Method for single molecule localization microscopy

Abstract

To provide a single molecule localization microscopy (SMLM) for optical super-resolution imaging based on the technique of point accumulation in nanoscale topography (PAINT).SOLUTION: By illuminating a sample with patterned illumination composed of spatial and temporal controlled illumination patterns across a visual field and continuously monitoring the visual field, approximate location of emitters capable of quickly adapting to patterned illumination can be identified. This method is referred to as "dynamic masking". The dynamic masking allows in parallel enhanced illumination of multiple regions over a focal surface associated with the approximate location of a single fluorescent molecule emitter for more accurate and efficient localization of a single emitter for PAINT.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to single molecule localization microscopy (SMLM) for optical super-resolution imaging based on the Point Accumulation for Imaging in Nanoscale Topography (PAINT) technique in nanoscale topography.

Background Art

[0002] In PAINT, a solution containing fluorescent dye molecule-labeled probes is applied to a biological sample. Subsequently, SMLM using PAINT is realized by recording a series of microscopic images of the sample. When the concentration of free probes is sufficiently low, the transient binding events of the probes can be detected as individual diffraction-limited spots on the background, which arise from the rapidly diffusing free probes. PAINT is basically limited by the free probe background and an additional background generated by probes that transiently bind to epitopes far from the focus.

Summary of the Invention

[0003] An object of the present invention is to achieve single molecule localization using PAINT at a relatively deep depth within a sample placed by a conventional method with a higher signal-to-background ratio and / or acquisition speed as compared to wide-field microscopy or confocal microscopy of the current state of the art.

[0004] While SMLM at a certain depth in conventionally placed samples is possible using PAINT with wide-field detection, it requires very sparse detection to limit contamination from background light, thereby limiting the acquisition speed. Confocal optical sectioning using a rotating disk or programmable array microscope (PAM) can significantly reduce out-of-focus background light, enabling faster acquisition. However, further improvements in background reduction or signal enhancement by these techniques would allow for a higher signal-to-background ratio for localization, enabling faster acquisition at a certain depth in conventionally placed samples for PAINT.

[0005] This improvement can be achieved by illuminating the sample with patterned illumination consisting of spatially and temporally controlled illumination patterns across the field of view. Continuous monitoring of the field of view allows for the rapid identification of approximate emitter locations to which patterned illumination can be adapted; this method is called "dynamic masking." Dynamic masking enables in-parallel enhanced illumination of multiple regions across the focal plane associated with the approximate location of a single fluorescent molecule emitter, for more accurate and efficient localization of a single emitter for PAINT.

[0006] Because patterned illumination requires high spatial resolution across the field of view, current technological solutions consist of spatial light modulators (SLMs) for generating patterned illumination.

[0007] In addition, if the SLM is located in both the excitation and emission paths of the conjugate image plane, the pixels corresponding to the region can function as confocal pinholes (similar to rotating disk confocals), thus enabling confocal sectioning and further increasing the signal-to-background ratio.

[0008] Furthermore, if synchrotron radiation from outside these regions is collected to generate another non-conjugated image, the subtraction of a well-scaled non-conjugated image from the conjugated image can be used to remove the out-of-focus background to match the noise limit (similar to PAM), further increasing the signal-to-background ratio.

[0009] For example, by dynamically sampling the emitter's Airy disk to generate additional localization information, the area of ​​enhanced illumination can be further optimized for other localization modalities beyond centroid estimation, thereby improving overall accuracy. [Brief explanation of the drawing]

