Laser emission based microscope devices and methods of using such devices for detecting laser emissions from a tissue sample are provided. The scanning microscope has first and second reflection surfaces and a scanning cavity holding a stationary tissue sample with at least one fluorophore/lasing energy responsive species. At least a portion of the scanning cavity corresponds to a high quality factor (Q) Fabry-Pérot resonator cavity. A lasing pump source directs energy at the scanning cavity while a detector receives and detects emissions generated by the fluorophore(s) or lasing energy responsive species. The second reflection surface and/or the lasing pump source are translatable with respect to the stationary tissue sample for generating a two-dimensional scan of the tissue sample. Methods for detecting multiplexed emissions or quantifying one or more biomarkers in a histological tissue sample, for example for detection and diagnosis of cancer, or other disorders/diseases are provided.

The invention relates to an optical arrangement, particularly for the detection beam path of a multi-spot scanning microscope, comprising a detection plane, in which a detector is positionable, comprising a dispersive device for spectrally splitting detection light. According to the invention, the optical arrangement is characterized in that a distorting optical unit is present for guiding the detection light into the detection plane, said distorting optical unit being arranged downstream of the dispersive device and upstream of a detection plane, and in that a rotating device is present for the relative rotation of a luminous field of the spectrally separated detection light and the distorting optical unit. The invention additionally relates to a multi-spot scanning microscope and a method for operating a microscope.

The present disclosure relates to method, computer programs with instructions, and apparatus for determining positions of two or more spaced-apart molecules in one or more spatial directions in a sample by means of a localization microscope. The present disclosure also relates to localization microscopes using such an apparatus. Light distributions arising due to interference of coherent light are used for determining the positions of the molecules. In the method, a plurality of light distributions are generated (S1) using a first light modulator having a plurality of switchable pixels. The first light modulator is arranged in an image plane of the localization microscope. Each light distribution has a local intensity minimum and regions with an intensity increase adjacent thereto. Each of the two or more molecules is illuminated (S2) with one light distribution. For each of the light distributions, photons emitted by the molecules are detected (S4) for different positionings of the light distribution. The light distributions are positioned (S3) independently of each other. Based on the photons detected for the different positionings of the light distributions, the positions of the molecules are finally derived (S5).

The disclosure relates to a method for scanning microscopy wherein a specimen is scanned simultaneously with a plurality of illumination spots of an excitation light. The light emitted by one specimen location irradiated with one illumination spot is detected independently of the light emitted by another specimen location illuminated with another illumination spot. A microscopic image of the specimen can be compiled from the emitted light detected for the different specimen locations. The method provides that the intensities of the different illumination spots are set independently of one another, and in that the illumination spots are guided over the specimen one after another in a scan line. The disclosure additionally relates to a scanning microscope.

An optical microscope device includes: a first illumination optical system including a light source that emits illumination light for illuminating a specimen an LCOS spatial light modulation element that controls a polarization state of the illumination light, a first illumination optical member that uniformly illuminates the LCOS spatial light modulation element, and a polarization optical element that controls a transmission state of the illumination light directed to the specimen from the LCOS spatial light modulation element in response to the polarization state of the illumination light; a second illumination optical system including a second illumination optical member that images a light flux from the first illumination optical system on a specimen surface; and an imaging optical system for imaging the specimen surface.

The disclosed technology brings histopathology into the operating theatre, to enable real-time intra-operative digital pathology. The disclosed technology utilizes confocal imaging devices image, in the operating theatre, “optical slices” of fresh tissue—without the need to physically slice and otherwise process the resected tissue as required by frozen section analysis (FSA). The disclosed technology, in certain embodiments, includes a simple, operating-table-side digital histology scanner, with the capability of rapidly scanning all outer margins of a tissue sample (e.g., resection lump, removed tissue mass). Using point-scanning microscopy technology, the disclosed technology, in certain embodiments, precisely scans a thin “optical section” of the resected tissue, and sends the digital image to a pathologist rather than the real tissue, thereby providing the pathologist with the opportunity to analyze the tissue intra-operatively. Thus, the disclosed technology provides digital images with similar information content as FSA, but faster and without destroying the tissue sample itself.

An apparatus for characterizing luminescent entities by excitation comprising: • a substrate (6) being in contact with a solution comprising luminescent entities; • a source of electromagnetic radiation (4) providing at least a primary beam of radiation (8); an objective (5); a first optical element (1) capable of transforming the intensity profile of the primary beam (8) into an arbitrary secondary intensity profile (distribution) (9); a second optical element (2) capable of separating (discriminating) radiation by wavelength; and a detector (7), where the arbitrary secondary intensity profile has at least an off-center circular continuous intensity distribution (33) focused on the back focal plane (12) of the objective forming a collimated beam (10) capable of creating an evanescent field on the side of the substrate where the solution comprising luminescent entities are located, where the evanescent field excites the luminescent entities thereby creating emission radiation separated by the second optical element (2) and captioned by the detector (7). The invention also relates to an apparatus comprising two optical elements providing a final third intensity profile (distribution) which is the convolution of two mathematical transformations corresponding to each of optical element one and four, respectively.

A system can be used for imaging volumetric samples using a camera array to capture images under different illumination patterns at different axial planes of focus, and at optionally varying lateral fields of view. The sequence of images taken under the different illumination patterns and optional varying lateral fields of view can be processed to generate an image representation of the sample at a focus plane of the sample. Multiple image representations at different focus planes are assembled to form a 3D volumetric representation of the sample.

An auxiliary apparatus for testing the confocality of a scanning and descanning microscope component group has a connector configured for connecting the auxiliary apparatus in a defined relative position to the scanning and descanning microscope component group, and an optical axis running at a fixed orientation with respect to the connector. Further, the auxiliary apparatus has an auxiliary detector with a plurality of auxiliary detection apertures in a plurality of auxiliary detection aperture positions that are arranged at distances in direction of the optical axis and laterally with respect to the optical axis; and an auxiliary light source providing auxiliary light through a plurality of auxiliary emission apertures in a plurality of auxiliary emission aperture positions arranged at distances in direction of the optical axis and laterally with respect to the optical axis.

Systems and methods for hyperspectral imaging are described. In one implementation, a hyperspectral imaging system includes a sample holder configured to hold a sample, an illumination system, and a detection system. The illumination system includes a light source configured to emit excitation light having one or more wavelengths, and a first set of optical elements that include a first spatial light modulator (SLM), at least one lens, and at least one dispersive element. The illumination system is configured to structure the excitation light into a predetermined two-dimensional pattern at a conjugate plane of a focal plane in the sample, spectrally disperse the structured excitation light in a first lateral direction, and illuminate the sample in an excitation pattern with the one or more wavelengths dispersed in the first lateral direction.