Apparatus for localizing single fluorescent molecules in a sample using single molecule localization microscopy - Patent Application 20070229633

Abstract

The present invention relates to an apparatus for localizing single fluorescent molecules contained in a sample using single molecule localization microscopy. The apparatus includes a scanning unit configured to scan the sample using a light beam at a first wavelength having a predetermined irradiance, an image acquisition unit configured to acquire a first image and a second image at a second wavelength, and a localization unit configured to localize the single fluorescent molecules in the second image. In addition, the apparatus includes a determination unit configured to determine one or more local densities of the single fluorescent molecules based on the first image, and a calculation unit configured to calculate one or more local irradiances of the light beam as a respective predetermined function of the one or more local densities of the single fluorescent molecules. The scanning unit is further configured to scan the sample using the one or more local irradiances of the light beam.

Description

[Technical field]

[0001] The present invention relates to methods and apparatus for localizing single fluorescent molecules using single molecule localization microscopy. [Background technology]

[0002] Single molecule localization microscopy (SMLM) is based on the delay of stochastic emission of molecules to achieve the emission of single fluorescent molecules. A sample containing fluorescent molecules is illuminated using an irradiance adjusted so that, on average, only one active molecule (emitting molecule) is present at any one time in the focal volume of the observation microscope. In the images acquired by SMLM, there are image spots called point spread functions (PSFs), each spot representing the image of a molecule. From this PSF, it is possible to measure the position of the molecule by determining the center of the spot using an algorithm. This position can be determined with an accuracy far beyond the diffraction-limited resolution of the microscope. This stochastic process is repeated for tens of thousands of images to obtain a final super-resolution image with high spatial resolution (up to 10 nm). For example, Lelek et al. 1 described the basic principles of SMLM, including different types of molecular labels, determination of the position of the PSF and the process for reconstructing a super-resolution image from the localization of the PSF.

[0003] In addition, Mailfert et al. 2 disclose an algorithm used to determine the PSF. Specifically, Mailfert et al. are concerned with an algorithm optimized to obtain higher spatial resolution for samples with a high density of fluorescent molecules. Specifically, the algorithm evaluates the probability of detection for each fluorescent molecule and the uncertainty of its localization so that inaccurate localizations can be filtered out when reconstructing a super-resolution image.

[0004] However, high spatial resolution depends on molecular detection, which in turn depends on the density of single fluorescent molecules. Specifically, the higher the density, the more difficult it is to detect and localize the molecules, and the lower the image quality (due to artifacts such as sample drift and overlapping PSFs). In contrast, a lower density of single fluorescent molecules allows for higher spatial resolution.

[0005] Furthermore, most SMLM techniques use Gaussian shaped illumination to illuminate the sample. However, Gaussian shaped illumination provides non-uniform excitation and limits the available field of view. Mau et al. 3 disclose a method to achieve uniform illumination. The system, called ASTER (Adaptive Scanning of Adjustable Excitation Region), includes a laser source with a Gaussian beam. The Gaussian beam is focused on two galvanometer scanning mirrors to control the illumination at the sample plane. The excitation beam maintains its position at the back focal plane, but angular rotation of the galvanometers results in a similar angle at the back focal plane and correspondingly a different position at the sample plane. By applying a specific pattern, such as a raster scan, the ASTER system can provide uniform excitation in an adjustable field of view. The Mau et al. system allows for more accurate determination of the PSF center, a wider field of view, and better efficiency compared to systems using conventional Gaussian shaped illumination that provide non-uniform excitation.

[0006] However, most biological samples contain non-uniform densities of molecules. Thus, the use of uniform illumination, such as the system used in Mau et al., may result in high spatial resolution in certain regions of a sample while providing low spatial resolution in other regions of the same sample. For example, sample regions with a high density of fluorescent molecules may have more artifacts than regions with a low density of fluorescent molecules.

[0007] As disclosed in Mailfert et al., density-adaptive localization algorithms have been developed to determine the PSF center for samples with high molecular density. However, the spatial resolution obtained from density-adaptive localization algorithms is still lower for high density compared to low density. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Lelek et.al,Single-molecule localization microscopy,Nature Reviews Method Primers,vol.1,39,2021 [Non-Patent Document 2] Mailfert et.al,A Theoretical High-Density Nanoscopy Study Leads to the Design of UNLOC,vol.113,3,2018 [Non-Patent Document 3] Mau et.al,Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields,Nature Communications,vol.12,3077,2021 Summary of the Invention [Means for solving the problem]

[0009] These problems are solved or alleviated by the claimed apparatus and methods.