[0010] [Figure 1] This figure shows a typical configuration of an SMLM based on different amounts of axial sectioning. [Figure 2] This figure shows the signal-to-background increase made possible by PAM, which enables noise-limited background reduction through axial sectioning, dynamic masking, and subtraction of the unconjugated image from the conjugated image. [Figure 3] This is a schematic diagram of the dynamic masking process. [Figure 4] This figure shows the dynamic masking process by illustrating the image acquired afterward, the determination of the emitter position, and the illumination pattern that changes due to the appearance and disappearance of the emitter. [Figure 5] This figure shows typical single-molecule localization images from wide-field microscopy and TIRF microscopy, and the configuration of enhanced illumination regions from a given image. [Figure 6] This figure shows a device for dynamic masking based on SLM(031) in the excitation pathway of a wide-field microscope. [Figure 7] This figure shows a device for dynamic masking that creates a confocal microscope by positioning SLM(031) in both the excitation and detection pathways of a fluorescence microscope. [Figure 8]This figure shows a device for dynamic masking in which SLM(031) is positioned in both the excitation and detection pathways of the fluorescence microscope to create a confocal microscope, and in addition, the unfocused background can be removed more effectively from the reconstructed optical sectioning image, as it is not simply discarded but recorded by a second camera. [Figure 9] This figure shows a device for dynamic masking in which an enhanced illumination pattern created by an SLM (031) and monitoring excitation light (032) are generated separately and coupled in the excitation path using a beam splitter / combiner (033). [Figure 10] This diagram shows a device for dynamic masking in which a holographic SLM is used. [Figure 11] This diagram illustrates the concept of a locally tilted microsheet, where (A) shows the tilted light cone emanating from the objective lens, and (B) shows the illumination off-center from the back focal plane of the objective lens. [Modes for carrying out the invention]

[0011] This specification describes a method for improving signal-to-background ratio for PAINT based on monitoring the dynamic appearance of a fluorescent probe across a field of view, combined with rapid enhanced excitation of corresponding regions including a transient probe, using dynamic patterned illumination. The present invention improves the signal-to-background ratio and therefore the acquisition speed by at least several times compared to conventional wide-field or confocal techniques for PAINT.

[0012] The following terms are used in the description of the method. SMLM (Single-Molecular Localization Microscopy) is a form of super-resolution microscopy in which individual fluorescent molecules are imaged individually due to their highly sparse dispersion, enabling localization with very high precision and accuracy. Subsequent imaging of many such molecules across multiple frames allows for the construction of images with higher resolution than that possible with conventional optical microscopy. Different forms of SMLM rely on alternative methods to achieve sufficient sparsity / isolation within each frame while allowing sampling of the entire field of view throughout the entire acquisition sequence. PAINT: Point accumulation in nanoscale topography; a form of SMLM in which the separation and concentration of fluorescent molecular emitters are achieved by transiently chemically bonding the emitters to the sample. PAM: Programmable Array Microscopy; a form of optical sectioning microscopy in which optical sectioning is achieved by projecting patterned light onto a sample using a programmable array, or spatial light modulator. TIRF: Total Internal Reflection Microscopy; a form of microscopy in which fluorescence is excited only in the non-transmissive evanescent wave region of the boundary layer of the microscope slide, thereby limiting all fluorescence to this small region. SLM: Spatial Light Modulator; a device that enables spatial modulation of the transmission or reflection of incident light, such as a liquid crystal array in an LCD screen. DMD: Digital Micromirror Device; a spatial light modulator device in which pixels are composed of small mirrors that can reflect in two distinct directions. LCoS: Liquid Crystal on Silicon; a spatial light modulator device in which pixels can rotate the polarization state of incident light in two directions. PSF: Point Spreading Function; the mechanical response function of an imaging system to a point light source.

[0013] In the drawings, the following reference numbers are used to indicate the characteristics described below. The same reference numbers are used in various drawings to refer to parts that perform similar or identical functions.

[0014] 001 Biological sample 002 Fluorescent molecular emitter 003 Free-diffusion fluorescent molecular emitter 004 Focused fluorescent molecular emitter 005 Objective lens 006 Sample chamber 007 Sample chamber of normal volume 008 Sample chamber of small volume 009 Sample chamber volume captured by TIRF 010 Sample chamber volume captured by optical sectioning microscopy 011 Optical cone 012 Confocal optical section 013 Dynamic masking process 014 Determination of emitter position 015 Region generation 016 Illumination control 017 Image acquisition 018 Patterned illumination 019 Newly emerged emitter observed by monitoring illumination 020 Emitter imaged by enhanced illumination 021 Disappeared emitter 022 Approximate position of emitter<"0000105">023 Region around the approximate position of emitter 024 Patterned illumination irradiated on the field of view 025 Cover glass 026 Main dichroic 027 Tube lens 028 Lens 029 Light source 030 Camera 031 Spatial light modulator (e.g., digital micromirror device) 032 Illumination unit 033 Beam combiner 034 Conjugate optical path 035 Non-conjugate optical path 036 Intermediate image plane 037 Light source for photoactivation 038 Holographic Wavefront Shaping SLM 039 Locally Inclined Microsheet 040 Back focal plane / entrance pupil 041 Lighting section

