Patent Description:
In recent years, a number of techniques have been developed to increase the resolution of light microscopy beyond the diffraction limit. Some of these techniques involve stochastically activating the fluorescence of a subset of molecules that are present in a field-of view, capturing an image of those molecules that are activated with a digital camera, transferring the image to a computer, and then reversibly or irreversibly deactivating the fluorescence of the molecules. This process is repeated for a large number of cycles until the pool of nearly all of the molecules present have been adequately sampled, which may take <NUM>,<NUM> cycles or more. Each image of this large number of images is then analyzed by fitting either single Gaussian distributions or overlapping Gaussians to fluorophore-image centroids or spots. The locations and probabilities of fit for each molecule are then determined by successively storing each image in the computer's memory, performing a cross-correlation of the fits of the Gaussians, and then storing the location and confidence of fit for each molecule. While acquisition of the images can be accomplished relative quickly, handling and processing such a large number of images is relatively slow because these techniques necessitate acquiring and processing a large amount of image data in order to obtain a relatively small number of pixel locations. For example, <NUM>,<NUM> cycles are used to capture <NUM>,<NUM> separate images which results in processing <NUM> gigabytes of data in order to generate a single <NUM> kilobyte final image. For these reasons, engineers, scientists, and microscope manufacturers continue to seek less computationally demanding systems and methods for handling and processing the images.

<CIT> and <CIT> each describe a camera and processor for a fluorescence microscopy system. <CIT> and <CIT> describe a similar system with summing individual images.

Various systems and methods for executing super-resolution microscopy of a specimen with most of the image processing performed in a camera of a fluorescence microscopy instrument are described. The camera may include one or more processors to execute machine-readable instructions that control excitation light output from a multi-channel light source, control capture of intermediate images of the specimen, and perform image processing of the intermediate images to produce a final super-resolution image of the specimen. The present invention provides a camera for fluorescence microscopy according to claim <NUM> and a method for producing a super-resolution image of a specimen according to claim <NUM>. Particular embodiments of the present invention are defined in the appended dependent claims.

Systems and methods for executing super-resolution microscopy of specimen within the camera of a fluorescence microscopy instrument greatly reduces the amount of data that is typically transferred to the instrument computer, which reduces the amount of data storage used by the computer and eliminates the need for a high-speed interface between the camera and the computer. In addition, by performing most of the image processing within the camera and avoiding a large number of data transfers from the camera to the computer, the overall performance of the fluorescence microscope instrument is improved.

<FIG> shows a schematic representation of an example fluorescence microscopy instrument <NUM> used to perform super-resolution microscopy. The instrument <NUM> includes a light source <NUM>, an excitation filter <NUM>, a dichroic mirror <NUM>, an objective lens <NUM>, an emission filter <NUM>, a camera <NUM>, and a computer <NUM>. The light source <NUM> can be configured with a number of separate lasers, each laser to emit a substantially monochromatic beam of light of a single wavelength or light within a narrow band of wavelengths of the electromagnetic spectrum. The light emitted by each laser is commonly referred to as a "channel. " For example, as shown in <FIG>, the source <NUM> emits two different beams of excitation light represented by differently patterned lines <NUM> and <NUM>. The beams <NUM> and <NUM> pass through the excitation filter <NUM>, which narrows the wavelength range of each beam. The dichroic mirror <NUM> reflects the beams <NUM> and <NUM> into the objective lens <NUM> which directs the beams into a specimen (not shown) disposed on a stage <NUM>. The specimen may be composed of a number of different components, many of which are labeled with fluorescent probes. The beam <NUM> can be an activation beam with a frequency that converts the fluorophores into an activate state, and the beam <NUM> can be an excitation beam with a different frequency that causes the fluorophores in the active state to fluoresce. A portion of the fluorescent light emitted from the fluorophores is collected and collimated by the objective lens <NUM> into a beam <NUM>. The dichroic mirror <NUM> allows transmission of the beam <NUM>, and the emission filter <NUM> removes stray excitation light from the beam <NUM>. The fluorescent light is captured by the camera <NUM> to create a single image of the specimen. As shown in <FIG>, the camera <NUM> is electronically connected to the light source <NUM> and the computer <NUM>. The camera <NUM> is configured to control operation of the light source <NUM> and to process captured images of the specimen as described below. In particular, the camera <NUM> controls operation of the light source <NUM> in order to execute super-resolution fluorescence microscopy described below. <FIG> also shows the camera <NUM> electronically connected to the computer <NUM>. In certain embodiments, the camera <NUM> executes super-resolution fluorescence microscopy by directing the capture and processing of all the images of the specimen into a final super-resolution image that is sent to the computer <NUM> for storage and/or display as described in greater detail below. Alternatively, the camera <NUM> and the computer <NUM> can split processing of the images as also described in greater detail below.

