WO2013008033A1

WO2013008033A1 - Improvements relating to fluorescence microscopy

Info

Publication number: WO2013008033A1
Application number: PCT/GB2012/051680
Authority: WO
Inventors: Angus John BAIN; Richard John MARSH
Original assignee: Ucl Business Plc
Priority date: 2011-07-13
Filing date: 2012-07-13
Publication date: 2013-01-17
Also published as: GB201111976D0

Abstract

Method of generating sub-wavelength resolution fluorescent images of a sample by 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 stimulated emission depletion induced by a continuous wave depletion light source, said stimulated emission depletion resulting in a spatial variation in the observed fluorescence lifetime and an evolution in the effective point spread function (PSF) of the microscope with time..

Description

Improvements relating to fluorescence microscopy

Field

This invention relates to fluorescence microscopy.

Background

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). There is a vast range of biological structures/systems where non-invasive observations below this length scale are inaccessible to conventional optical microscopy.

There has been considerable academic and commercial activity aimed at developing optical techniques that reveal structure on the loonm length scale and below. These fall into three categories:

1. Structured Illumination Techniques,

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)

2. Stochastic Image Reconstruction using photo-activatable molecules

These techniques include photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), which require specialised fluorescent probes and relatively long expose times (ca. 500s). See for example:

"Imaging Intracellular Fluorescent Proteins at Nanometer Resolution, E Betzig, G H Patterson, R Sougrat", O W Lindwasser, S Olenych, J S Bonifacino, M W Davidson, J Lippincott-Schwartz and H F Hess, Science Vol. 313 no. 5793 pp. 1642-1645 (2006) "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy

(STORM)", M J Rust, M Bates and X Zhuang, Nature Methods 3, 793-796 (2006) "Ultra high Resolution Imaging by Fluorescence Photoactivation Localization

Microscopy", S T Hess, T P K. Girirajan and M D Mason, Biophys. J 91 4258-4272, (2006)

3. Stimulated Emission Depletion (STED) Techniques

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:

"Breaking the diffraction resolution limit by stimulated emission: stimulated-emission- depletion fluorescence microscopy", S W Hell and J Wichmann, Optics Letters 19, 780- 782 (1994)

"Subdiffraction resolution in far-field fluorescence microscopy", T A Klar & S W Hell, Optics Letters 24, 954-956 (1999)

"Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission", T A Klar, S Jakobs, M Dyba, A Engler, S W Hell Proc Nat Acad Sci USA, 97 8206-8210 (2000)

"Process and Device for Optically Measuring a Point on a Sample with High Local Resolution", S W Hell J Wichmann, US 5,731,588 "STED-Fluorescent Light Microscopy with Two-Photon Excitation", S W Hell and K. Willig, US 2010/0176307

Summary

A fluorescence microscopy method according to embodiments of the present invention 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.

In embodiments, stimulated emission is induced with depletion light having a spatially varying intensity to cause spatial variation of the fluorescence emission in time.

In contrast to STED for example, in embodiments of the present invention a high degree of resolution is not dependent on a high degree of depletion. In embodiments, best resolution is achieved with typical depletion levels between 30-50%.

In this way, 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.

In embodiments, 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).

In embodiments, the image is a composite image formed by combining separate temporal slices within the fluorescence intensity decay.

In embodiments, 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. In embodiments, 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. In embodiments, 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.

As used herein, 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.

In embodiments, 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. Brief Description of the Drawings

So that the invention may be more fully understood, embodiments thereof will now be described, by way of example only, with reference to the accompanying figures, in 5 which:

Figure ι 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 (top) 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);

15 Figure 4 shows an example of reconstruction of a one dimensional image from five

'time windows' optimized for a single point fluorescence emission as described herein; Figure 5(a) shows an exemplary exact structure (true image);

Figure 5(b) is an exemplary measured image simulating the effect of the PSF on the image of 5(a);

0 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;

25 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.

Detailed Description

Image acquisition

0 Figure 1 shows a fluorescence microscopy apparatus 1 for collecting fluorescence

images according to an exemplary embodiment on the invention. As shown, 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

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.

In use, light from 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. For a sufficiently high numerical aperture lens, the size of the focal point can be focussed to a size of approximately half the wavelength of the focused light.

