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.

A method and an apparatus for imaging a sample (14). In the method, a first excitation radiation (5) is focused into a volume of the sample (14) and a caused first detection radiation (15) is captured and evaluated in respect of a form of its wavefront. A second excitation radiation (11) is manipulated on the basis of the evaluation results in order to correct the ascertained deviations of the wavefront. A region (20) to be imaged of the sample (14) is scanned by means of the second excitation radiation (11) and a second detection radiation (16) is captured as image data. The second excitation radiation (11) is directed in the form of at least two partial beams (11T) into the sample volume, into a respective spot (22) illuminated by the partial beam (11T) and the second detection radiations (16) respectively caused by the partial beams (11T) are captured separately.

A microscopic imaging method for creating a microscopic sample image (1A) of a sample (1) comprises the steps of arranging the sample (1) on a sampling crystal (10); irradiating the sample (1) with excitation laser pulses (2, 3) and generating sample response pulses (4) with a sample response field as a result of an interaction of the excitation laser pulses (2, 3) with the sample (1); irradiating the sampling crystal (10) with probe laser pulses (5) being temporally synchronized with the excitation laser pulses (2, 3) and spatially overlapped with the sample response pulses (4) in the sampling crystal (10), wherein the probe laser pulses (5) have a shorter wavelength than the excitation laser pulses (2, 3); detecting the sample response field by electric-field sampling with the sampling crystal (10), using the sample response pulses (4) and the probe laser pulses (5); and calculating the sample image (1A) based on the detected sample response field, wherein the excitation laser pulses (2, 3) have a wavelength in a range from mid-infrared to visible light and the sample response pulses (4) are created by a coherent interaction process induced in the sample (1) and with a fixed phase relationship relative to the excitation laser pulses (2, 3), the sampling crystal (10) is a non-centrosymmetric crystal, the irradiating step is repeated at multiple sample points (1A), wherein at each sample point (1A) the irradiating steps are successively repeated with multiple temporal probe delays of the probe laser pulses (5) relative to the excitation laser pulses (2, 3), at each probe delay, a sum or difference frequency pulse (6) of a sample response pulse (4) and a probe laser pulse (5) is generated, and at each probe delay, a spectral interference pulse (7) is created by a spectral interference of the sum or difference frequency pulse (6) and the current probe laser pulse, the detecting step includes sensing a polarization state of the spectral interference pulse (7) by an ellipsometer device (40) at each probe delay, wherein the local sample response field at the sample point (1A) is derived from the polarization states sensed at all probe delays, and the sample image (1A) is calculated based on the sample response field detected at the sample points (1A). Furthermore, a microscopic imaging apparatus is described.

Provided are a miniature endoscopic probe and a multi-photon endoscopy including the same.

A non-linear optical pumping detection apparatus and a non-linear optical absorption cross-section measurement method, which can simultaneously measure degenerate and non-degenerate two-photon absorption cross-section spectra. The measurement process is automatic, efficient and fast. The working wavelength band is from 380 nm to near infrared 1064 nm, and the non-linear performance measurement of the super-continuous wide spectra can be realized. A zoom optical system with a larger entrance pupil diameter is adopted as a weak signal acquisition lens. So the weak signal can be effectively extracted from background noise. Meanwhile, the mean square root diameter of an on-axis image point of the zoom optical system is 100 to 150 microns, the divergence angle 2α of the on-axis image point is 30.6 degrees, which well match the optical fiber coupling condition, thereby improving the coupling efficiency of the space light coupling into the optical fiber, and greatly improving the measurement sensitivity.

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 light sheet imaging system, such as a light sheet microscope, comprises an illumination arrangement for generating a light sheet for three-photon excitation of a fluorescent sample, and a fluorescence collection arrangement for collecting fluorescence generated in the sample as a result of three-photon excitation by the light sheet. The light sheet may be a non-diffractive, propagation-invariant light sheet. The light sheet may be formed from and/or comprise a Bessel beam. A method of light sheet imaging comprises using a light sheet for three-photon excitation of a fluorescent sample, and collecting fluorescence generated in the sample as a result of three-photon excitation of the sample by the light sheet. Such a method may be used for light sheet microscopy.

Provided herein is a macroscope comprising an objective apparatus comprising a multifocal widefield optics comprising a plurality of optical components configured to focus on a plurality of planes. Also provided herein are methods for analyzing a three-dimensional specimen, the method comprising obtaining, via a macroscope, synchronous multifocal optical images of a plurality of planes of the three-dimensional specimen, wherein the macroscope comprises an objective apparatus comprising a multifocal widefield optics comprising a plurality of optical components configured to focus on a plurality of planes. The three-dimensional specimen can be a biological specimen, such as brain.

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.

Fluorescent compounds that have aggregation-induced emission (AIE) characteristics. The compounds can be utilized as lipid droplet (LD)-specific bio-probes in cell imaging, with high photostability and brightness. For example, the compounds can be used for specific two-photon LDs staining in live cells and deep-tissues at ultralow concentrations. The compounds exhibit a large Stokes shift (>110 nm), high solid fluorescence quantum yields (up to 0.30), a good two-photon absorption cross-section (45-100 GM at 860 nm), high biocompatibility, and good photostability.