[0010] Instead of using uniform illumination and using optimization algorithms for localization of single fluorescent molecules, according to the invention the irradiance of the illuminating light beam is adapted to the density of the fluorescent molecules of the sample. Thus, for a sample containing low and high molecular density regions, the irradiance used to localize the molecules of the low density regions will be lower than the irradiance used to localize the molecules of the high density regions. According to an advantageous embodiment, the irradiance is adapted to the molecular density by modifying the diameter of the illuminating light beam, which allows optimization of the scanning efficiency.

[0011] A first aspect of the present invention provides an apparatus for localizing single fluorescent molecules contained in a sample using single molecule localization microscopy, comprising: a scanning unit configured to scan the sample using a light beam of a first wavelength having a predefined irradiance; an image acquisition unit configured to acquire a first image and a second image at a second wavelength; and a localization unit configured to localize the single fluorescent molecules in the second image, wherein the apparatus comprises: a determination unit configured to determine one or more local densities of the single fluorescent molecules based on the first image; and a calculation unit configured to calculate one or more local irradiances of the light beam as a predefined function of each of the one or more local densities of the single fluorescent molecules, characterized in that the scanning unit is further configured to scan the sample using the one or more local irradiances of the light beam.

[0012] Optionally, the local irradiance of one or more of the light beams is adapted to emit, on average, only one out of a single fluorescent molecule within a given focal volume in a given time frame.

[0013] Optionally, the apparatus is configured to perform a series of sequential processes for localizing single fluorescent molecules, and for each sequential process, the second image becomes the first image of the sequential process.

[0014] Optionally, the calculation unit is configured to calculate the one or more local irradiances based on a look-up table in which local irradiance values ​​are associated with local density values ​​of fluorescent molecules.

[0015] Optionally, the scanning unit includes a zoom for varying the diameter of the light beam and thereby its local irradiance.

[0016] Optionally, the determination unit is configured to determine a trajectory of the light beam, and the scanning unit is configured to scan the sample based on the trajectory, the trajectory including regions of the sample that contain single fluorescent molecules and avoiding regions of the sample that do not contain single fluorescent molecules.

[0017] A second aspect of the present invention provides a method for localizing single fluorescent molecules contained in a sample using single molecule localization microscopy, the method comprising: a) scanning the sample with a light beam at a first wavelength having a predetermined irradiance, the first wavelength being an excitation wavelength of the fluorescent molecule; b) acquiring a first image at a second wavelength, the second wavelength being an emission wavelength of the fluorescent molecule, and determining one or more local densities of single fluorescent molecules based on the first image; d) calculating one or more local irradiances of the light beam as a respective predetermined function of one or more local densities of single fluorescent molecules; d) scanning the sample using the one or more local irradiances of the light beam; f) acquiring a second image at the second wavelength; and g) localizing the single fluorescent molecules in the second image.

[0018] Optionally, the local irradiance of one or more of the light beams is adapted to emit, on average, only one out of a single fluorescent molecule within a given focal volume in a given time frame.

[0019] Optionally, steps c) to g) are performed multiple times sequentially, the second image being the first image of step c), and the sequential second images being obtained for each sequential time the steps are performed.

[0020] Optionally, the one or more local irradiances are calculated based on a look-up table in which local irradiance values ​​are associated with local density values ​​of fluorescent molecules.

[0021] Optionally, scanning the sample with one or more local irradiances of the light beam comprises varying the local irradiance by varying a diameter of the light beam.

[0022] Optionally, the method includes determining a trajectory of the light beam and scanning the sample based on the trajectory, the trajectory including regions of the sample that include single fluorescent molecules and avoiding regions of the sample that do not include single fluorescent molecules.

[0023] Optionally, the method further includes reconstructing a final image of the single fluorescent molecule based on the localization of the single fluorescent molecule in the second image and each of the sequential second images.

[0024] Embodiments of the present invention will now be described with reference to the accompanying drawings. [Brief description of the drawings]

[0025] [Figure 1] 1 is a state diagram of a fluorescent molecule, according to an example. [Diagram 2] 1 is a schematic diagram of an apparatus for localizing single fluorescent molecules, according to an example. [Figure 3a] 1 is an image of a fluorescent molecule, according to an example. [Figure 3b] 1 is an image of irradiance mapping, according to an example. [Figure 4] FIG. 1 is a flow diagram of a method for localizing a single fluorescent molecule, according to an example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] As shown in Figure 1, a fluorescent molecule first exists in the ground state S 0 (usually singlet). These fluorescent molecules absorb a photon at the excitation wavelength and enter the excited state S 1 (singlet multiplicity). The excited molecule can then return to its ground state by emitting a fluorescent photon or, with much lower probability, by undergoing a non-radiative transition to a "dark" triplet state T, which remains for a relatively long time (on the order of milliseconds to seconds). The return from the dark state to the ground state can be accelerated by light.