The fluorophores used to label components of the specimen typically have a number of different electronic states, an example of which is represented in <FIG> by an electronic band diagram <NUM>. When the fluorophores are introduced to the specimen, the fluorophores are attached to probes that bind to components of the specimen. The fluorophores are initially in a non-fluorescing, dark state which can be a ground state with electronic energy <NUM>. In super-resolution microscopy, intermediate images of the specimen are captured by the camera <NUM>. The camera <NUM> directs the light source <NUM> to emit the activation beam <NUM> with a frequency, va, for a brief period of time and with a very low intensity in order to stochastically convert a relatively small number of fluorophores into the active state with electronic energy <NUM>. The camera <NUM> then directs the light source <NUM> to turn "off" the beam <NUM> and emit the beam <NUM> with a frequency, ve, that converts only the subset of fluorophores already in the active state into a fluorescing state with electronic energy <NUM>. In the example of <FIG>, the fluorophores in the fluorescing state emit fluorescent excitation light with a frequency, vf, when transitioning to a lower energy <NUM> intermediate state followed by thermal relaxation back to the active state. In certain embodiments, when the camera <NUM> has finished capturing an image of the fluorescing fluorophores, the camera <NUM> directs the light source <NUM> to continue emitting the beam <NUM> for a period of time sufficient to cause the activated fluorophores to undergo hundred, thousands or more excitation/emission cycles represented by directional arrow <NUM>. The fluorophores under continued illumination by the excitation beam <NUM> ultimately transition to a bleached or an inactive state represented by energy level <NUM>. Alternatively, the light source <NUM> can emit a third beam of light (not shown) the converts the fluorophores from the active state into the inactive state. Converting fluorophores from the active state into the inactive state can be a complete or partial reconfiguring of the fluorophore into a molecule that is not able to transition to the active state or the fluorescing state when illuminated by either of the beams <NUM> and <NUM>.

The camera <NUM> can be a computing device that operates the lights captures, stores intermediate images, and processes the intermediate images to produce a final super-resolution image. <FIG> show schematic representations of example implementations of the camera <NUM>. In the example of <FIG>, the camera <NUM> includes one or more processors <NUM>, memory <NUM>, a detector <NUM>, a video or camera interface <NUM>, and one or more computer-readable mediums <NUM>. Each of these components is operatively coupled to one or more buses <NUM>. The processor <NUM> can be a single of multi-core processor with internal memory. The memory <NUM> can be main memory such as DRAM or SRAM or any other suitable memory. The detector <NUM> can be an array or photodetectors including an array of CMOS detectors or an array of CCD detectors. The interface <NUM> can be a Local Area Network ("LAN"), a wireless LAN, a <NUM> mobile wide area network ("WAN") or a WiMax® WAN. The computer-readable medium <NUM> can be any suitable non-transitory medium that participates in storing and providing machine-readable instructions to the processor <NUM> for execution. For example, the computer-readable medium <NUM> can be a magnetic disk, flash memory, an optical disk, or a magnetic disk drive. The computer-readable medium <NUM> can also store machine-readable instructions <NUM> directed to super-resolution fluorescent microscopy image processing described below. The computer-readable medium <NUM> may also store an operating system <NUM> and network machine-readable instructions <NUM>. The operating system <NUM> can be multi-user, multiprocessing, multitasking, multithreading, and real-time and can perform tasks such as recognizing input from input devices, recognizing input from a keyboard, a keypad, or a mouse; sending output to the computer <NUM>; keeping track of files and directories on the medium <NUM>; controlling peripheral devices such as disk drives, monitors and printers; and managing traffic on the one or more buses <NUM>. The network applications <NUM> includes various components for establishing and maintaining network connections, such as machine-readable instructions for implementing communication protocols including TCP/IP, HTTP, Ethernet, USB, and FireWire®. Alternatively, <FIG> shows an example of the camera <NUM> that includes a digital signal processor ("DSP") <NUM> coupled to the bus <NUM> to process the intermediate images to produce a final super-resolution image as described below. The DSP <NUM> may have multiple processors, multi-ported local memory and input/output control. The DSP <NUM> is able to transfer data to and from the memory <NUM> and the I/O controller while simultaneously processing data from local memory. In order to map machine-readable instructions into the DSP <NUM> architecture, blocks of data in memory <NUM> are broken into smaller subsets for processing in the DSP <NUM> processors. Alternatively, <FIG> shows an example of the camera <NUM> that includes a graphics processing unit ("GPU") <NUM> coupled to the bus <NUM> to process the intermediate images to produce a final super-resolution image as described below. The GPU <NUM> is a specialized computing device designed to rapidly manipulate and alter memory to accelerate the processing of images. Alternatively, the camera <NUM> can include application-specific integrated circuit ("ASIC") coupled to the bus <NUM> that is customized to process the intermediate images to produce a final super-resolution image as described below.

