Systems and methods for passive multi-directional illumination in light-sheet fluorescence imaging and microscopy are disclosed herein. An elliptical light shaping diffuser is placed in the illumination path between the source of a light-sheet and the illuminated sample. The light-sheet is diffused anisotropically along two directions perpendicular to its propagation direction, eliminating stripe artifacts in obtained images. The method includes converting a light-sheet into an elliptically diffuse light-sheet by passing it through an elliptical light shaping diffuser, illuminating a sample with the elliptically diffuse light-sheet. The system includes a light-sheet source, an elliptical light shaping diffuser adapted to convert the light-sheet into an elliptically diffuse light-sheet to illuminate the sample, typical microscopy optics and lenses, and image capturing elements.

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.

Some implementations of the disclosure relate to an imaging system, including: a sample holder to support a sample container having multiple sample locations; an optical stage having; an assembly comprising one or more actuators physically coupled to the sample holder to tilt the sample holder relative to the optical stage during imaging of the multiple sample locations to focus the optical stage onto a current sample location; a first light source to project a first pair of spots on the sample container; and a controller to control, based on a sample tilt determined from a first separation measurement of the first pair of spots from one or more images taken by an image sensor at one or more of the sample locations, the one or more actuators to tilt the sample holder along a first direction of the imaging or a second direction substantially perpendicular to the first direction.

Systems and methods herein provide improved, high-throughput multiphoton imaging of thick samples with reduced emission scattering. The systems and methods use structured illumination to modify the excitation light. A reconstruction process can be applied to the resulting images to recover image information free of scattering. The disclosed systems and methods provide high throughput, high signal-to-noise ratio, and high resolution images that are depth selective.

A method for measuring a position of a fluorophore includes configuring a set of compact light distributions, the set having at least one member, each light distribution characterized by a center, so that there is substantially zero intensity at the center of the set of compact light distributions. The method additionally includes moving the set of compact light distributions in relation to a set of hypothesized positions of the fluorophore, detecting, in a plurality of locations corresponding to the hypothesized set of positions, a set of images; and estimating the position of the fluorophore, by determining from the set of images a set of parameters describing the position of the fluorophore using an inverse problem method.

A lighting device for an imaging optical device such as a microscope is provided. The lighting device illuminates an object to be analyzed in an imaging optical device for microscopic analysis in at least two different contrasting techniques. The lighting device has light sources for the illumination, where the light sources are associated with a contrasting technique are controllable independently from each other.

The disclosure provides a method of generating an artificial fluorescent image of cells is provided. The method includes receiving a brightfield image generated by a brightfield microscopy imaging modality of at least a portion of cells included in a specimen, applying, to the brightfield image, at least one trained model, the trained model being trained to generate the artificial fluorescent image based on the brightfield image, receiving the artificial fluorescent image from the trained model

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.

A light source unit includes: a housing; a semiconductor laser that is disposed in the housing and that radiates excitation light; a first condenser optical system that condenses the excitation light; a dichroic mirror that selectively reflects the excitation light; a second condenser optical system that condenses the excitation light; a wavelength conversion member that performs wavelength conversion of the excitation light and emits wavelength-converted light; an emission section that outputs the wavelength-converted light transmitted through the second condenser optical system and the dichroic mirror; and a light blocking section that is disposed between an inner surface of the housing, the inner surface being in a traveling direction of the excitation light toward a reflection surface of the dichroic mirror, and a back surface, the back surface being an opposite side of the reflection surface, or is disposed on the inner surface of the housing.