[0027] When a sample labeled with fluorescent molecules is illuminated with an excitation wavelength, the sample initially emits strong fluorescent radiation. After a while, however, all of the molecules are effectively "trapped" in a dark state and thus "turned off." At that point, the fluorescence emission occurs only intermittently ("blinks"), and some molecules return to the ground state and emit light again ("turn on") before turning off again.

[0028] In SMLM, image acquisition is performed in this blinking regime after a primary phase with an initial intense emission of light.

[0029] According to the present invention and the ASTER technique, the illumination beam is scanned so that a given area of ​​the sample is illuminated for only a short time (50 μs to 500 μs). To achieve the blinking regime, a sufficient number of photons must reach the area of ​​the sample within the illumination time, and therefore the irradiance must be high enough. This number of photons (or equivalently, the irradiance value) depends on the number of fluorescent molecules in the area of ​​the sample. If the irradiance is too low, there will not be enough photons to turn off all the molecules and the single molecule state will not be achieved. However, this same irradiance may be sufficient in areas of the sample that contain fewer fluorescent molecules. In the method of the present invention, the irradiance is adapted to the local molecular density to achieve optimal resolution over the entire sample, despite a heterogeneous density of fluorescent molecules.

[0030] FIG. 2 is a schematic diagram of an apparatus 100 for localizing single fluorescent molecules contained in a sample 102 using single molecule localization microscopy (SMLM), according to one example.

[0031] The sample 102 may be made of, for example, cells (such as neurons) or tissue samples that contain fluorescently labeled molecules. For example, the fluorescent molecules may be labeled using SMLM fluorophores (such as photoresponsive, photoactivatable, phototransducible, spontaneously blinking or transiently blinking fluorophores). Once labeled, the fluorescent molecules then reversibly transition between their on and off emission states (i.e., blink) under illumination. In general, samples containing fluorescent molecules have a heterogeneous density of fluorescent molecules. Thus, certain regions of the sample 102 may have a high density of fluorescent molecules while other regions have a low density of fluorescent molecules. Additionally or alternatively, other regions may be devoid of fluorescent molecules.

[0032] As shown in Fig. 2, the apparatus 100 includes a scanning unit 104, an image acquisition unit 108, a determining unit 112, a computing unit 114, and a localizing unit 116. Each of the determining unit 112, the computing unit 114, and the localizing unit 116 can be implemented using hardware, software, and / or a combination thereof. For example, the hardware devices can be implemented using processing circuitry, such as, but not limited to, a processor, a central processing unit (CPU), a controller, an arithmetic logic unit (ALU), a digital signal processor, a microcomputer, a field programmable gate array (FPGA), a system on a chip (SoC), a programmable logic unit, a microprocessor, or any other device capable of responding to and executing instructions in a defined manner. The software can include computer programs, program codes, instructions, or some combination thereof, for independently or collectively instructing or configuring the hardware devices to operate as desired. The computer program and / or program code may include programs or computer readable instructions, software components, software modules, data files, data structures, and / or the like that may be implemented by one or more hardware devices, such as one or more of the hardware devices mentioned above. If the hardware device is a computer processing device (e.g., a CPU, a controller, an ALU, a digital signal processor, a microcomputer, a microprocessor, etc.), the computer processing device may be configured to execute the program code by performing arithmetic operations, logical operations, and input / output operations according to the program code. Each unit may also include one or more storage devices. The one or more storage devices may be tangible or non-transitory computer readable storage media, such as random access memory (RAM), read only memory (ROM), permanent mass storage device (such as a disk drive), solid state (e.g., NAND flash) device, and / or any other similar data storage mechanism capable of storing and recording data.The one or more storage devices may be configured to store computer programs, program codes, instructions, or some combination thereof, for one or more operating systems and / or for implementing the exemplary embodiments described herein. The computer programs, program codes, instructions, or some combination thereof may also be loaded into the one or more storage devices and / or one or more computer processing devices from a separate computer readable storage medium using a drive mechanism. Such separate computer readable storage medium may include a Universal Serial Bus (USB) flash drive, a memory stick, a Blu-ray / DVD / CD-ROM drive, a memory card, and / or other similar computer readable storage medium.

[0033] Alternatively, two or more of the determination unit 112, the calculation unit 114, and the localization unit 116 may be implemented by the same hardware and / or software. For example, the determination unit 112, the calculation unit 114, and the localization unit 116 may all be implemented by a single computer storage medium that can provide instructions to each unit to perform the functions described below.

[0034] The scanning unit 104 is configured to scan the sample 102 using a light beam 126 at a first wavelength having a predefined irradiance. In one example, the scanning unit can be an Adjustable Excitation Region Adaptive Scanning (ASTER) system as disclosed in Mau et al. The ASTER system 104 allows for uniform excitation while using a Gaussian beam.