The camera <NUM> alone or the camera <NUM> in combination with the computer <NUM> can execute any of various super-resolution fluorescent microscopy techniques that have been developed to allow imaging of fluorophore-labeled specimens at resolutions significantly below diffraction-limited resolution. These techniques are typically based on collecting a series of intermediate images of fluorescent light emitted from different subsets of fluorophore-labeled components of a specimen over time, provided the emitting fluorophores are separated from one another by distances greater than approximately <NUM>. In other words, provided the positions of the emitting fluorophores in the specimen can be resolvable by traditional optical microscopy, the positions of the fluorophores in a specimen can be determined, in certain cases, to a resolution of below <NUM>. However, because the fluorescent-emission signal can be interpreted only when the emitting fluorophores are sparsely arranged within the sample, a large number of intermediate images are produced from different subsets of sparse, stochastically distributed, activated fluorophores in order to construct a final super-resolution image of a fluorophore-labeled specimen. Each intermediate image captured by the camera <NUM> is a diffraction-limited image of a subset of sparsely arranged fluorophores. The light passing through the optical system of the camera <NUM> and the microscope causes the light to deviate from straight-line propagation and spread out somewhat in the image plane of the camera <NUM>, which is located at the detector <NUM>. The optical system can be the camera lens and other optical components of the microscope that direct and focus light from an object plane of the specimen onto the image plane of the camera <NUM>. When an optical system with a circular aperture receives plane waves output from a point source in the object plane, such as a fluorescent light emitting fluorophore, rather than there being a corresponding bright narrowly defined image point in the image plane, the light actually spreads out into a circular spot called an Airy disk composed of alternating light and dark rings. <FIG> show a representation of an example optical system <NUM> of a microscope that receives light output from a point source (x,y) <NUM> in an object plane <NUM> of a specimen. For example, the point source <NUM> can be a fluorescing fluorophore. The optical system <NUM> spreads the light out to produce a spot <NUM> in a corresponding image plane <NUM> of the camera. The light output from the point source <NUM> has an intensity I(x,y) that is transformed by the optical system <NUM> into the spot <NUM> centered about a point (x',y') <NUM> with a corresponding intensity distribution represented by a symmetrical Airy disk <NUM> over the spot <NUM>.

<FIG> shows an example representation of an Airy disk in one dimension within an image plane. Horizontal axis <NUM> is a line in the image plane passing through a point (x',y') in the image plane, such as the point <NUM> shown in <FIG>, and vertical axis <NUM> represents intensity. The Airy disk has a tall, relatively narrow central peak <NUM> with secondary peaks of decreasing height <NUM>-<NUM> extending outward away from the central peak. The height of the curve corresponds to intensity. Any point on the surface of the Airy disk corresponds to the intensity observed at a corresponding position on the image plane. In other words, an image produced by an optical system of a point source in the object plane appears as a central bright disk, corresponding to the central peak <NUM> of the Airy disk, with concentric rings of light of increasing radius corresponding to the rings or ridges surrounding the central peak.

The radius of the Airy disk determines the overlapping of neighboring Airy disks and therefore the diffraction limit of the image. <FIG> shows the diffraction limit associated with an optical system. Consider two points (x<NUM>,y<NUM>) <NUM> and (x<NUM>,y<NUM>) <NUM> in an object plane separated by a distance s<NUM> <NUM>. The images of these two points output from an optical system appear as two Airy disks <NUM> and <NUM> centered at image points (x'<NUM>,y'<NUM>) and (x'<NUM>,y'<NUM>). The spreading of light from point sources <NUM> and <NUM> into spots with intensity distributions represented by the disks <NUM> and <NUM> in the image plane is a diffraction-related phenomenon. When s<NUM> is sufficiently large that the corresponding distance s'<NUM> <NUM> between the centers of the disk <NUM> and <NUM> in the image plane separates the Airy disk so that the sum of the two Airy disks, represented in <FIG> by curve <NUM>, remains clearly bimodal, the images of the points <NUM> and <NUM> in the image plane can be distinguished from one another. However, when two points <NUM> and <NUM> in the object plane are separated by a sufficiently small distance s<NUM> <NUM> that the corresponding images <NUM> and <NUM> of the two points in the image plane overlap, with the sum of the two Airy disks, represented by curve <NUM>, merging into a single peak, the two points <NUM> and <NUM> cannot be distinguished from one another in the image plane. The minimum spacing, or maximum resolution, for traditional optical microscopy is generally regarded as: <MAT> where θ is the half-angle of the maximum cone of light that can enter or exit the optical system;.

