A microscope for imaging a sample is disclosed that may include at least one illumination objective arranged to eject an illumination light beam along an illumination path to illuminate the sample; an imaging objective arranged to receive detection light including at least a portion of the light ejected from the sample, wherein the detection light is propagated along a detection axis and the imaging objective has an imaging focal plane; an adjustment arrangement to linearly displace the illumination light beam and the imaging focal plane relative to each other along the detection axis; a sample holder arranged to receive a sample and having a portion which is transparent to the illumination light beam and to the detection light; and a holder support arranged to receive the sample holder and displace the sample holder relative to the imaging objective such that the imaging focal plane is positioned inside the sample holder.

A laser scanning microscope includes a light source configured to emit an illumination light beam. The illumination light beam has a transverse light intensity profile comprising an intensity minimum. The laser scanning microscope further includes a scanning device configured to scan the illumination light beam along a closed trajectory in a target area of a specimen, and a detector configured to detect fluorescence light emitted by a fluorophore within the target area of the specimen. The fluorophore is excited by the illumination light beam. The laser scanning microscope further includes a processor configured to determine an intensity distribution of the fluorescence light as a function of time and to determine a position of the fluorophore within the target area based on the intensity distribution of the fluorescence light.

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 of scanning a laser over a field of view, the method comprising: providing a laser to produce the laser beam; rasterizing the laser beam over a first sub-area of the field of view; deflecting the laser beam to a second sub-area of the field of view; and rasterizing the laser beam over the second sub-area of the field of view; and capturing image information produced by the laser beam so that, for each sub-area of the field of view, the rasterized laser beam defines a plurality of image segments; for each segment calculating an image correction and applying a correction to the laser according to the calculated image correction for the segment, and corresponding system.

A system for illuminating a microscopy specimen includes an illumination source configured to emit a light that travels along an illumination path to illuminate the microscopy specimen placed on an optical detection path of an optical microscope. The system also includes optical elements in the illumination path and configured to at least in part transform the light from the illumination source into a light sheet illuminating the microscopy specimen. The optical elements include an electronically tunable lens configured to vary a focal distance of the electronically tunable lens to dynamically vary a position of a waist of the light sheet illuminating the microscopy specimen. The optical elements include a deflector configured to vertically move the light sheet to illuminate the microscopy specimen at different horizontal planes.

A microscopy imaging system comprises a fluorescence lifetime imaging microscopy (FLIM) system comprising a pulsed light source configured to direct a plurality of excitation light pulses onto a sample, a photo detector configured to detect emitted fluorescent photons created by the plurality of excitation pulses interacting with the sample, and a FLIM data acquisition system configured to measure the time interval between the excitation light pulses and the detected emitted fluorescent photons, a scanning light microscopy (SLM) system comprising a SLM data acquisition system, a fast scanning mirror and a slow scanning mirror, wherein the mirrors are configured to scan the light pulses across the sample; and a data processing system communicatively connected to the FLIM and SLM systems. Microscopy imaging methods are also disclosed.

An optical assembly in a laser scanning microscope, having an optical scanning unit providing a first pupil plane, a first beam deflecting device, made of a first scanner arranged on the first pupil plane, for scanning excitation radiation in a first coordinate direction, a first focusing device generating a second pupil plane, optically conjugated to the first pupil plane, and a second beam deflecting device for deflecting the excitation radiation. The second deflecting device is arranged on the second pupil plane. A second focusing device to generate a third pupil plane, is optically conjugated to the first pupil plane and the second pupil plane. A third beam deflecting device is arranged on the third pupil plane, and a variable beam deflecting device is provided to switch an optical beam path between a first beam path and a second beam path.

Systems, methods, and apparatus for differential phase contrast microscopy by transobjective differential epi-detection of forward scattered light are provided. In some embodiments, a microscope objective comprises: a housing with mounting threads at a second end; optical components defining an optical axis, comprising: an objective lens mounted at a first end, configured to collect light from a sample placed in a field of view, the plurality of optical components create a pupil plane at a first distance along the optical axis at which rays having the same angle of incidence on the objective lens converge at the same radial distance from the optical axis; a photodetector within the housing offset from the optical axis at a second distance along the optical axis; and another photodetector within the housing at second distance along the optical axis and offset from the optical axis in the opposite direction from the first photodetector.

A confocal microscope device for scanning a two-dimensional array of illumination beams over a target surface and scanning a corresponding two-dimensional array of emission beams stimulated by the array of illumination beams on to a sensor of an imaging device. The device comprises first scanning optics operable to scan the array of illumination beams over the target surface along a first axis and scan the array of emission beams over the sensor along the first axis. The device further comprises second scanning optics operable to deflect, on a second axis, the array of illumination beams as they are scanned over the target surface along the first axis, such that uneven stimulation of the target surface by the array of illumination beams due to interference of the illumination beams is reduced, and deflect, on the second axis, the array of emission beams as they are scanned over the sensor of the imaging device along the first axis such that uneven stimulation of the sensor by the array of emission beams due to interference of the emission beams is reduced.

Diffraction-based imaging systems are described. Aspects of the technology relate to imaging systems having one or more sensors inclined at angles with respect to a sample plane. In some cases, multiple sensors may be used that are, or are not, inclined at angles. The imaging systems may have no optical lenses and are capable of reconstructing microscopic images of large sample areas from diffraction patterns recorded by the one or more sensors. Some embodiments may reduce mechanical complexity of a diffraction-based imaging system. A diffractive imaging system comprises a light source, a sample support configured to hold a sample along a first plane, and a first sensor comprising a plurality of pixels disposed in a second plane that is tilted at an inclined angle relative to the first plane. The first sensor is arranged to record diffraction images of the light source from the sample.