[0015] Figure 1 shows a typical SMLM configuration for PAINT, illustrating the microscope objective lens (005), the sample under inspection (001) with a fluorescent emitter (002) temporarily coupled to it, and the sample chamber (006) having a buffer volume (007) containing a free fluorescent molecule emitter (003). Background reduction methods include reducing the buffer height (008) and reducing axial illumination using total internal reflection fluorescence (TIRF) microscopy or light sheet microscopy. With TIRF microscopy, detection is limited to within approximately 100 nm of the coverslip glass (009). With light sheet microscopy, detection deep within the sample and sectioning on the order of approximately half the wavelength of the excitation light (010) are possible, but it requires non-standard and uncommon sample placement, positioning the sample at the focus of a more complex optical configuration consisting of two orthogonal, closely spaced objective lenses.

[0016] Uniform illumination of a sample allows for the recognition of transient fluorescent molecular emitters that appear across the entire field of view. However, even if there is no information obtained outside the region containing the fluorescent molecular emitters, uniform illumination can also cause background excitation and fading. The novel method described herein involves irradiating with enhanced illumination to illuminate only the region containing the transient focused fluorescent molecular emitter, thereby preventing unwanted excitation of the unfocused fluorescent molecular emitter, which consists of a free diffusion probe and a probe bound to an unfocused epitope.

[0017] Improving the signal-to-background ratio for SMLM can be achieved using a microscope capable of illuminating the sample with spatially and temporally controllable patterned illumination across the field of view. Figure 2 shows the illumination of a sample for the method disclosed herein. Spatially controlled patterned illumination is focused on a set of focused emitters (004) within the optical section (012) of a biological sample (001). The resulting patterned illumination consists of a set of light cones (011) centered on the corresponding emitters. This patterned illumination enables parallel enhanced illumination of multiple regions across the focal plane related to the position of a single fluorescent molecule emitter in an adaptive and highly dynamic manner. The patterned illumination can be used based on a method called “dynamic masking” introduced herein, which masks regions that do not contain the focused fluorescent molecule emitter, thereby reducing the overall illumination of the sample and reducing the background. In this way, the sample is illuminated in a highly efficient manner so that regions containing information useful for SMLM are preferentially illuminated. As a result, a series of images including individual fluorescent emitters are detected with a higher signal-to-background ratio for optimal single-molecule localization, while overall fading is minimized.

[0018] Figure 3 shows a schematic of the dynamic masking method (013). Dynamic masking uses continuous (or strobe) video-rate monitoring (017) of the illuminated field of view to locate the location of the fluorescent molecule emitter (014), followed by on-the-fly image processing to rapidly generate a region around the approximate location of the emitter (015). The region is then applied to spatial and temporal control of the illumination (016) to construct patterned illumination (018) consisting of regions of enhanced illumination that locally enhance the excitation of transient fluorescent molecule emitters, thereby increasing the signal-to-background detection for single-molecule localization within the biological sample (001). The patterned illumination is rapidly and continuously updated to account for both the appearance and disappearance of single molecules across the entire field of view.

[0019] Figure 4 illustrates the dynamic masking process in detail. At the start of the experiment, the field of view is monitored using a predetermined level of uniform illumination. The subsequent images are analyzed, and the approximate location (022) of the estimated fluorescent molecule emitter (019) is determined using current technology image processing algorithms. Next, a region (023) of a size determined by the resolution limit of the microscope is created within patterned illumination (024) at the approximate location of the fluorescent molecule emitter, enabling enhanced excitation while minimizing the total illumination of the sample.

[0020] Since the position of the molecular emitter is determined for each subsequently acquired image, the appearance of emitter (019) in the detected image sequence leads to the adaptation of patterned illumination across the field of view (024), adding a new region of enhanced illumination (023) corresponding to the emitter's position (022) and enhancing the fluorescence emission from that emitter (020). Conversely, the disappearance of the fluorescent molecular emitter (021) leads to the removal of the corresponding region of enhanced illumination. Determining the approximate position of the emitters allows for the optimization of the response time of the dynamic masking process using multiple images of the image sequence, and can account for various effects, including molecular flashing and signal-to-noise detection. Thus, the patterned illumination determined by the dynamic masking process reduces the aforementioned background resulting from defocused localization and free-diffusing fluorescent molecular emitters. In addition, minimizing sample illumination reduces the overall fading rate of the entire pool of fluorescent molecular emitters.