The minimum spacing, or maximum resolution, in the input image corresponds to spacing between Airy disk at which the first left-hand zero point of the right-hand disk coincides with the first right-hand zero point of the left-hand disk. The minimum separation, or maximum resolution, of any two adjacent fluorescing fluorophores that can be imaged corresponds to about <NUM> for optical microscopy systems. The minimum spacing, or maximum resolution, is referred to as "the diffraction limit," since the Airy disk images of point sources in the image plane arise as a result of diffraction.

In order to ensure that the density of fluorophores simultaneously activated at any point in time is such that any pair of activated fluorophore is separated by at least <NUM>, the camera <NUM> operates the light source <NUM> to emit the activation beam <NUM> with a very low intensity so that very few photons with energy ℏva reach the object plane. The few photons that do reach the object plane only excite a subset of the fluorophores and the fluorophores that are excited are stochastically distributed over the object plane so that the likelihood of any two photons activating two fluorophores separated by less than <NUM> is very low. As a result, the image captured by the camera <NUM> is composed of a sparse distribution of ideally nonoverlapping Airy disks.

<FIG> shows an example object plane <NUM> of a sparse stochastic distribution of four fluorescing fluorophores <NUM> and a representation of a process used to produce a resulting super-resolution image <NUM> of the four fluorescing fluorophores. The sparse stochastic distribution of fluorescing fluorophores in the object plane <NUM> is produced by exciting the specimen with a very low intensity beam <NUM> as described above. The optical system <NUM> of the camera <NUM> receives light output from each fluorescent point source in the object plane <NUM>, such as fluorescent point source (x,y) <NUM>, and spreads the light out to produce four corresponding spots in a corresponding image plane <NUM>, such as spot <NUM>. The intensity distribution of each spot in the image plane <NUM> is characterized by an Airy disk that corresponds to the fluorescing point source <NUM>, such as an Airy disk at the spot <NUM>. The camera <NUM> processes the image by curve fitting a two-dimensional Gaussian distribution to each spot. For example, curve <NUM> represents a Gaussian distribution curve fit to the spot <NUM>. The (x', y') coordinates associated with the maximum of each Gaussian distribution are taken as the centroid coordinates of each spot. The resulting super-resolution image <NUM> is produced by assigning an intensity value to each centroid obtained in the spot approximation. For example, centroid coordinates (x'<NUM>, y'<NUM>) correspond to the maximum of the Gaussian distribution <NUM>, and the intensity value associate with the centroid coordinates (x'<NUM>, y'<NUM>) <NUM> forms a pixel in the image <NUM> that corresponds to the fluorescing fluorophore located at the point <NUM> in the object plane <NUM>. The resulting image <NUM> is a super-resolution, intermediate image of sparse, stochastically distributed pixels that correspond to the point sources in the object plane <NUM>. The camera can store the image data.