Acquisition of fluorescence after single or two-photon pulsed excitation is carried out with the sample continually illuminated by the depletion light source to de-excite a proportion of the fluorescent marker molecules through stimulated emission. 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. Those skilled in the art will appreciate that various known techniques and apparatus for recording the time evolution of the fluorescence intensity may be readily incorporated into the apparatus and methods described herein. Examples are Time Correlated Single Photon Counting (TCSPC) and phase sensitive detection using high repetition rate (ca. io⁸s^_1) pulsed or high frequency modulated excitation, described for example in "Picosecond Fluorescence Lifetime Imaging Microscopy by TCSPC imaging" W Becker A Bergmann, K Koenig and U Tiralpur Proc. SPIE 4262, 410 (2001), "Analysis of excited state processes by phase modulation fluorescence spectroscopy", J R

Lakowicz and A Baiter Biophys. Chem. 16, 99-115 (1982) and "Fluorescence lifetime- resolved imaging microscopy", R M Clegg and P C Schneider in Fluorescence

Microscopy & Fluorescent Probes ed. J Slavik 15-33 New York Plenum Press (1996). For a given focal depth in the sample, in for example the case of TCSPC detection, the resulting data comprises a 2 dimensional (spatial) array of detected photon counts and their coincidence (arrival) times. Those skilled in the art, cognizant of the present disclosure, will appreciate that this data ( photon count per time bin) could be used to calculate the fluorescence decays at each pixel, and that summation of time bins for each pixel would produce an intensity image. In embodiments of the invention, 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).

Image processing

To facilitate understanding of the image processing techniques described herein, consider first the simplified case of a point source of fluorescence in a conventional (e.g. confocal) microscope with scanning along one dimension (a line). In the far field limit light of wavelength λ can only be focused to an area whose radius is c.a. λ/ 2. A scanning microscope will therefore not only excite and detect fluorescence from the region of the sample at the centre of the laser focus, but also from adjacent regions albeit with a reduced probability. The quantitative description of this effect is encapsulated in the point spread function (PSF). Under optimal conditions this causes a point source of fluorescence (i.e. an object much smaller then the focused spot size) located at position x₀ to appear spread out from this point with an intensity distribution of fluorescence described by P^PSF(x)

[1]

(where A is a dimensionless coefficient chosen such that the area covered by P^PSF(x) is normalised to unity). The intensity of fluorescence can be described by,

[2]

The full width half maximum (FWHM) of the intensity distribution is determined by

[3]

The dimension x can be in units of length or number of pixels in an image with x₀ 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 time dependence of the fluorescence intensity at time f after pulsed excitation at t=o, I(t) is a function of the fluorescence lifetime (f/) of the fluorescent marker molecule:

[4]

Where I(t=o) is the fluorescence intensity detected within a short time window immediately following the maximum intensity of the pulsed excitation source. The fluorescence lifetime is the same for all identical molecules in equivalent environments. However in the presence of a continuous wave depletion field laser field the rate of population decay from the excited state is increased due to stimulated emission and the detected fluorescence will show an increased decay rate (yielding a shorter apparent fluorescence lifetime). In embodiments, the intensity of the focused depletion field has a spatial (Gaussian) variation of the form

[5]

where ω' is the beam radius.

The stimulated depletion rate at position x is linearly dependent on ID (X)

[6]

k_D {x) = BI_D' {x) where B is a constant of proportionality. As a consequence probe molecules at different positions within the PSF will display different fluorescence decay rates. For very short time periods after excitation (much less then the normal fluorescence lifetime) the majority of the observed fluorescence will be emitted from molecules positioned close to the centre of the PSF. However for time periods which are comparable to, or longer than, the normal fluorescence lifetime the fluorescence will increasingly arise primarily from molecules located toward the edges of the PSF which experience lower degrees of stimulated emission depletion.

The effective fluorescence lifetime in the presence of depletion t^D(x) is given by

[7]

Here the depletion intensity I'D(X) is expressed as its spatial dependence (relative to ι at the centre x₀) and a magnitude that will result in a specific fraction of the molecules being removed (i¾ and x is the distance from the centre of the PSF. For theoretical simplicity it is assumed that the beam radius of the focused depletion field ω' is the same as ω (eqs.i-2), this is not a fundamental requirement.

A computer-simulated example of the evolution of the effective PSF in one dimension for a point source is shown in Figure 2, where ω=ω'. 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 x₀ is at pixel 11. The FWHM of the initial (diffraction limited) PSF corresponds to 6.67 pixels.

According to embodiments of the invention, the time resolved florescence intensities are divided into images corresponding to separate temporal slices or 'time windows' within the intensity decay. Combination of these images in the correct proportions (a linear superposition) leads to a reduction in the observed PSF. For example, subtracting some of the fluorescence distribution from the 2.5f_/ time 'window' in figure 2 from the t=o distribution would reduce the signal from the edges of the PSF by a greater degree than the central region leading to a narrowing of the PSF albeit at the expense of signal intensity. The resulting (one dimensional) image is given by:

[8]

where n is the number of 'time windows', ti are their corresponding time values and the 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.

