A super resolution technique, intended mainly for fluorescence microscopy, acquires the three-dimensional position of an emitter, through a hybrid method, including a number of steps.

In a first step the two-dimensional position of an emitter is acquired, using a technique, named in this application as an Abbe’s loophole technique., In this technique a doughnut, or a combination of distributions, having a zero intensity at the combined center of the distributions, is projected onto the sample containing the emitter, under conditions wherein the doughnut null is moved towards the emitter to reach a position in which the emitter does not emit light.

In a second step, an axial measurement is obtained using a 3D shaping method, characterized by the fact that the emitted light is shaped by an additional optical module creating a shape of the light emitted by the emitter, this shape being dependent of the axial position and means to retrieve the axial position from the shape.

A method for acquiring microscope-images of sample being larger than a field of view of a microscope, the method comprising creating a continuous relative movement between a sample and the microscope, wherein the optical axis of a microscope objective is substantially perpendicular to the vector of the relative movement, illuminating a part of the sample through the microscope objective, wherein the illuminated part of the sample is smaller than the field of view and forms an illumination slit, moving the illumination slit in a scanning direction across the field of view, and detecting light from the sample collected by the microscope objective, wherein the sample is moved in the same direction as the scanning direction or in a direction perpendicular to the scanning direction while the illumination slit is moved across the field of view.

A method of performing imaging includes operating a light sheet projection module in a first state during a first measurement process and using a first primary objective for illumination of a specimen using a light sheet and detection of a first fluorescent emission. The method also includes operating the light sheet projection module in a second state during a second measurement process and using a second primary objective for illumination of the specimen using the light sheet and detection of a second fluorescent emission.

Real-time autofocus. In an embodiment, a scanning apparatus includes an imaging sensor, a focusing sensor, an objective lens, and processor(s) configured to analyze image data captured by the imaging and focusing sensors, and move the objective lens. Real-time autofocus during scanning of a sample is achieved by determining a true-Z value for the objective lens for a point on a sample and for each of a plurality of regions on the sample. The true-Z values and/or surfaces calculated therefrom are used to determine a predicted-Z value for an unscanned region of the sample. The objective lens is adjusted to the predicted-Z value at the beginning of the unscanned region. After scanning the region, a true-Z value is determined for the region and compared to the predicted-Z value. A rescan of the region is initiated if the comparison exceeds a predetermined threshold.

A microscope system comprises: a light source; an objective lens; a stage; a two-dimensional image sensor that captures an image of a specimen placed on the stage; a focusing device that changes distance between the objective lens and the stage; and a control circuit, wherein the control circuit executes, during a movement period in which the stage moves in a direction orthogonal to an optical axis of the objective lens, focus control for controlling the focusing device based on focus evaluation information detected during the movement period, and exposure control for controlling an exposure period of the two-dimensional image sensor, and executes light emission control that causes the light source to emit light with different light emission intensities during the exposure period and during a focus evaluation period in which the focus evaluation information is detected.

For checking the confocality of a scanning and descanning microscope assembly comprising a light source providing illumination light focused into a focal area in a focal plane, a detector detecting light coming out of the focal area and having a detection aperture to be arranged in a confocal fashion with respect to the focal area, and a scanner, an auxiliary detection aperture of an auxiliary detector arranged in the focal plane is scanned with the focal area of the illumination light to record a first comparison intensity distribution of the illumination light registered by the auxiliary detector, and the detection aperture of the detector is scanned with auxiliary light that exits out of an auxiliary emission aperture of an auxiliary light source concentrically arranged with respect to the auxiliary detection aperture in the focal plane to record a second comparison intensity distribution of the auxiliary light registered by the detector.

An instrument and method for scanning all or part of a large specimen mounted on a specimen holder takes a plurality of measurements of each pixel in the whole or part of the specimen being scanned at a plurality of exposure values. A computer controls the movement of the specimen holder during scanning and again of the detector to produce a digitized image of all or part of the specimen with larger dynamic range than the dynamic range of the detection system. In a further embodiment, the instrument can scan two successive, identical strips at a different exposure values and combine the images from the two scans into one digitized image having a larger dynamic range than the dynamic range of the detection system.

Certain examples disclose systems and methods for imaging a target. An example method includes: a) activating a subset of light-emitting molecules in a wide field area of a target using an excitation light; b) capturing one or more images of the light emitted from the subset of the molecules illuminated with the excitation light; c) localizing one or more activated light emitting molecules using one or more single molecule microscopic methods to obtain localization information; d) simultaneously capturing spectral information for the same localized activated light emitting molecules using one or more spectroscopic methods; e) resolving one or more non-diffraction limited images of the area of the target using a combination of the localization and spectral information for the localized activated light emitting molecules; and 0 displaying the one or more non-diffraction limited images.

An optical system of an optical analysis device is easily evaluated with high accuracy. There is provided a method of evaluating an optical analysis device including an optical system A capable of forming a confocal volume C at a focal position by condensing excitation light B, the method including the steps of: placing, at the focal position of the optical system A, a phantom sample in which two or more types of solid members having different fluorescent substance concentrations are arranged adjacent to each other; irradiating the phantom sample 1 with excitation light through the optical system A while relatively moving the confocal volume C formed by the optical system A and the phantom sample in an arrangement direction of the solid members; detecting fluorescent light generated in the solid members placed in the confocal volume C; and evaluating the optical system A based on the detected fluorescent light.

Generating a composite image of a non-flat surface includes: acquiring, using a microscope, multiple images of different areas of the non-flat surface, where each image includes a region of overlap with at least one adjacent image, the microscope having sufficient resolution to image in three dimensions a microstructure on the non-flat surface having a lateral dimension of 10 microns or less and a height of 10 nm or less; determining, for each of the images, a set of rigid body parameters relating a position and orientation of the test object in the image to a common coordinate system, where the set of rigid body parameters is determined by fitting the resolved microstructure in the overlap region in the image with the corresponding microstructure in the overlap region of the adjacent image; and combining the images based on the sets of rigid body parameters to generate a composite image.