[0035] As shown in FIG. 2, the ASTER system 104 includes a laser 120 that emits a light beam 126 with a Gaussian shaped illumination profile. The laser emits at a first wavelength that corresponds to the excitation wavelength of a fluorophore used to label the molecules contained in the sample 102. For example, the laser 120 can be a diode-pumped solid-state laser emitting at 638 nm. An excitation filter 132 is used to select the excitation wavelength of the laser 120. In this example, the ASTER system 104 includes multiple mirrors for the practicality and compactness of the system. In addition, the ASTER system 104 includes a variable afocal system 160 that can be used as an optical zoom to change the diameter of the light beam 126. Details of the variable afocal system 160 are shown in the inset 160a of FIG. 2. The variable afocal system 160 includes two variable-focus liquid lenses 170a, 170b with focal lengths that can vary from -28 mm to 28 mm, and a 100 mm focal length f'. 3 The lenses 170a and 170b include one fixed positive lens 171 having a focal length f'. 1 , f' 2 By changing the angle .theta., the diameter of the light beam 126 can be changed to obtain a diameter that is 0.1 to 10 times the initial diameter of the light beam 126.

[0036] In addition, the ASTER system 104 includes a microscope 140 that includes an objective lens 144. The microscope 104 also includes a tube lens 177 having a focal length of 200 mm such that the light beam 126 is focused at the back focal plane 142 of the objective lens 144 of the microscope 144. The microscope 140 may be, for example, an inverted microscope. The microscope may be part of both the ASTER system 104 (i.e., the scanning unit 104) and the image acquisition unit 108 described below. The sample 102 is placed on a cover slip of the microscope 142.

[0037] Furthermore, the ASTER system 104 includes a galvanometer scanning mirror 122. The galvanometer scanning mirror 122 is controlled by a waveform generator (not shown). Although only one galvanometer scanning mirror 122 is shown in FIG. 2 for ease of representation, in practice, two galvanometer scanning mirrors 122 are used. Between the two galvanometer scanning mirrors 122, which are placed in a plane conjugate with the back focal plane 142 of the objective lens 144 of the microscope 140, a lens 172 with a focal length of 100 mm is used to focus the Gaussian light beam 126 (which provides non-uniform excitation as shown in FIG. 2). Specifically, an angular shift applied to the galvanometer scanning mirror 122 induces a similar angular shift in the back focal plane 142 of the objective lens 144, which induces a position shift of the light beam 126 in the sample 102 plane. A lens 173 with a focal length of 100 mm is placed after the two galvanometer scanning mirrors 122. This configuration allows for a collimated beam to be obtained for scanning large XY areas. After focusing the light beam 126 in the back focal plane 142 of the objective lens 144, a time-averaged flat-top excitation profile at the sample 102 is obtained (as shown in FIG. 2). Thus, fast scanning of the sample 102 with the Gaussian light beam 126 in a defined pattern, such as a raster scan or Archimedes' spiral, produces a globally homogenous illumination.

[0038] Furthermore, the ASTER system 104 includes a translation stage 130, which may be a motorized translation stage or the like, and which includes a dielectric mirror 180. To expand the light beam 126, two fixed lenses 174, 175 with respective focal lengths of -50 mm and 200 mm are used, each located on each side of the dielectric mirror 180. The translation stage 130 allows for the change of the polar angle of the light beam 126 in the back focal plane 142 of the objective lens 144 of the microscope 140. Thus, the translation stage 130 can be used to change from epi-fluorescence excitation to oblique illumination and total internal reflection fluorescence excitation, which are different schemes used to study samples with SMLM, each using different polar angles.

[0039] As indicated above, the ASTER system 104 is configured to scan the sample 102 using a light beam 126 at a first wavelength having a predefined irradiance. The predefined irradiance may be a low irradiance to minimize photobleaching of fluorescent molecules. For example, the predefined irradiance may be determined by a user based on an estimated average density of fluorescent molecules. Alternatively, the predefined irradiance may be retrieved from a look-up table that includes irradiance values ​​associated with an estimated average density of fluorescent molecules and / or a type of molecule and / or fluorophore.

[0040] The image acquisition unit 108 of the device 100 is configured to acquire a first image 110a at a second wavelength. As explained above, under illumination, the fluorescent molecules reach an excited state, then return to their ground state by emitting fluorescent photons, and then become excited again. Alternatively, when scanning the fluorescent molecules, the fluorescent molecules may reach a dark state without emitting radiation. To be in a single molecule state in a high density region, the majority of the molecules need to be "off" (i.e., trapped in a dark state). Therefore, the image acquisition unit 108 is configured to acquire an image when the majority of the fluorescent molecules are "off". Since the probability of reaching the triplet state is lower than the probability of reaching the ground state, there is a delay between the start of scanning and image acquisition. The delay ranges from hundreds of milliseconds to several seconds. In addition, after some time under illumination, the fluorescent molecules reach an excited state again. Therefore, the image acquisition unit 108 acquires an image before the majority of the fluorescent molecules return to "on". Additionally, the scanning unit 104 scans another area of ​​the sample before the fluorescent molecules are turned back "on." Thus, the scanning unit 104 scans a region of the sample in a time range of approximately 50 μs to 500 μs.

