A pulsed beam of NIR excitation light is projected into a sample (345) at an oblique angle and scanned by a scanning element through a volume in the sample. 2-photon excitation excites fluorescence within the sample. The fluorescence is imaged onto an intermediate image plane that remains stationary regardless of the orientation of the scanning element. The image is captured by a linear array of light detecting elements (392) or a linear portion of a rectangular array. At any given position of the scanning element, the linear array (or portion) images all depths simultaneously. A plurality of images are captured for each of a plurality of different orientations of the scanning element. The orientation of the scanning element is controlled to move in a two dimensional pattern, which causes the beam of excitation light to sweep out a three dimensional volume within the sample.

Method for determining the characteristics of a system for generating at least one pattern of light, the method comprising: a) providing a desired pattern of light, b) expressing the amplitude and the phase of the output pulse of the system as a function of the input laser pulse and in function of the characteristics of the system to obtain a calculated output pulse, the input laser pulse having a duration below or equal to 1 nanosecond, c) determining at least one characteristic of the system by minimizing a distance between the calculated output pulse and the desired output laser pulse.

An optical phantom produces a time-resolved diffuse reflectance spectrum and includes: a light source; a spatial light modulator; and an optical delay line including optical fibers of different length that produce different time-of-flight distributions, such that different time-of-flight distributions are combined and produce phantom light having the time-resolved diffuse reflectance spectrum.

A system, including a structured illumination stage to provide a spatially modulated imaging field is provided. The system further includes a spatial frequency modulation stage to adjust the frequency of the spatially modulated imaging field, a sample interface stage to direct the spatially modulated imaging field to a sample, and a sensor configured to receive a plurality of fluorescence emission signals from the sample. The system also includes a processor configured to reduce a sample scattering signal and to provide a fluorescence emission signal from a portion of the sample including the spatially modulated imaging field. A method for using the above system to form an image of the sample is also provided.

A super-resolution scanning confocal polarization contrast microscope is provided. The microscope has a laser light source (1), sample stage (10) for mounting a sample 6 and detector (8). A polarization controller (3) is used to set the polarization state of the light beam to any one of a defined set of different polarization states. A spatial light modulator (5) modulates the light beam in amplitude and/or phase to focus a sub-diffraction-limit central spot on the sample together with unwanted sidebands. A scanning confocal scheme is used with a pin hole 9 in front of the detector (8) so that only that portion of the light is detected which has comes from the central spot, while rejecting light that has been scattered by the sample from the sidebands. Polarization contrast images with sub-diffraction limit resolution can thus be acquired.

Methods are provided to identify spatially and spectrally multiplexed probes in a biological environment. Such probes are identified by the ordering and color of fluorophores of the probes. The devices and methods provided facilitate determination of the locations and colors of such fluorophores, such that a probe can be identified. In some embodiments, probes are identified by applying light from a target environment to a spatial light modulator that can be used to control the direction and magnitude of chromatic dispersion of the detected light; multiple images of the target, corresponding to multiple different spatial light modulator settings, can be deconvolved and used to determine the colors and locations of fluorophores. In some embodiments, light from a region of the target can be simultaneously imaged spatially and spectrally. Correlations between the spatial and spectral images over time can be used to determine the color of fluorophores in the target.

Methods, systems and kits are described herein for detecting the results of an assay. In particular, the methods, systems and devices of the present disclosure rely on a difference between the diffusion rates of a reporter molecule and an analyte of interest in order to quantify an amount of analyte in a microfluidic device. The analyte may be a secreted product of a biological micro-object.

A metrology system includes an illumination source to generate an illumination beam, a multi-channel spectral filter, a focusing element to direct illumination from the single optical column to a sample, and at least one detector to capture the illumination collected from the sample. The multi-channel spectral filter includes two or more filtering channels having two or more channel beam paths. The two or more filtering channels filter illumination propagating along the two or more channel beam paths based on two or more spectral transmissivity distributions. The multi-channel spectral filter further includes a channel selector to direct at least a portion of the illumination beam into at least one selected filtering channel to filter the illumination beam. The multi-channel spectral filter further includes at least one beam combiner to combine illumination from the two or more filtering channels to a single optical column.

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.

In an illumination system (12, 13) for a scatterometer, first and second spatial light modulators lie in a common plane and are formed by different portions of a single liquid crystal cell (260). Pre-polarizers (250) apply polarization to first and second radiation prior to the spatial light modulators. A first spatial light modulator (236-S) varies a polarization state of the first radiation in accordance with a first programmable pattern. Second spatial light modulator (236-P) varies a polarization state of the second radiation accordance with a second programmable pattern. A polarizing beam splitter (234) selectively transmits each of the spatially modulated first and second radiation to a common output path, depending on the polarization state of the radiation. In an embodiment, functions of pre-polarizers are performed by the polarizing beam splitter.