<FIG> shows an example of super-resolution fluorescence microscopy that can be used to obtain a final super-resolution image a specimen. The specimen is labeled with fluorophores of sufficient density to ensure that, when the positions of the fluorophores are accurately determined, those positions will together produce an image of a structure, component, or organelle of interest to the fluorescence microscopist. Then, the specimen is immobilized and a number of intermediate images are generated from the specimen by, for each intermediate image, activating a small subset of the fluorophores and exciting fluorescent emission from the activated fluorophores, as described above with reference to <FIG>. Only subsets of the fluorophores are activated in order to sufficiently separate fluorophores from one another to satisfy the above-discussed separation constraint. Initially, the fluorophores are in a non-fluorescing, dark state. The specimen is weakly illuminated with a frequency of light that converts a subset of the fluorophores from the dark state to an active state. Activation of a small subset of the fluorophores is stochastic in nature. Activation is carried out with a weak illumination in order to ensure that the average spacing between fluorophores is significantly greater than the diffraction-limited distance (i.e., <NUM>), so that no two activated fluorophores are sufficiently closely spaced that their Airy disk images overlap to the extent that the central peaks cannot be resolved, as discussed above with reference to <FIG>, and therefore centroids for the fluorophore positions cannot be accurately computed. The specimen is then illuminated with excitation light that causes the activated fluorophores to fluoresce. Following data collection for an intermediate image, the active fluorophores are then illuminated with a bright light of the specific wavelength most effective to bleach the active fluorophores, so that they cannot be again activated and do not fluoresce during data collection for subsequent intermediate images as described above with reference to <FIG>. As shown in <FIG>, for example, each of intermediate images <NUM>-<NUM> are produced by collecting data from a different set of sparsely arranged, activated fluorophores. In other words, each of the intermediate images <NUM>-<NUM> is obtained and processed as described above with reference to <FIG>. The intermediate images are then summed together <NUM> to produce a final, composite super-resolution image <NUM> that reveals a fluorophore-labeled structure, organelle, cellular component, or other feature <NUM> in the specimen.

<FIG> shows a control-flow diagram <NUM> of an example method of super-resolution microscopy executed by a camera. With this method, intermediate image data acquisition, image processing to produce a final super-resolution image, sending and receiving data from the computer, and operation of the light source is carried out by the camera. In block <NUM>, the method directs the computer to prompt a fluorescent microscope operator to supply a resolution quality for a final super-resolution image or the method directs the computer to prompt the operator for a threshold parameter Nth, where Nth represents the total number of intermediate images to be captured to produce the final super-resolution image. The computer activates the camera and sends the resolution quality or threshold parameter Nth to the camera. The camera then executes the operations now described with reference to blocks <NUM>-<NUM>. In block <NUM>, an intermediate image index N is initialized to zero. In the while-loop of blocks <NUM>-<NUM>, a number of intermediate images, as discussed above with reference to <FIG>, are produced. In each iteration of the while-loop of steps <NUM>-<NUM>, a next set of fluorophores is activated, in block <NUM>, with the density of the activated fluorophores less than or equal to the maximum resolvable density discussed above with reference to <FIG>. In block <NUM>, fluorescent emission from the activated fluorophores is excited, and a next data set is collected, over time. In block <NUM>, an intermediate image is produced from the collected data by analyzing the data to find the centroids of the point-source images, as discussed above with reference to <FIG>. In block <NUM>, the intermediate image is summed with previously accumulated or recorded intermediate images to produce a super-resolution image, as described above with reference to <FIG>, or the image index is incremented. In block <NUM>, when sufficient data has been accumulated to generate a final image of adequate resolution, or the number of intermediate images produced exceeds the threshold Nth, the method proceeds to block <NUM>, otherwise the method proceeds to block <NUM> and blocks <NUM>-<NUM> are repeated. In block <NUM>, the activated fluorophores are brightly illuminated by light of an appropriate wavelength to bleach the activated fluorophores, removing that set of fluorophores from subsequent intermediate images. In block <NUM>, a final super-resolution image is produced, as discussed above with reference to <FIG>, and the super-resolution imaging process terminates by transmitting the final image to the computer. In block <NUM>, the computer stores the final image.

<FIG> shows a control-flow diagram <NUM> of an example method of super-resolution microscopy executed by a camera and a computer of a microscopy instrument. With this method, execution of task is divided between the camera and the computer. In particular, intermediate image acquisition and control over operation of the light source is carried out by the camera, while a final super-resolution image is selected by the computer. In block <NUM>, the method directs the computer to prompt a fluorescent microscope operator to supply a resolution quality for a final super-resolution image or the method directs the computer to prompt the operator for a threshold parameter Nth, where Nth represents the total number of intermediate images to be captured to produce the final super-resolution image. In block <NUM>, an intermediate image index N is initialized to zero. In the while-loop of blocks <NUM>-<NUM>, execution of the method switches over to the camera with a number of intermediate images is to be produced by the camera, as discussed above with reference to <FIG>, and the data associated with the images is accumulated or recorded in memory by the camera. In each iteration of the while-loop of steps <NUM>-<NUM>, a next set of fluorophores is activated, in block <NUM>, with the density of the activated fluorophores less than or equal to the maximum resolvable density discussed above with reference to <FIG>. In block <NUM>, fluorescent emission from the activated fluorophores is excited, and a next data set is collected, over time. In block <NUM>, an intermediate image is produced from the collected data by analyzing the data to find the centroids of the point-source images, as discussed above with reference to <FIG>. In block <NUM>, the intermediate image is summed with previously accumulated intermediate images to produce a super-resolution image, as described above with reference to <FIG>. In block <NUM>, the super-resolution image is sent from the camera to the computer. In block <NUM>, the computer increments the index N. In block <NUM>, when sufficient data has been accumulated to generate a final image of adequate resolution, or the number of intermediate images produced exceeds the threshold Nth, the method proceeds to block <NUM>, otherwise the method proceeds to block <NUM> in which activated fluorophores are brightly illuminated by light of an appropriate wavelength to bleach the activated fluorophores, removing that set of fluorophores from subsequent intermediate and blocks <NUM>-<NUM> are repeated. In block <NUM>, accumulated intermediate image data is further processed to produce a final super-resolution image, as discussed above with reference to <FIG>. In block <NUM>, the computer stores the final image and the super-resolution imaging process terminates.

