Abstract: A microfluidic flow cell subassembly, which may be assembled into a flow cell having fluidic connections outside of the main substrate, is described for encapsulating a sample to allow for subsequent controlled delivery of reagents to the sample, such as multiplexed in situ biomarker staining and analysis. Methods for fabricating the subassembly and assembled flow cell are also provided. The method includes the steps of adhering a gasket layer to a substrate layer, wherein the gasket layer may contain enclosed features and adhering an adherent layer to the gasket layer. The subassembly may be sealed against a solid support to form a flow cell.

Abstract: Methods for detecting multiple targets in a biological sample are provided. The methods includes contacting the sample with a first probe; physically binding the first probe to a first target; observing a first signal from the first probe; applying a chemical agent to modify the first signal; contacting the sample with a second probe; physically binding the second probe to a second target; and observing a second signal from the second probe. The methods disclosed herein also provide for multiple iterations of binding, observing, signal modification for deriving information about multiple targets in a single sample. An associated kit and device are also provided.

Abstract: A method is provided for scaling an image taken at a given exposure time to a selected exposure time. The method generally comprises: determining a dark pixel intensity of an imaging device; acquiring a first image at a given exposure time; and adjusting a pixel intensity of one or more pixels in the first image, based at least in part on the dark pixel intensity, for a second exposure time that is different from the given exposure time.

Abstract: A closed loop automated method for staining of a biological sample is provided. The method comprises providing a biological sample, staining at least a portion of the biological sample by flowing in a reagent, monitoring one or more optical characteristics of the biological sample, and calculating a figure of merit based on at least one of the optical characteristics. An automated device for iterative staining of a biological sample is also provided.

Abstract: An open top microfluidic device comprising a microfluidic slide carrier and one or more multiplexing stations is provided which allows sequential staining and imaging without the need for using or removing a coverslip on a mounted biological sample.

Abstract: Methods for detecting multiple targets in a biological sample are provided. The methods includes contacting the sample with a first probe; physically binding the first probe to a first target; observing a first signal from the first probe; applying a chemical agent to modify the first signal; contacting the sample with a second probe; physically binding the second probe to a second target; and observing a second signal from the second probe. The methods disclosed herein also provide for multiple iterations of binding, observing, signal modification for deriving information about multiple targets in a single sample. An associated kit and device are also provided.

Abstract: A method for processing and imaging a first and second plurality of samples, comprising processing at least one sample from the first plurality of samples, imaging the at least one sample from the first plurality of samples, while being capable of simultaneously processing at least one sample from the second plurality of samples; and imaging the at least one processed sample from the second plurality of samples.

Abstract: Some systems described herein include a frequency dependent phase plate for generating multiple phase-contrast images of a sample, each from a different frequency range of light, each phase-contrast image for frequency range of light formed from light diffracted by the sample interfered with undiffracted light that has a frequency-dependent baseline relative phase shift from the phase plate. In some embodiments, the multiple phase-contrast images may be used to generate a quantitative phase image of a sample. The phase-contrast images or the produced quantitative phase image may have sufficient contrast for label-free auto-segmentation of cell bodies and nuclei.

Abstract: A screening module configured to screen at least a portion of a biological sample disposed on an analysis surface is provided. The screening module comprises a laser source a scanning unit comprising one or more scanning devices, wherein the scanning devices are configured to rotate in an oscillatory scanning motion about an axis of rotation to scan the analysis surface in at least one direction, wherein the scanning unit is physically coupled to the laser source, and a detection unit comprising one or more detection devices.

Abstract: A microfluidic flow cell subassembly, which may be assembled into a flow cell having fluidic connections outside of the main substrate, is described for encapsulating a sample to allow for subsequent controlled delivery of reagents to the sample, such as multiplexed in situ biomarker staining and analysis. Methods for fabricating the subassembly and assembled flow cell are also provided. The method includes the steps of adhering a gasket layer to a substrate layer, wherein the gasket layer may contain enclosed features and adhering an adherent layer to the gasket layer. The subassembly may be sealed against a solid support to form a flow cell.