[0021] Patterned illumination is dynamically updated to account for the transient appearance and disappearance of fluorescent molecular emitters, either by continuous monitoring outside the approximate location of the fluorescent molecular emitter, or by short monitoring snapshots that alternate with enhanced illumination over the region containing the fluorescent molecular emitter.

[0022] Field-of-view monitoring and patterned illumination updates can be performed dynamically at a speed that matches the timescale of the visibility of the fluorescent probe, which may be limited by factors such as transient coupling, flickering, and / or photofading.

[0023] Figure 5 shows a typical example of an SMLM experiment using PAINT. Panel A shows a wide-field image of an individual CD44fab-Vio667 (Miltenyi Biotec) probe bound to formaldehyde-fixed blood cells (a single representative frame from sequential acquisition). Panel B shows a representative frame of the same sample using TIRF microscopy. Panel C shows a field-wide region containing the fluorescent molecule emitter localized by SMLM, created by applying dilation to Panel B after simple thresholding. In this typical example, the area covered by these regions accounts for only 1.8% of the field of view. By limiting enhanced illumination to these regions in subsequent images, higher signal-to-background imaging can be achieved for single-molecule localization.

[0024] [Embodiment 1] In one embodiment of the present invention, dynamic masking is performed by adding an SLM to the excitation pathway of a wide-field fluorescence microscope.

[0025] Figure 6 shows an example of this embodiment in which a digital micromirror device (DMD) is used as an SLM in the excitation path of a microscope, with one oriented micromirror reflecting the excitation light on the sample and the other oriented micromirror discarding the excitation light into a beam dump. Different SLMs, such as LCoS, may be used instead of the DMD, in which case the functionality remains the same, but the overall arrangement of the optical path is different. For simplicity, an example using a DMD is shown in the figure.

[0026] The excitation light source (029) illuminates a DMD (031) located in the conjugate image plane defined by the microscope's objective lens (005) and tube lens (027). Patterned excitation light from the DMD is coupled in a standard epifluorescence configuration within the objective lens using a main dichroic (026) and projected through the microscope's objective lens (005) onto a biological sample (001) placed on a coverslip glass (025). The fluorescence is imaged by a camera (030) using a standard wide-field emission path.

[0027] In this embodiment, a DMD is used to establish both monitoring illumination and enhancement illumination areas. The percentage of time a given DMD pixel reflects light back to the sample is called the pixel's duty cycle. Sample monitoring is achieved by running a low duty cycle pattern across the corresponding pixels of the DMD, while enhancement illumination is achieved by running a high duty cycle pattern.

[0028] The average area of ​​enhanced illumination is expected to be about 1% of the field of view. Including the excitations required to monitor the field of view for the emergence of new molecules, wide-field excitation B w and dynamic masking B DM The following relationship can be obtained regarding the amount of background light expected from this.

[0029]

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[0030] Here, f is the percentage of the field of view illuminated by the enhanced illumination, and m is the relative excitation dose of the monitoring illumination intensity compared to the intensity of the enhanced region. For typical values ​​of f=0.01 and m=0.04, a reduction of 1 / 20th of the background excitation is achieved.

[0031] [Embodiment 2] In one embodiment of the present invention, patterned illumination is directly produced by a light source consisting of an array of individual light sources whose intensity can be independently modulated. Such a light source eliminates the need for an SLM. In the embodiment described above, shown in Figure 6, such an array of light sources is positioned at the DMD(031) position in the image plane of the microscope.

[0032] [Embodiment 3] In an improved version of the first embodiment, the SLM is located not only in the conjugate image plane of the excitation pathway but also in the conjugate image plane of the emission pathway. This optical arrangement creates a confocal imaging device that, similar to a rotating disk confocal microscope, allows a significant portion of the out-of-focus background fluorescence from the sample to be rejected by the SLM's pixels acting as confocal pinholes. With an SLM located in the conjugate image plane and using dynamic masking, the signal-to-background ratio of the detection is higher than that obtained with a rotating disk.

