Optical sensing techniques and devices based on detection of fluorescent emissions at different optical wavelengths by nonlinear optical absorption of different excitation beams at different excitation wavelengths that interact with fluorescently-labeled structures within the sample to cause nonlinear optical absorption of two or more photons at each excitation wavelength. The fluorescent light at different fluorescent emission wavelengths by nonlinear optical absorption of excitation light at a particular excitation wavelength is spectrally separated into different optical channel output beams along different optical channel optical paths at different designated fluorescent imaging wavelength bands and the fluorescent light at different fluorescent imaging wavelengths within each designated fluorescent imaging wavelength is detected. This two-stage spectral separation in obtaining fluorescent images at different fluorescent imaging wavelengths in different fluorescent imaging wavelength bands enables highly sensitive hyperspectral imaging based on two-photo or multi-photon nonlinear absorption.

A multi-photon imaging system includes a laser module having a first channel for outputting a two-photon excitation laser pulse and a second channel for outputting a three-photon excitation laser pulse. The system further includes a first optical path for guiding the two-photon laser pulse from the first channel of the laser module and a second optical path for guiding the three-photon laser pulse from the second channel of the laser module. A microscope is also provided for simultaneously receiving the two-photon laser pulse from the first optical path and the three-photon laser pulse from the second optical path, and simultaneously, or with well controllable delays, delivering the two-photon laser pulse and the three-photon pulse to a target volume. The system further includes a photodetector configured to collect photons generated within the target volume in response to simultaneous excitation of the target volume by both the two-photon laser pulse and the three-photon laser pulse.

A microscope having three-dimensional imaging capability and a three-dimensional microscopic imaging method are provided, the microscope including: at least one excitation device configured to generate a detectable contrast in a detection target region of a sample which is to be detected, in an excitation principal axis direction; at least one detection device, configured to detect the contrast as generated from the detection target region of the sample in a detection principal axis; and at least one movement mechanism, configured to generate a relative movement of the sample relative to the excitation device and the detection device; the relative movement is in a direction neither parallel to nor perpendicular to the excitation principal axis direction or the detection principal axis direction.

Devices and methods for recording dynamics of cellular and/or biochemical processes, including a device including one or more dispersive elements configured to receive a pulsed laser beam with a spectrum of different wavelengths and disperse the spectrum of the pulsed laser beam; and one or more first elements configured to receive the dispersed spectrum of the pulsed laser beam, and generate a multiphoton excitation area in a biological sample by re-overlapping in time and space the dispersed spectrum of the pulsed laser beam on an area in the biological sample, wherein the device is configured to record at high speed changes of cellular and biochemical processes of a population of cells of the biological sample based on generation of the multiphoton excitation area in the biological sample.

A microscope directs light through an excitation objective to generate a lattice light sheet (LLS) within a sample. A detection objective collects signal light from the sample in response to the LLS and images the collected light onto a detector. Second and third light beams are imaged onto focal planes of the excitation objective and detection objective, respectively. One or more wavefront detectors determine wavefronts of light emitted from the sample and through the excitation objective in response to the imaged second light beam and emitted from the sample through the detection objective in response to the imaged third light beam. A wavefront of the first light beam is modified to reduce a sample-induced aberration of the LLS within the sample, and a wavefront of the signal light emitted from the sample is modified to reduce a sample-induced aberration of the signal light at the detector.

A microscope system including: a source of light; a sample objective configured for focusing the light at a focal plane within a sample; a remote focus unit configured for changing a position of the focal plane along an axis perpendicular to the focal plane; one or more optical element configured for directing the focused light to a location within the focal plane; and a detector configured for detecting light emitted from the focal plane within the sample; wherein the one or more optical element is located after the remote focus unit along a beam path of the light from the source to the sample objective, such that the changing the position of the focal plane along the axis is performed before the directing the focused light to the location within the focal plane.

A pulsed light generation apparatus includes a dispersing unit for dispersing pulsed light for respective wavelengths, a polarization dependent type spatial light modulator for modulating the dispersed pulsed light in respective wavelengths, and a combining unit for combining wavelength components of the pulsed light output from the spatial light modulator. A polarization plane of the pulsed light input to the spatial light modulator is inclined with respect to a polarization direction in which the spatial light modulator has a modulation function. The spatial light modulator causes a time difference between a first polarization component of the pulsed light along the polarization direction and a second polarization component of the pulsed light intersecting with the first polarization component.

A system for displaying one or more images in three dimensions. The system has a three dimensional illumination volume containing a gas that emits one or more types of visible light when at certain multi-photon excited states. The system includes lasers (e.g. lasers with beams outside of the visible wavelengths) that can be directed to intersect in the illumination volume to excite particles of the gas to a multi-photon excited state to emit visible light. Scanning the beam intersection (or multiple beam intersections) through the illumination volume generates three dimensional images.

A microscope directs light through an excitation objective to generate a lattice light sheet (LLS) within a sample. A detection objective collects signal light from the sample in response to the LLS and images the collected light onto a detector. Second and third light beams are imaged onto focal planes of the excitation objective and detection objective, respectively. One or more wavefront detectors determine wavefronts of light emitted from the sample and through the excitation objective in response to the imaged second light beam and emitted from the sample through the detection objective in response to the imaged third light beam. A wavefront of the first light beam is modified to reduce a sample-induced aberration of the LLS within the sample, and a wavefront of the signal light emitted from the sample is modified to reduce a sample-induced aberration of the signal light at the detector.

A microscope system including: a source of light; a sample objective configured for focusing the light at a focal plane within a sample; a remote focus unit configured for changing a position of the focal plane along an axis perpendicular to the focal plane; one or more optical element configured for directing the focused light to a location within the focal plane; and a detector configured for detecting light emitted from the focal plane within the sample; wherein the one or more optical element is located after the remote focus unit along a beam path of the light from the source to the sample objective, such that the changing the position of the focal plane along the axis is performed before the directing the focused light to the location within the focal plane.