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WO2013008033A1 - Améliorations concernant la microscopie à fluorescence - Google Patents

Améliorations concernant la microscopie à fluorescence Download PDF

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Publication number
WO2013008033A1
WO2013008033A1 PCT/GB2012/051680 GB2012051680W WO2013008033A1 WO 2013008033 A1 WO2013008033 A1 WO 2013008033A1 GB 2012051680 W GB2012051680 W GB 2012051680W WO 2013008033 A1 WO2013008033 A1 WO 2013008033A1
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Prior art keywords
fluorescence
fluorescence microscopy
depletion
light source
emission
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English (en)
Inventor
Angus John BAIN
Richard John MARSH
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UCL Business Ltd
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UCL Business Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • 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
    • 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
    • G02B21/008—Details of detection or image processing, including general computer control
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison

Definitions

  • This invention relates to fluorescence microscopy.
  • Optical imaging techniques such as confocal microscopy have been developed which can resolve objects or observe contrast on a distance scale restricted to approximately half the wavelength of the illuminating source. In the visible region of the spectrum this is on the order of a quarter to a third of a micron (250-330nm).
  • confocal microscopy In the visible region of the spectrum this is on the order of a quarter to a third of a micron (250-330nm).
  • Structured (wide-field) illumination uses patterned excitation to excite the sample, the measurement of the fringes arising from the interference between the illumination pattern and the sample and post-exposure image analysis for enhanced image resolution.
  • the technique enhances the resolution to half the diffraction limit. See for example: “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy” M G L Gustafsson, Journal of Microscopy Volume 198, Issue 2, 82-87, (2000)
  • PAM photoactivated localization microscopy
  • PROM stochastic optical reconstruction microscopy
  • STED creates a sub-micron fluorescent spot by the overlap of the initial exciting beam (PUMP) with a depletion (DUMP) laser (pulsed or continuous wave) which is "shaped" to provide a 'doughnut' intensity profile.
  • the DUMP removes close to 100% of the fluorescent molecules outside the "hole” through stimulated emission.
  • the PUMP- DUMP combination is scanned over the sample to produce the image.
  • the drawbacks of STED are [i] the expense and complexity of the DUMP beam-shaping optics and [ii] the on-sample DUMP powers that are required to obtain high resolution. To obtain an effective point spread function on the order of the diameter of the "hole” in the focused DUMP beam requires close to 100% population removal elsewhere.
  • Such DUMP powers are in the regions of GWcm-2 , this is an intensity where the onset of photochemical damage and sample heating become significant risks. See for example:
  • a fluorescence microscopy method comprises inducing stimulated emission to spatially modify the time evolution of fluorescence emission. Data is acquired relating to the fluorescence emission and the data is processed to form an image.
  • stimulated emission is induced with depletion light having a spatially varying intensity to cause spatial variation of the fluorescence emission in time.
  • a high degree of resolution is not dependent on a high degree of depletion.
  • best resolution is achieved with typical depletion levels between 30-50%.
  • image enhancements may be achieved with low depletion powers, far lower than in STED.
  • Low on-sample powers are particularly advantageous when imaging biological structures/systems, as high powers may damage the sample.
  • methods and apparatus according to the present invention provide for super resolution microscopy.
  • Super resolution refers to the ability to resolve objects and/or observe contrast on a distance scale below that afforded by conventional optical imaging such as confocal microscopy (described for example in US 3,013,467).
  • the image is a composite image formed by combining separate temporal slices within the fluorescence intensity decay.
  • processing the data to form an image comprises forming a linear combination of time slices within the fluorescence intensity decay.
  • the number, sign, relative magnitude and temporal width of time slices in said linear combination may be determined by determining a linear combination of time slices of a point spread function which yields an optimum point spread function.
  • Time slices of the point spread function may be obtained by measurement of a sub-wavelength test sample.
  • processing the data comprises forming different linear combinations of time slices for respective different regions of the sample, thereby to form said composite image.
  • the invention also provides a fluorescence microscopy apparatus for acquiring data for time-varying fluorescence emission, comprising: a depletion light source to spatially modify the time evolution of the fluorescence emission by way of stimulated emission; and a data processing apparatus to process data acquired for said fluorescence emission to form an image
  • the depletion light source may comprise a continuous wave (CW) light source.
  • the fluorescence microscopy apparatus may comprise an excitation light source in the form of a pulsed excitation light source.
  • the excitation light source and the depletion light source are arranged so that the excitation beam and the depletion beam are spatially coincident at the sample.
  • the depletion light source may be configured to operate in the fundamental transverse mode.
  • the term "light” includes visible and also non-visible light such as infrared light and ultra-violet light.
  • Embodiments of the present invention provide for spatial (3 dimensional) modification of the time evolution of fluorescent images using stimulated emission with a continuous wave light source.
