Methods and systems for three-dimensional lightsheet imaging
AssigneeEnumerix, Inc.
InventorsJanice Lai, David Stern
Life of this patent
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Abstract
Disclosed herein are light sheet imaging systems for imaging fluorescent samples. Also disclosed herein are sample holder systems for high throughput light sheet imaging of multiple three-dimensional samples without user intervention. Further disclosed herein are automated image processing methods to identify and quantify fluorescent particles within three-dimensional image sets without user intervention or user bias.
Claims19
A light sheet imaging system comprising:
- a plurality of containers comprising a plurality of samples, wherein each sample of the plurality of samples comprises a distribution of fluorescent particles and is contained within a container of the plurality of containers;
- a collimated illumination source configured to emit a collimated beam along a beam path;
- a Powell lens positioned in the beam path after the collimated illumination source and configured to expand the collimated beam along a single axis;
- a first cylindrical lens positioned in the beam path after the Powell lens and configured to re-collimate the beam along a single axis, thereby producing a collimated beam having a short axis orthogonal to a direction of propagation and long axis orthogonal to the short axis and the direction of propagation;
- and
- a second cylindrical lens positioned in the beam path after the first cylindrical lens and configured to focus the beam along the short axis to a focal plane;
- wherein the light sheet imaging system is configured to scan a set of cross-sections of each sample of the plurality of samples within each container of the plurality of containers.
A method comprising:
- generating a count of signal positive particles of a distribution of fluorescent particles, upon:
- collecting a three-dimensional image data set comprising a plurality of cross-section images of a container containing the distribution of fluorescent particles, the plurality of cross-section images generated upon scanning the container with a light sheet at a frequency from 0.1 to 20 kilohertz;
- identifying a plurality of substantially spherical fluorescent particles in the three-dimensional image data set;
- dividing one or more cross-section images of the plurality of cross-section images into a plurality of image grids;
- applying an intensity threshold independently to one or more image grids of the plurality of image grids;
- and
- identifying one or more signal positive particles from the plurality of substantially spherical fluorescent particles, wherein the one or more signal positive particles have intensities greater than the intensity threshold.
A method of analyzing a plurality of samples, the plurality of samples comprising at least 24 samples each comprising a distribution of fluorescent particles, the method comprising automatically scanning a set of cross-sections of each sample of the plurality of samples with a laser beam, thereby generating emission light in each sample of the plurality of samples, wherein the laser beam is a sheet of laser light.
Dependencies are inferred from the claim text.