In alternative embodiments, the image capture and production of intermediate images can be carried out by the camera, while control of the light source, accumulation of the intermediate image data, and processing the accumulated image data to produce a final image are carried out by the computer. <FIG> shows a control-flow diagram <NUM> of an example method of super-resolution microscopy executed by a camera and a computer of a microscopy instrument. In block <NUM>, the method directs the computer to prompt a fluorescent microscope operator to supply a resolution quality for a final super-resolution image or the method directs the computer to prompt the operator for the threshold parameter Nth. In block <NUM>, an intermediate image index N is initialized to zero. In each iteration of the while-loop of blocks <NUM>-<NUM>, a next set of fluorophores is activated, in block <NUM>, with the density of the activated fluorophores less than or equal to the maximum resolvable density discussed above with reference to <FIG>. In block <NUM>, fluorescent emission from the activated fluorophores is excited. In blocks <NUM> and <NUM>, execution of the method switches to the camera. In block <NUM>, an image of the specimen is captured, and in block <NUM>, the image is processed to produce an intermediate image by analyzing the image data to find the centroids of the point-source images, as discussed above with reference to <FIG>. In block <NUM>, the computer stores and sums the intermediate image created by the camera with previously accumulated intermediate images to produce a super-resolution image, as described above with reference to <FIG>. In block <NUM>, the image index is incremented. In block <NUM>, when sufficient data has been accumulated to generate a final image of adequate resolution, or the number of intermediate images produced exceeds the threshold Nth, the method proceeds to block <NUM>, otherwise the method proceeds to block <NUM> in which activated fluorophores are brightly illuminated by light of an appropriate wavelength to bleach the activated fluorophores, removing that set of fluorophores from subsequent intermediate images and blocks <NUM>-<NUM> are repeated. In block <NUM>, accumulated intermediate image data is further processed to produce a final super-resolution image, as discussed above with reference to <FIG>. In block <NUM>, the computer stores the final image and the super-resolution imaging process terminates.

Claim 1:
A camera (<NUM>) for fluorescence microscopy, wherein the camera (<NUM>) is configured to receive light emitted from a subset of light emitters within a specimen, to capture an image of the subset of light emitters, when the specimen is exposed to a beam of light emitted by a light source (<NUM>) or to beams of light emitted by a number of lasers,
wherein the camera (<NUM>) includes a processor (<NUM>) and a video or camera interface (<NUM>), the processor (<NUM>) being configured to process each captured image into an intermediate image (<NUM>, <NUM>) for each subset of light emitters, the processing comprising analyzing the captured image and determining centroid coordinates of each spot produced by one of the light emitters, wherein coordinates of a maximum of an intensity distribution produced by a light emitter are associated with the centroid coordinates of a spot, and
wherein the processor (<NUM>) of the camera (<NUM>) is configured to provide the intermediate images (<NUM>, <NUM>), and to sum accumulated intermediate images (<NUM>, <NUM>) to produce a final super-resolution image (<NUM>) of the specimen from the accumulated intermediate images and to send the final super-resolution image (<NUM>) of the specimen, via the video or camera interface (<NUM>), to a computer (<NUM>), or
wherein the processor (<NUM>) of the camera is configured to send the intermediate images, via the video or camera interface (<NUM>), to a computer (<NUM>), the computer (<NUM>) being configured to store and sum accumulated intermediate images with previously accumulated intermediate images to produce a final super-resolution image (<NUM>) of the specimen from the accumulated intermediate images.