  • linear combinations of time segments of the resulting modified fluorescent images resulting yield a composite image with increased (sub-diffraction limit) spatial resolution. Determination of the number, sign, relative magnitude and temporal width of the images can be determined by the combination of segments of the evolving point spread function, i.e. time sliced fluorescent images for a sample with sub diffraction limited spatial structure. The combination of images that yield the minimum (or optimum with regards to image contrast and signal to noise) point spread function are used in the reconstruction of the time resolved fluorescence images.
  • Figure ⁇ is a schematic drawing of a fluorescence microscopy apparatus according to an exemplary embodiment of the invention.
  • Figure 2 illustrates an example of spatial variation of the fluorescence signal of a point l o obj ect for four time 'windows';
  • Figure 3 shows five exemplary 'time window' distributions corresponding to o, o.5tf , tf, 2tf and 3tf, where tf is the fluorescence lifetime;
  • Figure 3 (bottom) shows, by way of example, an improved PSF constructed from the time windows distributions of Figure 3 (top);
  • Figure 4 shows an example of reconstruction of a one dimensional image from five
  • Figure 5(b) is an exemplary measured image simulating the effect of the PSF on the image of 5(a);
  • Figures 5(c)-s(e) show exemplary simulated images showing the effect of the PSF and continuous wave stimulated emission depletion after 1, 2 and 3 fluorescence lifetimes respectively;
  • Figure 5(f) shows the result of combining 'time slice images in the proportions described below, recovering much of the true image
  • Figure 6 shows an example of the axial (z) dependence of the reconstructed PSF for a single point source compared with the normally observed distribution.
  • FIG. 1 shows a fluorescence microscopy apparatus 1 for collecting fluorescence
  • the fluorescence microscopy apparatus includes a fluorescence microscope in the form of a scanning confocal microscope 2.
  • Confocal microscope 2 includes an objective lens 3, and a first light source in the form of a pulsed excitation light source 4.
  • the apparatus 1
  • the 35 also include a second light source comprising a continuous wave (CW) depletion light source 5.
  • the excitation light source 4 generates an excitation beam and the depletion light source 4 generates a depletion beam at a different wavelength to the excitation beam.
  • a dichroic mirror (not shown) is provided which is reflective at one of the wavelengths and transmissive at the other, and arranged so that the depletion beam is spatially co-incident with the excitation beam at the sample.
  • the fluorescence microscopy apparatus 1 also includes a detection system in the form of time-resolved fluorescence detection system 6, and a data processing apparatus 7 such as a PC or other computing apparatus, to process the acquired data.
  • the two light sources 4, 5 is sent coaxially through the microscope objective lens to focus in the sample at the same point.
  • the focal point is then scanned across the sample and fluorescence is collected as the fluorescent marker molecules are excited and de-excited at rates determined by the intensities of the two beams.
  • the size of the focal point can be focussed to a size of approximately half the wavelength of the focused light.
  • the wavelength of the depletion light may be chosen as to coincide with the red edge of the molecular emission spectrum of the fluorescent marker, thus causing de-excitation without significant re-excitation.
  • the time-resolved fluorescence detection system 7 records the time evolution of the fluorescence intensity following excitation at each point (pixel) as the focused excitation source and depletion source is moved from pixel-to-pixel across the sample.
  • TCSPC Time Correlated Single Photon Counting
  • phase sensitive detection using high repetition rate ca. io 8 s _1
  • high repetition rate ca. io 8 s _1
  • the resulting data comprises a 2 dimensional (spatial) array of detected photon counts and their coincidence (arrival) times.
  • this data photon count per time bin
  • time slices of the fluorescent image in the presence of the depletion light are formed by the summation of equivalent time bins for each pixel. As is described in more detail hereinbelow, the time slices are combined in linear combination to form a combined image with improved characteristics.
  • the number of pixels in each direction may be chosen so that the focused spot size in any dimension extends over several pixels with the pixel size smaller than the desired spatial resolution. Additionally the temporal range (e.g. number of time bins in
  • TCSPC may be chosen to extend over several fluorescent lifetimes. A sufficient number of time bins must be used to yield the desired resolution (see below).
  • the full width half maximum (FWHM) of the intensity distribution is determined by
  • the dimension x can be in units of length or number of pixels in an image with x 0 the location of the point source.
  • the FWHM will depend on the method of excitation, the wavelength of light and the detection optics (e.g. microscope objective and, where present, confocal optics and pinhole).
  • the fluorescence lifetime is the same for all identical molecules in equivalent environments.
  • the intensity of the focused depletion field has a spatial (Gaussian) variation of the form
  • the stimulated depletion rate at position x is linearly dependent on ID (X)
  • the depletion intensity I'D(X) is expressed as its spatial dependence (relative to ⁇ at the centre x 0 ) and a magnitude that will result in a specific fraction of the molecules being removed (i3 ⁇ 4 and x is the distance from the centre of the PSF.
  • ⁇ ' the beam radius of the focused depletion field ⁇ ' is the same as ⁇ (eqs.i-2), this is not a fundamental requirement.
  • the four "time windows" of Figure 2 correspond to o, 1.25, 2.5 and 5.0 times the fluorescence lifetime (in the absence of depletion) .