[0041] The image acquisition unit 108 includes a dichroic mirror 106, which is used to separate the excitation wavelength (received from the scanning unit 104) and the emission wavelength (received from the sample 102) so that the emission from the sample 102 can be collected by the image acquisition unit 108. The image acquisition unit 108 includes a camera 128 (such as an sCMOS camera) that acquires a first image 110 as the sample 102 is scanned at a predefined irradiance. The image acquisition unit 108 also includes a fixed lens 176 with a focal length of 180 mm or 200 mm to focus the light beam 126 at the camera 128. The second wavelength is the emission wavelength of the fluorophore used to label the molecules of the sample 102. FIG. 3a shows an example of a first image 110a. The first image 110a presents a spot or point spread function (PSF), each PSF representing a single fluorescent molecule. As indicated above, the sample 102 may exhibit a heterogeneous density of fluorescent molecules. Fig. 3a shows that the sample 102 exhibits high density regions 212a, low density regions 214a, medium density regions 216a, and regions 218a lacking fluorescent molecules. Thus, a single irradiance (predefined irradiance) is used to scan the sample 102, and the irradiance is not optimized for each region of the sample 102. Thus, the first image 110a exhibits overlap PSFs, which are clusters of fluorescent molecules emitting simultaneously in a given time frame within a given focal volume (i.e., field of view) in regions of the sample 102 that have a high density of fluorescent molecules.

[0042] The determining unit 112 of the apparatus 100 is configured to determine one or more local densities of single fluorescent molecules based on the first image 110a. Specifically, the determining unit 112 determines the density of each region of the sample 102 as shown in the first image 110a. For example, the determining unit 112 determines a distribution of dots (representing PFS) in the first image 110a. In one example, the determining unit 112 can determine the regions of interest that are regions that contain fluorescent molecules (other regions are free of fluorescent molecules). For example, as shown in FIG. 3a, the regions that contain PSFs (i.e., show dots) are the regions of interest, and other regions (i.e., dark regions) do not contain fluorescent molecules. The determining unit 112 can further determine the density of sub-regions of the region of interest. For example, as shown in FIG. 3a, the sub-regions 212a, 214a, and 216a are sub-regions of the region of interest of the first image 110a, each of which has a high density, a low density, and a medium density of fluorescent molecules, respectively. Based on the determination of the local density of each sub-region of the sample 102, the determination unit 102 can determine a trajectory of the light beam 126 of the scanning unit 104. For example, the trajectory can be determined to include only the region of interest and to avoid regions that do not include fluorescent molecules. For example, the trajectory would avoid the region 218a shown in FIG. 3a.

[0043] The calculation unit 114 of the apparatus 100 is configured to calculate one or more local irradiances of the light beam 126 as a predefined function of each of the one or more local densities of single fluorescent molecules determined by the determination unit 112. In particular, the calculation unit 114 can receive the determined densities from the determination unit 112 for each subregion of the region of interest and calculate an associated irradiance value that can be used to scan each subregion of the sample 112 (corresponding to a subregion of the first image 110a). For example, the calculation unit 112 can calculate the irradiance of each subregion based on a look-up table in which local irradiance values ​​are associated with local density values ​​of fluorescent molecules. Alternatively, the calculation unit can use an analytical function to calculate the irradiance of each subregion. Then, for subregions with a low density of fluorescent molecules, a low irradiance is calculated. Similarly, for subregions with a high density of fluorescent molecules, a high irradiance is calculated, and for subregions with a medium density of fluorescent molecules, a medium irradiance is calculated. For example, the computation unit 112 calculates a high irradiance for the sub-region 212a shown in FIG. 3a, a medium irradiance for the sub-region 216a, and a low irradiance for the sub-region 214a.

[0044] The scanning unit 104 can then scan the sample 102 by using the optimized scanning settings, i.e., based on the local irradiance calculated by the calculation unit 114, preferably based on the trajectory determined by the determination unit 112. In particular, to optimize the scanning time, the scanning unit 104 scans only the region of interest (and avoids regions without fluorescent molecules) while using the irradiance associated with each sub-region of the sample 102. In particular, since the diameter of the light beam 126 is larger at lower irradiances, a larger area can be scanned when using a small light beam 126. Thus, for sub-regions with a low density of fluorescent molecules, the scanning unit 104 scans the sample 102 with a low irradiance calculated by the scanning unit 104. Similarly, for sub-regions with a high density of fluorescent molecules, the scanning unit 104 scans the sample 102 with a high irradiance, and for sub-regions with a medium density of fluorescent molecules, the scanning unit 104 scans the sample 102 with a medium irradiance.