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[0033] Figure 7 shows an example of this embodiment using a DMD as an SLM. Here, the DMD (031) is located in both the excitation and emission paths, and a dichroic mirror (026) that separates the excitation light and the synchrotron radiation is positioned between the DMD and the camera (030). Synchrotron radiation from the microscope's objective lens (005) is first imaged onto the DMD using a tube lens (027), and then imaged onto the camera using a set of relay lenses (028).

[0034] [Embodiment 4] In a further improvement of the above embodiment, non-conjugate, non-focused light is captured by the detector rather than simply discarded.

[0035] Figure 8 shows an example of this embodiment using a DMD as an SLM. Here, the non-conjugate, non-focused light reflected by the DMD (031) is imaged by a second camera in an additional non-conjugate path (035) to the existing conjugate path (034), similar to the optical arrangement of a PAM.

[0036] Parallel confocal detection across the field of view can lead to considerable crosstalk between confocal pixels, increasing the out-of-focus background. This is a common limitation of rotating disk confocal microscopy and is particularly noticeable when imaging thick or densely labeled samples. Detection of rejected non-conjugate light by a camera provides a spatial image of the out-of-focus background fluorescence, which can serve as an indicator of the residual background of the conjugate image. Weighted subtraction of the non-conjugate image from the conjugate image makes it possible to remove the residual background of the optical sectioning image to noise limits.

[0037] When applying a dynamic masking method to PAM, it is preferable for the pixels of the DMD forming the enhanced illumination region to use a 50% duty cycle to allow for proper subtraction of the out-of-focus background. However, for maximum intensity, a 100% duty cycle can be used for the enhanced illumination region. Even in this case, accurate optical sectioning is guaranteed in the surrounding region. However, in the case of enhanced illumination regions with a 100% duty cycle, light is not projected onto the non-conjugate detector for these regions, so direct local measurements of the out-of-focus background cannot be obtained from the non-conjugate image. Nevertheless, with some additional image processing, a local non-conjugate estimate can be derived based on adjacent pixels in the non-conjugate image, allowing for the subtraction of an approximate contribution of the out-of-focus background from the enhanced illumination region.

[0038] Compared to SMLM acquisition using a conventional PAM configuration, the use of dynamic masking in PAM is,

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[0039] [Embodiment 5] In another embodiment, monitoring illumination is generated at a different location from the SLM, and the SLM is used solely to generate the area of ​​enhanced illumination.

[0040] Figure 9 shows an example of this embodiment using a DMD as an SLM. This example is similar to the wide-field microscope configuration shown in Figure 6, but with the addition of two further elements (032) and (033). Monitoring illumination (032) is generated at a different location from the DMD (031) and its corresponding light source (029). Both illuminations are then coupled in the excitation path of the microscope using a beam combiner (033). This beam combiner can be selected to provide a specific ratio of monitoring illumination to enhancement illumination from the DMD. Alternatively, the coupled beams may have different polarizations or be spectrally shifted, such as by using a polarizing beam combiner or a dichroic beam combiner.

[0041] [Embodiment 6] In the improved version of the above embodiment shown in Figure 9, monitoring (032) is performed using an optical sectioning method such as a rotating disk confocal, slit scanning confocal, or structured illumination, while enhanced illumination is generated separately using an SLM and coupled into the excitation path using a suitable beam combiner.

[0042] [Embodiment 7] In another embodiment of the present invention, an optical element is added to the synchrotron radiation path between the SLM and the detector to obtain additional axial information for individual fluorescent molecule emitters. Such axial information can be generated, for example, by adding a cylindrical lens or diffracting element to predictably distort the PSF of the particles, thereby providing information about their position above or below the focal plane. Dynamic masking using enhanced illumination, in principle, fits the distortions produced by these methods. This is because the region surrounding the particles is large enough that it only increases the signal from the individual emitters without significantly altering the distortion itself that underlies the axial determination. Another option would be to use an image splitter element to form multiple images on different focal planes of the detector.

[0043] These optical elements may be combined with the background subtraction capability of Embodiment 4. It is preferable that the axial information optical elements are included in both the conjugate and non-conjugate optical paths to cause equivalent distortion to the out-of-focus light in the non-conjugate image as in the conjugate image. The placement of the axial information optical elements in front of the non-conjugate detector does not need to be very precise, because the non-conjugate image records only out-of-focus light, and the precise distortion of the out-of-focus light by a slightly misaligned axial information optical element is not important.