  • the degree of population removal Fd is 0.333.
  • the distributions have been normalized to yield an area of unity.
  • the location of the point object x 0 is at pixel 11.
  • the FWHM of the initial (diffraction limited) PSF corresponds to 6.67 pixels.
  • the resulting (one dimensional) image is given by:
  • n is the number of 'time windows'
  • ti are their corresponding time values
  • d are dimensionless coefficients which determine the contribution of each 'time window' to the reconstructed PSF P( x ) res .
  • Figure 3 shows the results of adding five such 'time windows' together in the appropriate proportions and the resulting narrowing of the PSF.
  • the coefficients of the superposition are 1, 5.85501,-10.028, 11.1026,-7.33873 respectively.
  • the reconstructed point spread function has a FWHM of 2 pixels.
  • the coefficients of the superposition can be determined in a number of ways. In this simple case they are set such that the values of the superposition at 4 points at the edge of the PSF are zero (pixels 2, 3, 4 and 6 in figure 3) by numerical solution of the resulting simultaneous equations. Alternatively the coefficients can be optimised using an iterative algorithm, for example to minimise the standard deviation of the PSF.
  • the measured fluorescence intensities at any particular point (pixel) will consist not only of the molecules at that point but also a contribution from adjacent points (pixels) weighted by the PSF evaluated for the distance between these points (pixels) and the central point.
  • the one dimensional 'image' can therefore be represented by a linear superposition of individual (single pixel) PSFs and intensities.
  • the total fluorescence signal measured in the n th pixel I n tot is given by
  • N x is the actual number of fluorescence events within that pixel.
  • the range of the summation (.Xmm-Xmox) would ideally be the entire range of pixels but can easily be truncated to a point where the relative contribution from the PSF is negligible.
  • the fluorescence lifetime of the probe molecules has a spatial variation dependent on the depletion intensity at that point.
  • the contribution to the total intensity at any one pixel from adjacent pixels in the above sum will have a time dependence given by
  • Figure 4 shows the results of such a combination for a 1 dimensional image made from 9 points of varying intensities.
  • the 'time windows' used for the reconstruction are the same and used in the same proportions as for the optimisation of the single point PSF, shown in Figure 3. Shown for comparison is the underlying spatial structure (the 'true image') and what would be observed using a normal (diffraction limited) PSF.
  • Circles show the observed fluorescence distribution due to the PSF.
  • Triangles show the reconstructed image using the same five 'time windows' using the same weightings as in Figure 3
  • the approach can be extended to a normal two dimensional image by describing the normal PSF and the relative depletion intensity as functions of both dimensions.
  • the coefficients for any reconstruction depend only on the intrinsic PSF of the instrument, the spatial variation of the depletion intensity (ID (X) ), the pixel resolution and the degree of depletion Fd.
  • the first three are independent of the image data and are either constant or experimentally controllable.
  • the degree of depletion depends on the intensity of the depletion beam, the particular probe molecule chosen and to some degree the local environment of the probe (e.g. fluorescence lifetime variations, molecular orientation and rotational diffusion rates). If Fa is unchanging between different samples or images there is no need to re-evaluate the coefficients. If Fd does change (even between different regions of a sample) this does not prevent the reconstruction of an improved resolution image.
  • sub-wavelength resolution fluorescent images can be realised by the linear combination of fluorescent images recorded in a series of time windows following pulsed excitation of fluorescent markers in the sample in the presence of a stimulated emission depletion induced by a continuous wave light source.
  • This gives rise to a spatial variation in the observed fluorescence lifetime and an evolution in the effective point spread function (PSF) of the microscope with time.
  • Knowledge of this evolution e.g. by measurement of a sub wavelength test sample, or by theoretical modelling
  • time slices (segments) of the evolving PSF can be combined to yield a minimised (sub-diffraction limit) PSF.
  • optimised (sub-diffraction limit) PSF minimised (sub-diffraction limit)
  • Exemplary embodiments of the invention described herein achieve super resolution in fluorescence microscopy through imaging the modifications to the time and spatial dependence of fluorescent probe emission in the presence of stimulated emission depletion induced by a continuous wave light source.
  • One embodiment of the invention can be realised by the addition of the depletion laser to a fluorescence lifetime imaging microscopy (FLIM) apparatus, together with a data processing apparatus to analyse the information provided by the intensity-space-time data provided by the FLIM system.
  • FLIM fluorescence lifetime imaging microscopy
  • a low power (ca 0.1 W) continuous wave depletion light source may be used.
  • the CW depletion light source may provide an unstructured (conventional) spatial profile (e.g: fundamental transverse Gaussian mode TEMoo), which in embodiments is similar to that of the pulsed light source used to excite the fluorescent probe by single, or multi-photon excitation.
  • a sub-diffraction limited fluorescent spot is not created (as in STED microscopy) but rather reconstructed by analysis of the space and time variation of the fluorescence emitted in the presence of the depletion field as recorded by for example a fluorescence lifetime imaging (FLIM) microscope. Spatial resolution is not critically determined by the degree of depletion and on-sample powers are estimated to be at least an order of magnitude below that of the typical STED doughnut.