[0045] To vary the irradiance for scanning the sample 102, the scanning unit 104 varies the diameter of the light beam 126. As indicated above, the ASTER system 104 includes a variable afocal system 160 configured to change the diameter of the light beam 126. To increase the irradiance, the variable afocal system 160 is used to decrease the diameter of the light beam 126, and to decrease the irradiance, the variable afocal system 160 is used to increase the diameter of the light beam 126. For example, FIG. 3b shows an irradiance mapping 220 used to scan the sample 102, where the irradiance has been determined based on the first image 110a of FIG. 3a. To scan the sub-region 212a with a high density of fluorescent molecules, a small diameter light beam 126 is used, to scan the region 214a with a low density of fluorescent molecules, and a medium-sized diameter light beam 126 is used to scan the sub-region 216a with a medium density of fluorescent molecules.

[0046] While scanning the sample using the optimized scanning settings (i.e., optimized irradiance and trajectory), the image acquisition unit 108 can then acquire a second image 110b. The second image is acquired at a second wavelength. Similar to the first image 110a, the second image also presents a PSF. However, due to the adaptation of the irradiance to the density of the fluorescent molecules in each sub-region of the sample 102, the second image presents less overlapping PSFs than the first image 110a. In other words, a single fluorescent molecule condition has been achieved. In particular, by adapting the irradiance of the scanning unit 104 to the density of the fluorescent molecules in each sub-region, clusters of fluorescent molecules emitting simultaneously can be avoided, and a single fluorescent molecule emitting in the focal volume at a given time can be obtained. In addition, it is possible to increase the scanning efficiency by using a large light beam 126 to scan areas with a low density of fluorescent molecules, as well as by scanning only the area of ​​interest and avoiding areas without fluorescent molecules.

[0047] The localization unit 116 of the device 100 is configured to localize the single fluorescent molecule in the second image. The localization unit 116 may be a computer program that extracts an intensity profile for each PSF of the second image to localize the single fluorescent molecule. The localization unit 116 may use the von Diezmann method to determine the localization of the single fluorescent molecule from the intensity profile of each PSF. 4 The single molecule localization method described in can be used. Specifically, the localization unit 116 can analyze the PSF of the second image, which fits a Gaussian model to each intensity profile and calculates an estimate of the center of each fluorescent molecule. Since the second image is acquired in a single molecule state, the second image does not present overlapping PSFs, even in areas with a high density of fluorescent molecules. Therefore, it is possible to precisely localize the PSF.

[0048] To obtain a final image with super-resolution, the process performed by the device 100 described above can be repeated multiple times. For example, the process can be performed thousands or tens of thousands of times. However, photobleaching causes variations in the density of fluorescent molecules (due to changes in the fluorophores used to label the fluorescent molecules, which causes bleaching or permanent loss of fluorescence). Therefore, when repeating the process, it may be necessary to adapt the irradiance each time the process of the device 100 described above is repeated. To adapt the irradiance, each successive process is performed by using the second image 110b as the first image 110a. In particular, the determining unit 112 determines the local density of single fluorescent molecules based on the second image 110b. The calculating unit 114 then calculates the local irradiance of the light beam 126 of the scanning unit 104 as a predefined function of each local density of fluorescent molecules determined based on the second image 110b. Further, the scanning unit 104 scans the sample 102 using an irradiance corresponding to the local density of the fluorescent molecules determined based on the second image 110b. Finally, a third image is acquired by the acquisition unit 108, and the localization unit 116 localizes the single fluorescent molecules in the third image. The sequential process can be repeated by using the third image to localize the fluorescent molecules in the fourth image, and so on. Thus, thousands or tens of thousands of images of the sample 102 can be acquired to localize the single fluorescent molecules. The localization can then be rendered as a super-resolution image 150 of the single fluorescent molecule. For example, the localization unit can be implemented by software that localizes the single fluorescent molecule in the second image 110b, the third image, the fourth image, ... the 1000th image, etc. The software can have a grid defined with a bin size similar to the accuracy of the localization. In each bin, the localizations are counted and converted to pixel intensities, from which the super-resolution image 150 is reconstructed.

[0049] Advantageously, the iterative process described above allows the adjustment of the irradiance after each scan, not just after the acquisition of the first image. In particular, the irradiance can be adapted each time a new image is acquired. This allows the accuracy of the localization of the PSF to be improved, since the density is estimated each time a new image is acquired. Since the molecules do not emit light simultaneously, the density is measured several times and the irradiance is adapted, ensuring an optimization of the localization of the PSF. In other words, by using a sequential process, the density determination becomes more accurate and therefore the localization can be optimized.