[0044] [Embodiment 8] In another embodiment of the present invention, the regions of enhanced illumination within the patterned illumination are optimized for other SMLM localization modes beyond centroid estimation by dynamic subsampling of individual PSFs of the fluorescent molecular emitter, by adjusting the size, shape, and position of each corresponding region of enhanced illumination.

[0045] For example, the PSF of a molecule can be dynamically sampled by slightly displacing the center of the illumination region relative to the center of the fluorescent molecule. This generates additional localization information in the form of triangulation based on the relative intensity of different illumination positions. The displacement must be selected such that the increase in triangulation information is greater than the decrease in signal, thereby ensuring improved localization accuracy.

[0046] Similarly, three-dimensional (3D) triangulation may be performed by adding slight depth displacements due to focal displacement within the sample, or by using an SLM that forms a digital hologram in which the depth of individual focal spots can vary. Such 3D triangulation would further enable the extraction of the axial position of the emitter.

[0047] [Embodiment 9] In most of the embodiments described above, the SLM may be a holographic phase-based SLM. This technique allows for more efficient use of excitation light, thereby providing higher intensity to the enhanced illumination area.

[0048] Figure 10 shows an example of this embodiment in which the excitation light (029) is modulated by a holographic SLM (038) located substantially on a plane conjugate to the back focal plane of the objective lens.

[0049] The phase mask introduced by the SLM generates an illumination pattern on the image plane or a plane conjugate to the image plane (036), which can be calculated using the Fourier transform or other algorithms. It may also be reasonable to remove the SLM from the conjugate plane to ensure that the zeroth-order light is not focused on the focal plane. Alternatively, diffuse light may be used to illuminate the SLM, removing the zeroth-order light from the focal plane. It is also conceivable to use the zeroth-order light as monitoring light to illuminate the entire field of view, and the first-order light as patterned illumination to localize molecules. The advantage of placing the SLM in a position conjugate to the back focal plane of the objective lens is that the power of the light can be concentrated on the location of the molecules to be analyzed. Therefore, the total intensity required to illuminate the SLM can be orders of magnitude lower than when the entire field of view of the target is illuminated and only individual locations are switched on.

[0050] [Embodiment 10] In another embodiment of the present invention, molecules are illuminated by a tilted light cone.

[0051] Figure 11 shows the tilted light cone (Panel A) on the sample and the required illumination area at the back focal plane of the objective lens (Panel B). This can be achieved in various ways. For example, the area illuminated on the SLM is imaged off-center (041) of the back focal plane (040), rather than at the center. The resulting patterned illumination then consists of a light beam tilted relative to the object plane. The tilt angle can be from 30 to approximately 90 degrees with respect to the object plane normal. Thus, the target fluorescent molecule emitter is illuminated at the focal plane by the tilted light cone (039). This already provides local sectioning and background intensity reduction. Fluorescent molecule emitters not on the object plane can be recognized because they are only excited when displaced laterally from the focal point along the tilt direction.

[0052] A method for forming a series of images of a biological sample using a light microscope with a resolution-limited objective lens (005) for the purpose of single-molecule localization microscopy is described. This method may include exciting emitters (002) in parallel within the field of view of the objective lens with excitation light from at least one first light source (029), and then detecting the light emitted in parallel from the emitters as a result of the excitation light with at least one first detector (030) to acquire an image. Then, based on the acquired image, the approximate positions (022) of the emitters can be determined. Regions corresponding to the approximate positions of the emitters can be generated, each region (023) being localized in each emitter within a subset and having characteristic dimensions comparable to the resolution limit of the microscope. A first patterned illumination (024) from at least one light source may be adapted based on the regions to illuminate multiple emitters in parallel in subsequent images. A series of repetitions of the above steps constitute a "dynamic masking" process (013), in which the patterned illumination may be updated to take into account the appearance and disappearance of individual emitters within the field of view of the acquired image.

[0053] This method may further include creating patterned illumination consisting of enhanced illumination within a region corresponding to an approximate location of the emitter, the enhanced illumination region may receive more excitation light than the rest of the field of view, and the region may be arranged to illuminate multiple emitters in parallel.