For real images 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

[9]

Where 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. As discussed above, in the presence of the depletion light 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

[10] W =∑N_M+X exp(- ²/2«²)xexp(- tA^D( )) where t^D(x) is calculated as in the previous example. The spatial distribution of fluorescence in each 'time window' is thus well described. In reconstructing a high resolution image, the measured image consists of a summation of single point PSFs and intensities. Therefore the optimal combination of 'time windows' calculated to minimise the PSF of a single point described above will produce the minimum PSF for all the points sources that contribute to the fluorescence image. The image formed from this combination will be identical to that which would have been produced from a conventional instrument with a PSF identical to that of the 'optimised' PSF. 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.

In Figure 4, squares indicate the 'true' image of the object of intensity ratio 4:3:2.

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.

[11]

y

l (t) =∑ ∑N_n+x+xy exp(- (x² ₊ y² )/2a>² )xexi>(- t/t^D (x,y))

[12]

t^D {x, y) = t_f [l - F_d ]/[l + [l_D' {x, y) - l]F_d ]

[13]

For simplicity it has been assumed the PSF and the spatial intensity profile of the continuous wave depletion laser have radial symmetry but this is not essential. In non- radial symmetry the PSF and the depletion laser intensity functions are simply the products of two one-dimensional functions that separately describe the intensity variation in each dimension. Alternatively either or both can be replaced by measured values. Figure 5 shows simulations of the described process for a specified 2

dimensional image consisting of 32x32 pixels. Again the 'time-windows' and the coefficients for the reconstruction are identical to those calculated in figure 3 for optimization of a single point source. Displayed are the 'true image', four of the five 'time window' images used in the reconstruction, showing the 'blurring' of the image by the instrument PSF and the reconstructed image by combining the component images in the previously calculated proportions. Calculation of all the 'time window' images and the reconstructed image can be rapidly calculated on a standard PC. 3-Dimensional imaging

Extending the process for three dimensions follows exactly the same principle as in the transition from one to two dimensions. In this case, the PSF and depletion intensity variations are described in 3 dimensions. There is a choice in how to optimize the image recombination. For example either by minimization of the cross-sectional area of the recombined PSF at the focal plane for maximum resolution in the x-y plane (the axial or z dependence could be neglected in these circumstances). Alternatively the recombined PSF could be optimized for minimum volume (corresponding to the greatest localization of the source). A further approach could be solely to minimize the PSF in the z (axial direction) to localize detected emission to a sample surface for example. Each of these methods could be applied separately to a fluorescence image without having to reacquire new data. Thus, in embodiments, a high degree of control on the shape of the effective PSF can be achieved, which is not available to other methods of achieving sub-diffraction resolution.

It is important to note that optimisation of the PSF in the x-y plane also leads to improvement in the axial direction (z axis). The intensity of the depletion beam decreases above and below the laser focal region and hence leads to a longer effective lifetime for molecules emitting in these regions. Later time windows will therefore contain a higher signal from the regions above and below the focal plane as well as regions that have a larger radial distance from the focal point. A combination of time window images intended to improve the resolution in one of these dimensions should in principle lead to a similar improvement in the other. Considering only the axial (z) direction, the PSF has a Lorentzian dependence about the beam waist

[14]

P (z) = ^A'

Where the parameter k is the inverse of the square of the Rayleigh range of the focused Gaussian beam [11] and_^ ' is a constant of proportionality (c.f. equation [1]). For simplicity the PSF and the spatial variation in the depletion intensity are assumed to be the same as above (this is also not essential). Figure 6 shows the resultant PSF in the axial direction by combining the 'time window' 1 dimensional images in the

combinations used in Figure 3. The fitted k values (solid lines) for the two functions are 3.26 and 0.125 respectively indicating an effective reduction in the Rayleigh range by a factor of 5.10. The result shows a decrease in the PSF that is actually greater than in the radial direction with a trade-off in a reduction in contrast.

In embodiments, 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. Here the calculated coefficients will no longer represent the optimum reconstructed PSF for these regions. Simulations have shown that if this variation is small a slight loss of image contrast is observed in the reconstructed image. If this variation is significant then the resolution of the reconstructed image will be lower than that expected (but still better than the standard image) for a uniform Fd throughout the sample. This issue can be overcome by subdividing the overall image into sections e.g. quadrants. By comparison of these with the corresponding regions the image produced in the absence of depletion, the 'local' values of Fd can be obtained together with the optimum coefficients for each quadrant. The total image reconstruction now becomes the result of four separate 'local' reconstructions. As will be understood from the foregoing, in various embodiments of the present invention 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) can be used to determine the coefficients by which time slices (segments) of the evolving PSF can be combined to yield a minimised (sub-diffraction limit) PSF. These coefficients are then used to recombine equivalent time slices of the fluorescence image produced by the sample under investigation to yield a composite image with enhanced (sub-diffraction limited) spatial resolution.

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.

In embodiments, 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. According to embodiments, 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.