[0050] In another example, the calculation unit 114 is further configured to calculate the scan time as a predetermined function of each of one or more local densities of single fluorescent molecules. Thus, for each of the calculated irradiances associated with the local densities, a scan time can be associated. As a result, the scanning unit can be further configured to scan the sample using the calculated scan times, with each scan time being associated with an irradiance for each local density. In particular, the higher the density, the shorter the scan time can be. Conversely, the lower the density, the longer the scan time can be. For example, for local areas with low densities of molecules, a low irradiance can be used to scan these areas. The scan time can be longer because it takes longer for the molecules to "turn on". In contrast, the higher the density, the more molecules there are in the sample, so the shorter the time it takes for the molecules to "turn on". In other words, for the same scan time, a high density has a higher probability of having molecules that emit light than a low density.

[0051] Although the apparatus 100 is shown and described above as using the ASTER system as the scanning unit 104, the invention is not limited to the disclosed scanning unit 104. Other scanning units can be used without departing from the scope of protection of the claimed invention.

[0052] 4 is a flow diagram depicting a method 300 for localizing a single fluorescent molecule contained in a sample 102 using single molecule localization microscopy. For example, the method 300 can be performed by the apparatus 100 described above with reference to FIG.

[0053] In block 302, the sample 102 is scanned using a light beam 126 at a first wavelength having a predetermined irradiance, the first wavelength being an excitation wavelength of the fluorescent molecules. In one example, the sample 102 is scanned by an ASTER system 104, as described with reference to FIG.

[0054] In block 304, a first image 110a is acquired at a second wavelength, the second wavelength being an emission wavelength of the fluorescent molecules. The first image 110a is acquired while the sample 102 is scanned at the first wavelength.

[0055] In block 306, one or more local densities of single fluorescent molecules are determined based on the first image 110a. For example, the sample 102 may have a heterogeneous density of fluorescent molecules, and thus the first image 110a may exhibit regions with high and low densities of fluorescent molecules and / or regions without fluorescent molecules. In one example, a trajectory of the light beam 126 may be determined. The trajectory includes regions of the sample 102 that contain single fluorescent molecules and avoids regions of the sample 102 that do not contain single fluorescent molecules.

[0056] In block 308, one or more local irradiances of the light beam 126 are calculated as a predefined function of each of one or more local densities of single fluorescent molecules. For example, the one or more local irradiances can be calculated based on a look-up table in which local irradiance values ​​are associated with local density values ​​of fluorescent molecules. The one or more local irradiances of the light beam 126 are calculated such that, on average, only one of the fluorescent molecules emits light in a given time frame (single molecule state). The single molecule state can prevent overlapping PSFs.

[0057] In block 310, the sample 102 is scanned using the calculated local irradiance or irradiances of the light beam 126. For example, areas of the sample 102 having a high density of fluorescent molecules are scanned by using the calculated high irradiance, and areas of the sample 102 having a low density of fluorescent molecules are scanned by using the calculated low irradiance. The sample 102 can be scanned based on the determined trajectory. Thus, only areas of the sample 102 containing fluorescent molecules are scanned. Additionally or alternatively, the local irradiance or irradiances can be obtained by varying the diameter of the light beam 126.

[0058] In block 312, a second image 110b is acquired at a second wavelength. The second image 110b is acquired while scanning the sample using the calculated local irradiance or irradiances of the light beam 126. Because the irradiance is optimized, the overlap PSF is minimized and few artifacts are visible in the second image 110b.

[0059] Single fluorescent molecules are localized in the second image 110b in block 314. Because the second image 110b is acquired in a single molecule state, it is possible to localize single fluorescent molecules with high precision.

[0060] The method 300 can be performed multiple times to obtain a super-resolution image. However, photobleaching causes variations in the density of single fluorescent molecules. Thus, the steps of blocks 306-314 can be performed multiple times sequentially, and each time the method is repeated, the second image 110b becomes the first image 110a, and sequential second images are obtained in which the single fluorescent molecules are localized. In addition, a final image of the single fluorescent molecule can be reconstructed based on the localization of the single fluorescent molecule in each of the second image and sequential second images.

[0061] According to the method 300 with reference to Fig. 4, instead of using uniform illumination to scan the sample 102, illumination adapted to the density of fluorescent molecules is used. As a result, it is possible to achieve a single fluorescent state even in high-density areas. Thus, by achieving a single fluorescent molecule state, it is possible to obtain images with fewer artifacts and localize the PSF with high accuracy while increasing the scanning efficiency.