[0054] Regarding the method described above, an array of individual light sources may be used as the first light source, and patterned lighting can be created by varying the intensity of the individual light sources.

[0055] This method may also include modulating light from a first light source using a spatial light modulator (SLM, 031) to establish patterned illumination.

[0056] This method may include imaging the synchrotron radiation from the emitter onto the SLM, and then detecting the subsequently modulated synchrotron radiation using a first detector. Thus, modulating the detected light from the emitter with the same subset of SLM pixels that modulate the light from the first light source onto the sample would enable confocal detection of the emitter by rejecting out-of-focus light from the remaining SLM pixels in the detected image.

[0057] The above method using confocal detection may further include detecting the out-of-focus light rejected by the SLM by imaging that light onto at least one of the unused areas on the surfaces of the first and second detectors. The method may further include removing the remaining out-of-focus light from the confocal image by scaled subtraction of the detected out-of-focus light.

[0058] The above method may also include sampling the point spreading function (PSF) of each emitter in parallel by time modulation and spatial modulation of patterned illumination across the emitters.

[0059] The method may also include creating an inclined optical cone (039) with respect to the optical axis over each emitter by illuminating the back focal plane (040) of the microscope's objective lens off-center (041).

[0060] While various details have been described in conjunction with the exemplary embodiments outlined above, various alternatives, modifications, variations, improvements, and / or substantial equivalents, whether publicly known or not currently foreseeable, may become apparent upon consideration of the above disclosure. Therefore, the exemplary embodiments described herein are for illustrative purposes only and are not limiting.

Claims

1. A method for forming images of a series of biological samples using a light microscope having a resolution-limited objective lens (005) for the purpose of single-molecule localization microscopy, Exciting the emitter (002) in parallel within the field of view of the objective lens by excitation light from at least one first light source (029), The light emitted in parallel from the emitter as a result of the excitation light is detected by at least one first detector (030) to acquire an image. Based on the acquired image, the approximate position (022) of the emitter is determined, To generate a region corresponding to the approximate position of the emitter, wherein each region (023) is localized to each emitter in the subset, and each region has characteristic dimensions comparable to the resolution limit of the microscope. Based on the aforementioned region, a first patterned illumination (024) from at least one light source is applied to illuminate multiple emitters in parallel in the following image, and The above steps constituting the "dynamic masking" process (013) are repeated in succession, and the patterned illumination is updated based on the appearance and disappearance of the individual emitters in the field of view of the acquired image. A method that includes this.

2. The method according to claim 1, further comprising creating a patterned illumination consisting of enhanced illumination within the region corresponding to the approximate position of the emitter, wherein the region of enhanced illumination receives more excitation light than the rest of the field of view, and the region is arranged to illuminate a plurality of emitters in parallel.

3. The first light source is an array of individual light sources, By changing the intensity of each of the aforementioned light sources, patterned lighting can be created. The method according to claim 1 or 2, further comprising:

4. The method according to claim 1 or 2, further comprising modulating the light from the first light source using a spatial light modulator (SLM, 031) to establish the patterned illumination.

5. The synchrotron radiation from the emitter onto the SLM is imaged, and then the subsequently modulated synchrotron radiation is detected using the first detector, thereby, The same subset of pixels of the SLM modulates the light from the first light source onto the sample, modulating the detected light from the emitter, and enabling confocal detection of the emitter by rejecting out-of-focus light from the detection image by the remaining pixels of the SLM. The method according to claim 4, further comprising:

6. The unfocused light rejected by the SLM is detected by imaging the light onto at least one of the unused areas on the surfaces of the first detector and the second detector. The scaled subtraction of the detected out-of-focus light removes the remaining out-of-focus light from the confocal image. The method according to claim 5, further comprising:

7. The method according to claim 1 or 2, further comprising sampling the point spreading function of each emitter in parallel by time modulation and spatial modulation of the patterned illumination across the emitters.

8. The method according to claim 3, further comprising creating an inclined optical cone (039) with respect to the optical axis over each of the emitters by illuminating the back focal plane (040) of the objective lens of the microscope off-center (041).

9. The method of claim 4, further comprising creating an inclined optical cone (039) with respect to the optical axis over each of the emitters by illuminating the back focal plane (040) of the objective lens of the microscope off-center (041).

Images