Many modifications and variations will be evident to those skilled in the art, that fall within the scope of the following claims:

Claims

1. Fluorescence microscopy method, comprising:

inducing stimulated emission to spatially modify the time evolution of fluorescence emission;

acquiring data relating to said fluorescence emission; and processing the data, thereby to form an image.

2. Fluorescence microscopy method as claimed in claim 1, comprising recording a plurality of images corresponding to separate temporal slices within the fluorescence intensity decay.

3. Fluorescence microscopy method as claimed in claim 1 or claim 2, wherein processing the data comprises forming a linear combination of time slices within the fluorescence intensity decay, thereby to form said image.

4. Fluorescence microscopy method as claimed in claim 3, wherein the number, sign, relative magnitude and temporal width of time slices in said linear combination is determined by determining a linear combination of time slices of a point spread function which yields an optimum point spread function.

5. Fluorescence microscopy method as claimed in claim 4, wherein time slices of the point spread function are obtained by measurement of a sub-wavelength test sample.

6. Fluorescence microscopy method as claimed in any of claims 3 to 5, wherein processing the data comprises forming different linear combinations of time slices for respective different regions of the sample, thereby to form said image.

7. Fluorescence microscopy method as claimed in any preceding claim, wherein said data is processed to minimise a point spread function in at least one dimension.

8. Fluorescence microscopy method as claimed in any preceding claim, comprising:

illuminating fluorescent probes with depletion light, thereby to induce said stimulated emission; concurrently illuminating the fluorescent probes with excitation light to induce fluorescence;

acquiring data relating to fluorescence emission from fluorescence probes exposed to said excitation light and said depletion light.

9. Fluorescence microscopy method as claimed in any preceding claim, comprising illuminating fluorescent probes in a sample with depletion light to induce said stimulated emission, wherein said data is acquired for fluorescence emission in a region of the sample exposed to said depletion light.

10. Fluorescence microscopy method as claimed in any preceding claim, comprising providing depletion light to induce said stimulated emission, said depletion light having a spatially varying intensity to cause spatial variation of said fluorescence emission in time.

11. Fluorescence microscopy method as claimed in any preceding claim, wherein said fluorescence emission is induced by pulsed excitation of fluorescent probes in a sample in the presence of said stimulated emission.

12. Fluorescence microscopy method as claimed in any preceding claim, wherein said stimulated emission is induced by a continuous wave (CW) light source.

13. Fluorescence microscopy method as claimed in any preceding claim, wherein said time evolution is modified in at least two dimensions.

14. Fluorescence microscopy method as claimed in claim 13, wherein said time evolution is modified in three dimensions.

15. Fluorescence microscopy apparatus for acquiring data relating to 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.

16. Fluorescence microscopy apparatus as claimed in claim 15, wherein said processing data comprises forming a linear combination of time slices within the fluorescence intensity decay.

17. Fluorescence microscopy apparatus as claimed in claim 16, wherein said linear combination is selected to minimise a point spread function in at least one dimension.

18 Fluorescence microscopy apparatus as claimed in claim 16 or claim 17, wherein the number, sign, relative magnitude and temporal width of time slices in said linear combination is determined by determining a combination of time slices of a point spread function which yields an optimum point spread function.

19. Fluorescence microscopy apparatus as claimed in claim 17 or claim 18, wherein time slices of the point spread function are obtained by measurement of a sub- wavelength test sample.

20. Fluorescence microscopy apparatus as claimed in any of claims 15 to 19, wherein said depletion light source comprises a continuous wave (CW) light source.

21. Fluorescence microscopy apparatus as claimed in any of claims 15 to 20, wherein the depletion light source is configured to provide depletion light having a spatially varying intensity to cause spatial variation of the fluorescence emission in time.

22. Fluorescence microscopy apparatus as claimed in any of claims 15 to 21, comprising an excitation light source to illuminate fluorescent probes in a sampl excitation light.

23. Fluorescence microscopy apparatus as claimed in any claim 22, wherein the depletion light source is configured to illuminate fluorescent probes in a sample with depletion light to induce said stimulated emission, wherein the fluorescence microscopy apparatus is configured to acquire data for fluorescence emission in a region of the sample exposed to said depletion light.

24. Fluorescence microscopy apparatus as claimed in claim 22 or claim 23, wherein the excitation light source comprises a pulsed excitation light source.

25. Fluorescence microscopy apparatus as claimed in any of claims 22 to 24, wherein the excitation light source is configured to produce an excitation beam and the depletion light source is configured to produce a depletion beam, and wherein 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.

26. Fluorescence microscopy apparatus as claimed in any of claims 15 to 25, wherein the depletion light source is configured to provide light having an unstructured spatial profile.

27. Fluorescence microscopy apparatus as claimed in any of claim 26, wherein the depletion light source is configured to operate in the fundamental transverse mode.