[0062] Although the invention has been shown and described in detail with the help of preferred embodiments, the invention is not limited to the disclosed examples. A person skilled in the art can deduce other variants without departing from the scope of protection of the claimed invention. For example, different lenses can be used with different focal lengths. In addition, a variable focus lens (specifically a variable focus liquid lens) is not essential, but it makes it possible to obtain a fast zoom. In other examples of the invention, the irradiance can be varied by using a variable attenuator or by controlling the light source. However, these further methods for varying the irradiance do not allow optimizing the scanning time. In another example, instead of scanning only the area of ​​the sample that contains the fluorescent molecules, the entire sample can be scanned (for example, by using a raster scan). Furthermore, the fluorescent molecules may exhibit more complex dynamics than those presented in FIG. 1 and may be, for example, photoconverting molecules.

Claims

1. 1. An apparatus (100) for localizing single fluorescent molecules contained in a sample (102) using single molecule localization microscopy, comprising: a scanning unit (104) configured to scan the sample using a light beam (126) of a first wavelength having a predetermined irradiance; an image acquisition unit (108) configured to acquire a first image (110a) and a second image (110b) at a second wavelength; a localization unit (116) configured to localize the single fluorescent molecule in the second image; Including, a determining unit (112) configured to determine one or more local densities of the single fluorescent molecules based on the first image; a calculation unit (114) configured to calculate one or more local irradiances of the light beam as a predetermined function of each of the one or more local densities of the single fluorescent molecules; Including, 10. An apparatus (100), wherein the scanning unit is further configured to scan the sample using the one or more local irradiances of the light beam.

2. 2. The apparatus of claim 1, wherein the one or more local irradiances of the light beams are adapted to cause, on average, only one of the single fluorescent molecules to emit light within a predetermined focal volume in a predetermined time frame.

3. 3. The apparatus of claim 1, configured to perform a series of sequential processes for localizing the single fluorescent molecule, wherein for each sequential process, the second image becomes the first image of the sequential process, and the scanning unit is further configured to scan the sample using the one or more local irradiances calculated based on the one of the one or more local densities determined from the second image.

4. The apparatus of claim 1 or 2, wherein the calculation unit is configured to calculate the one or more local irradiances based on a look-up table in which local irradiance values ​​are associated with local density values ​​of fluorescent molecules.

5. 3. The apparatus of claim 1, wherein the scanning unit includes a zoom (124) for varying the diameter of the light beam and thereby the local irradiance thereof.

6. 3. The apparatus of claim 1, wherein the determination unit is configured to determine a trajectory of the light beam, and the scanning unit is configured to scan the sample based on the trajectory, the trajectory including regions of the sample that contain the single fluorescent molecule and avoiding regions of the sample that do not contain the single fluorescent molecule.

7. 1. A method (300) for localizing a single fluorescent molecule contained in a sample (102) using single molecule localization microscopy, comprising: a) scanning the sample with a light beam (126) at a first wavelength having a predetermined irradiance, the first wavelength being an excitation wavelength of the fluorescent molecules; b) acquiring a first image (110a) at a second wavelength, said second wavelength being an emission wavelength of said fluorescent molecules; c) determining one or more local densities of the single fluorescent molecules based on the first image; and d) calculating one or more local irradiances of the light beam as a predetermined function of each of the one or more local densities of the single fluorescent molecules; d) scanning the sample using the one or more local irradiances of the light beam; f) acquiring a second image (110b) at said second wavelength; g) localizing the single fluorescent molecule in the second image; and The method (300).

8. 8. The method of claim 7, wherein the one or more local irradiances of the light beam are adapted to cause, on average, only one of the single fluorescent molecules to emit light within a predetermined focal volume in a predetermined time frame.

9. 9. The method of claim 7 or 8, wherein steps c) to g) are performed sequentially multiple times, the second image becoming the first image of step c), sequential second images being obtained for each sequential time the steps are performed, the process further comprising scanning the sample using the one or more local irradiances calculated based on the one of the one or more local densities determined from the second image.

10. 9. The method of claim 7 or 8, wherein the one or more local irradiances are calculated based on a look-up table in which local irradiance values ​​are associated with local density values ​​of fluorescent molecules.

11. 9. The method of claim 7 or 8, wherein scanning the sample using the one or more local irradiances of the light beam comprises varying the local irradiances by varying a diameter of the light beam.

12. 9. The method of claim 7 or 8, comprising determining a trajectory of the light beam and scanning the sample based on the trajectory, wherein the trajectory includes regions (212a, 214a, 216a) of the sample that contain the single fluorescent molecule and avoids regions (218a) of the sample that do not contain the single fluorescent molecule.

13. 10. The method of claim 9, further comprising reconstructing a final image (150) of the single fluorescent molecule based on the localization of the single fluorescent molecule in the second image and each of the sequential second images.

Images