This disclosure provides methods and apparatuses for sorting target particles. In various embodiments, the disclosure provides a cassette for sorting target particles, methods for sorting target particles, methods of loading a microchannel for maintaining sample material viability, methods of quantifying sample material, and an optical apparatus for laser scanning and particle sorting.

Biological research requires isolation and analysis of material, for example, RNA, DNA and protein, from tissue samples. The methods and compositions described herein allow for high resolution imaging of large and intact tissue samples, and subsequent isolation of material in a precise and location dependent-manner. The methods and compositions described herein may be used, for example, for biomarker discovery, identification of cell populations, pathology analysis, and generation of expression data in specific regions of interest.

Particulate sensing systems or processes identify particulates suspended in an air sample by irradiating the air sample with UV light and measuring light from individual particles in the air sample. Two photodiodes having different wavelength sensitivity may be used to measure the fluorescent light emitted from a single particle, and a type of the particle may be identified using outputs from photodiodes. Repeating the process for multiple particles may produces distributions that further distinguish or identify particulate types.

Provided herein are devices, systems, and methods for characterizing a biological sample in vivo or ex vivo in real-time using time-resolved spectroscopy. A light source generates a light pulse or continuous light wave and excites the biological sample, inducing a responsive fluorescent signal. A demultiplexer splits the signal into spectral bands and a time delay is applied to the spectral bands so as to capture data with a detector from multiple spectral bands from a single excitation pulse. The biological sample is characterized by analyzing the fluorescence intensity magnitude and/or decay of the spectral bands. The sample may comprise one or more exogenous or endogenous fluorophore. The device may be a two-piece probe with a detachable, disposable distal end. The systems may combine fluorescence spectroscopy with other optical spectroscopy or imaging modalities. The light pulse may be focused at a single focal point or scanned or patterned across an area.

Systems and methods include a fluorescence measurement apparatus. A single-wavelength light source generates an excitation light source. A sample holder holds a sample and includes a surface transparent to the excitation light source. Mounts attached to the single-wavelength light source(s) or the sample holder change an incident angle of the excitation light source on the surface. Optical components positioned in a path of a fluorescence emission emitted from the surface guide the fluorescence emission to a detector that obtains spectra from at least first and second angles-of-incidence. A device records spectra obtained by the detector from the first and second angles-of-incidence, normalizes and analyzes intensities of the spectra, subtracts a first spectrum corresponding to the first angle-of-incidence from a second spectrum corresponding to the second angle-of-incidence to obtain a difference, identifying a sample type of the sample based on an API gravity mapped to the difference.

In one aspect, a method of sorting cells in a flow cytometry system is disclosed, which includes illuminating a cell with radiation having at least two optical frequencies shifted from one another by a radiofrequency to elicit fluorescent radiation from the cell, detecting the fluorescent radiation to generate temporal fluorescence data, and processing the temporal fluorescence data to arrive at a sorting decision regarding the cell without generating an image (i.e., a pixel-by-pixel image) of the cell based on the fluorescence data. In other words, while the fluorescence data can contain image data that would allow generating a pixel-by-pixel fluorescence intensity map, the method arrives at the sorting decision without generating such a map. In some cases, the sorting decision can be made with a latency less than about 100 microseconds. In some embodiments, the above method of sorting cells can have a sub-cellular resolution, e.g., the sorting decision can be based on characteristics of a component of the cell. In some embodiments in which more than two frequency-shifted optical frequencies are employed, a single radiofrequency shift is employed to separate the optical frequencies while in other such embodiments a plurality of different radiofrequency shifts are employed.

Apparatus include divergence optics removably coupled to receive a probe beam in a first imaging mode to cause the probe beam to diverge before impinging on a first area of a target surface, and to not receive the probe beam in a second imaging mode to cause the probe beam to impinge on a second area of the target surface smaller than the first area, collection optics configured to receive, in response to the probe beam, luminescence light emitted from the first area and spectral light emitted from the second area, and an optical detector coupled to the collection optics, wherein the optical detector includes a luminescence imaging detector portion and a spectral imaging detector portion adjacent to the luminescence imaging detector portion, wherein the luminescence imaging detector portion is configured to receive the luminescence light emitted from the first area and the spectral imaging detector portion is configured to receive the spectral light from the second area.

A particle analysis system comprising: a light detector that acquires light generated by irradiating a particle with excitation light; and an information processing unit that outputs a spectral plot including spectrum information of an autofluorescence population specified in a two-dimensional plot of measurement data each of which corresponds to the acquired light and spectrum information of the measurement data and that records the spectrum information of the autofluorescence population as an autofluorescence reference spectrum in a fluorescence separation process.

A system (100) and a method for detecting fluorescence is disclosed. The system (100) essentially comprises a labelled sample wherein said labelled sample emits an electromagnetic radiation of a defined wavelength when irradiated by a LASER beam of a commensurate wavelength, a source (102) for emitting said LASER beam, oriented as to aim at said labelled sample, a chamber for holding said labelled sample during said LASER irradiation, a reflective layer (108) positioned to reflect said electromagnetic radiation, and a detector (112) positioned to detect and amplify said electromagnetic radiation. The method essentially comprises the steps of providing a labelled sample wherein said labelled sample emits an electromagnetic radiation of a defined wavelength when irradiated by a LASER beam of a commensurate wavelength, providing a source (102) for emitting said LASER beam, oriented as to aim at said labelled sample, providing a chamber for holding said labelled sample during said LASER irradiation, providing a reflective layer (108) positioned to reflect said electromagnetic radiation, providing a detector (112) positioned to detect and amplify said electromagnetic radiation, irradiating said sample with said LASER beam and analyzing said amplified electromagnetic radiation from said detector (112) with a signal processing block (114).

In a right-handed XYZ coordinate system, a dichroic-mirror array of the present disclosure includes a first group in which m (m≥2) dichroic mirrors DA1 to DAm are arranged parallel to each other along a positive direction of an X axis and a second group in which n (n≥2) dichroic mirrors DB1 to DBn are arranged parallel to each other along a negative direction of the X axis. Incident surfaces of the DA1 to DAm and incident surfaces of the DB1 to DBn are perpendicular to an XZ plane. A slope of straight lines with normal lines of the incident surfaces of the DA1 to DAm projected onto the XZ plane are negative, and a slope of straight lines with normal lines of incident surfaces of DB1 to DBn projected onto the XZ plane are positive.