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Abstract

L'invention concerne un procédé de génération d'images fluorescentes à résolution sous-longueur d'onde d'un échantillon par une combinaison linéaire d'images fluorescentes enregistrées dans une série de fenêtres temporelles à la suite d'une excitation pulsée de marqueurs fluorescents dans l'échantillon en présence d'une décroissance d'émission stimulée induite par une source lumineuse à décroissance continue d'ondes, ladite décroissance d'émission stimulée se traduisant par une variation spatiale de la durée de vie observée de fluorescence et une évolution de la fonction effective d'étalement de point (PSF) du microscope avec le temps.
PCT/GB2012/051680 2011-07-13 2012-07-13 Améliorations concernant la microscopie à fluorescence Ceased WO2013008033A1 (fr)

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CN103279926A (zh) * 2013-05-15 2013-09-04 中国航空工业集团公司沈阳空气动力研究所 一种tsp/psp旋转部件测量的模糊修正方法
WO2014169197A1 (fr) * 2013-04-12 2014-10-16 Duky University Systèmes et procédés pour microscopie avec contraste de phase à super-résolution et éclairage structuré
WO2015055900A2 (fr) 2013-10-14 2015-04-23 Bioaxial Sas Procédé et dispositif de mesure optique
WO2016092161A1 (fr) 2014-12-09 2016-06-16 Bioaxial Sas Procédé et dispositif de mesure optique
US10238279B2 (en) 2015-02-06 2019-03-26 Duke University Stereoscopic display systems and methods for displaying surgical data and information in a surgical microscope
US10694939B2 (en) 2016-04-29 2020-06-30 Duke University Whole eye optical coherence tomography(OCT) imaging systems and related methods
US10835119B2 (en) 2015-02-05 2020-11-17 Duke University Compact telescope configurations for light scanning systems and methods of using the same
CN112162079A (zh) * 2020-09-09 2021-01-01 中国科学院过程工程研究所 一种无人值守式熔体热物性参数的测试系统装置及测试方法

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014169197A1 (fr) * 2013-04-12 2014-10-16 Duky University Systèmes et procédés pour microscopie avec contraste de phase à super-résolution et éclairage structuré
US9864183B2 (en) 2013-04-12 2018-01-09 Duke University Systems and methods for structured illumination super-resolution phase microscopy
CN103279926A (zh) * 2013-05-15 2013-09-04 中国航空工业集团公司沈阳空气动力研究所 一种tsp/psp旋转部件测量的模糊修正方法
WO2015055900A2 (fr) 2013-10-14 2015-04-23 Bioaxial Sas Procédé et dispositif de mesure optique
EP3926387A1 (fr) 2013-10-14 2021-12-22 Bioaxial SAS Système de super résolution d'images de fluorescence basé sur la diffraction conique incluant un algorithme de reconstruction bayésien
WO2016092161A1 (fr) 2014-12-09 2016-06-16 Bioaxial Sas Procédé et dispositif de mesure optique
US10835119B2 (en) 2015-02-05 2020-11-17 Duke University Compact telescope configurations for light scanning systems and methods of using the same
US10238279B2 (en) 2015-02-06 2019-03-26 Duke University Stereoscopic display systems and methods for displaying surgical data and information in a surgical microscope
US10694939B2 (en) 2016-04-29 2020-06-30 Duke University Whole eye optical coherence tomography(OCT) imaging systems and related methods
CN112162079A (zh) * 2020-09-09 2021-01-01 中国科学院过程工程研究所 一种无人值守式熔体热物性参数的测试系统装置及测试方法

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