05-06 2025-11-13 Mango, Inc. Profondeur étendue de champ pour images à haute résolution sur la base d'images décalées de sous-pixels
US12498325B2 (en) 2020-10-22 2025-12-16 S.D. Sight Diagnostics Ltd. Accounting for errors in optical measurements

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6222664B1 (en) * 1999-07-22 2001-04-24 Agilent Technologies Inc. Background reduction apparatus and method for confocal fluorescence detection systems
US6740868B1 (en) * 1999-11-29 2004-05-25 Leica Microsystems Heidelberg Gmbh Scanning microscope and a method of scanning a specimen
WO2004061435A1 (fr) * 2002-12-26 2004-07-22 Applied Precision, Llc Systeme et procede d'illumination de cellules vivantes pour imagerie
US20060237666A1 (en) * 2005-04-25 2006-10-26 Olympus Corporation Multiphoton-excitation laser scanning microscope
US20060239983A1 (en) * 2000-07-31 2006-10-26 Piero Anversa Methods and compositions for the repair and/or regeneration of damaged myocardium
US20060245021A1 (en) * 2003-09-18 2006-11-02 Dan Davidovici Multiphoton fluorescence microscope with plane array detector
US20070057211A1 (en) * 2005-05-25 2007-03-15 Karsten Bahlman Multifocal imaging systems and method
EP1855141A1 (fr) * 2006-05-11 2007-11-14 Olympus Corporation Microscope à balayage laser et procédé d'observation au microscope

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6222664B1 (en) * 1999-07-22 2001-04-24 Agilent Technologies Inc. Background reduction apparatus and method for confocal fluorescence detection systems
US6740868B1 (en) * 1999-11-29 2004-05-25 Leica Microsystems Heidelberg Gmbh Scanning microscope and a method of scanning a specimen
US20060239983A1 (en) * 2000-07-31 2006-10-26 Piero Anversa Methods and compositions for the repair and/or regeneration of damaged myocardium
WO2004061435A1 (fr) * 2002-12-26 2004-07-22 Applied Precision, Llc Systeme et procede d'illumination de cellules vivantes pour imagerie
US20060245021A1 (en) * 2003-09-18 2006-11-02 Dan Davidovici Multiphoton fluorescence microscope with plane array detector
US20060237666A1 (en) * 2005-04-25 2006-10-26 Olympus Corporation Multiphoton-excitation laser scanning microscope
US20070057211A1 (en) * 2005-05-25 2007-03-15 Karsten Bahlman Multifocal imaging systems and method
EP1855141A1 (fr) * 2006-05-11 2007-11-14 Olympus Corporation Microscope à balayage laser et procédé d'observation au microscope

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KUO CT, ET AL: "Postnatal deletion of Numb/Numblike reveals repair and remodeling capacity in the subventricular neurogenic niche", CELL, vol. 127, no. 6, 15 December 2006 (2006-12-15), pages 1253 - 1264, XP002619441 *
MILIND RAJADHYAKSHA ET AL., J. OF INVEST. DERM., vol. 104, no. 6, 1995, pages 1 - 7
MILIND RAJADHYAKSHA ET AL., LASER FOCUS WORLD, 1997, pages 119 - 127

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12393010B2 (en) 2013-08-26 2025-08-19 S.D. Sight Diagnostics Ltd. Distinguishing between entities in a blood sample
US11796788B2 (en) 2015-09-17 2023-10-24 S.D. Sight Diagnostics Ltd. Detecting a defect within a bodily sample
US11914133B2 (en) 2015-09-17 2024-02-27 S.D. Sight Diagnostics Ltd. Methods and apparatus for analyzing a bodily sample
US11733150B2 (en) 2016-03-30 2023-08-22 S.D. Sight Diagnostics Ltd. Distinguishing between blood sample components
US12196664B2 (en) 2016-03-30 2025-01-14 S.D. Sight Diagnostics Ltd. Distinguishing between blood sample components
US12174175B2 (en) 2016-05-11 2024-12-24 S.D. Sight Diagnostics Ltd. Performing measurements on a sample
US11808758B2 (en) 2016-05-11 2023-11-07 S.D. Sight Diagnostics Ltd. Sample carrier for optical measurements
US12181463B2 (en) 2016-05-11 2024-12-31 S.D. Sight Diagnostics Ltd. Performing optical measurements on a sample
CN111788471B (zh) * 2017-11-14 2023-12-12 思迪赛特诊断有限公司 用于光学测量的样品载体
US11921272B2 (en) * 2017-11-14 2024-03-05 S.D. Sight Diagnostics Ltd. Sample carrier for optical measurements
US11609413B2 (en) 2017-11-14 2023-03-21 S.D. Sight Diagnostics Ltd. Sample carrier for microscopy and optical density measurements
CN111788471A (zh) * 2017-11-14 2020-10-16 思迪赛特诊断有限公司 用于光学测量的样品载体
US12196940B2 (en) 2017-11-14 2025-01-14 S.D. Sight Diagnostics Ltd. Sample carrier for microscopy and optical density measurements
US11614609B2 (en) 2017-11-14 2023-03-28 S.D. Sight Diagnostics Ltd. Sample carrier for microscopy measurements
US12189112B2 (en) 2019-12-12 2025-01-07 S.D. Sight Diagnostics Ltd. Artificial generation of color blood smear image
US12436101B2 (en) 2019-12-12 2025-10-07 S.D. Sight Diagnostics Ltd. Microscopy unit
WO2022061191A1 (fr) * 2020-09-20 2022-03-24 The Regents Of The University Of California Microscopie à éclairage à feuilles transversales (transim)
US12498325B2 (en) 2020-10-22 2025-12-16 S.D. Sight Diagnostics Ltd. Accounting for errors in optical measurements
WO2025235411A1 (fr) * 2024-05-06 2025-11-13 Mango, Inc. Profondeur étendue de champ pour images à haute résolution sur la base d'images décalées de sous-pixels

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