Abstract: A method for the imaging of protein expression and location in biological samples using optical segmentation is provided. The steps comprise acquiring a fluorescent image of a biological sample, analyzing the image and generating a masking pattern corresponding to a specific structure within the biological sample, transforming the masking pattern into the spatial coordinates of a digital micro-mirror device (DMD) which may then be projected onto the biological sample and obtaining a masked fluorescent image. Also provided is an image analysis system for imaging of protein expression and location in biological samples using optical segmentation.

Abstract: Imaging systems and methods for generating images of a sample wherein the system comprises an illumination source for illuminating the sample; an image viewing subsystem for capturing images from the sample; one or more liquid crystal panels and one or more birefringent elements, which are positioned between the sample and the image viewing subsystem, so that the images from the sample pass through the liquid crystal panels and the birefringent elements before reaching the image viewing subsystem; a device that changes one or more polarization states of the liquid crystal panels; and a controller that is configured to cause the device to change the polarization states of the liquid crystal panels.

Abstract: Systems and methods described herein employ multiple phase-contrast images with various relative phase shifts between light diffracted by a sample and light not diffracted by the sample to produce a quantitative phase image. The produced quantitative phase image may have sufficient contrast for label-free auto-segmentation of cell bodies and nuclei.

Abstract: Methods for detecting multiple targets in a biological sample are provided. The methods includes contacting the sample with a first probe; physically binding the first probe to a first target; observing a first signal from the first probe; applying a chemical agent to modify the first signal; contacting the sample with a second probe; physically binding the second probe to a second target; and observing a second signal from the second probe. The methods disclosed herein also provide for multiple iterations of binding, observing, signal modification for deriving information about multiple targets in a single sample. An associated kit and device are also provided.

Abstract: Methods for scaling an image taken an optimal exposure time to a selected exposure time, generally comprising, determining a dark pixel intensity of an imaging device; acquiring a first image at an optimal exposure time; and adjusting a pixel intensity of one or more pixels in the first image, based at least in part on the dark pixel intensity, for a second exposure time that is different from the optimal exposure time.

Abstract: Automated methods and devices that facilitate iterative staining of biological samples from imaging applications are provided. The methods include the steps of providing a small volume flow cell containing a biological sample, applying a stain to the biological sample, combining at least two precursor reagents to form an activated destaining agent and wherein the activated destaining agent decomposition rate is greater than or similar to the destaining reaction rate, flowing the destaining agent over the biological sample at a flow rate that is greater than the decomposition rate of the activated destaining agent, and releasing the sample from the flow cell wherein the integrity of the sample is intact. The process of staining, combining and flowing may be iteratively repeated.

Abstract: A closed loop automated method for staining of a biological sample is provided. The method comprises providing a biological sample, staining at least a portion of the biological sample by flowing in a reagent, monitoring one or more optical characteristics of the biological sample, and calculating a figure of merit based on at least one of the optical characteristics. An automated device for iterative staining of a biological sample is also provided.

Abstract: A method of autofocusing includes capturing first, second and third images of a sample, at respective first, second and third sample distances and respective first, second and third lateral positions determined with respect to an objective; determining a quantitative characteristic for the first, second and third images; determining a primary sample distance based upon at least the quantitative characteristics for the first, second, and third images; and capturing a primary image of the sample at the primary sample distance and at a primary lateral position that is offset from the first, second and third lateral positions.

Abstract: Imaging systems and methods for generating images of a sample wherein the system comprises an illumination source for illuminating the sample; an image viewing subsystem for capturing images from the sample; one or more liquid crystal panels and one or more birefringent elements, which are positioned between the sample and the image viewing subsystem, so that the images from the sample pass through the liquid crystal panels and the birefringent elements before reaching the image viewing subsystem; a device that changes one or more polarization states of the liquid crystal panels; and a controller that is configured to cause the device to change the polarization states of the liquid crystal panels.

Abstract: The invention provides method for locating one or more substantially circular-shaped tissue sample positioned on a solid support. The method involves the steps of transmitting light of a preselected wavelength onto a tissue sample, wherein the light induces the tissue sample to autofluoresce, identifying the center location of the tissue sample using the autofluoresced light, correlating the coordinates of the center location of the tissue sample on the solid support using an x, y-coordinate system, and mapping the coordinates of the tissue sample on the solid support to differentiate tissue sample containing regions from blank regions on the solid support. In a second aspect, the invention provides an apparatus for locating one or more substantially circular-shaped tissue sample positioned on a solid support.