METHODS AND SYSTEMS FOR THREE-DIMENSIONAL LIGHTSHEET IMAGING
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.
1 - 64 . (canceled)
65 . A 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 respective container of the plurality of containers;
a collimated illumination source configured to emit a collimated beam along a beam path;
a set of optical elements positioned along the beam path and configured to elongate the collimated beam along an axis;
a reservoir comprising a set of transparent sides through which light from the beam path is configured to enter;
a tube holder configured to retain a subset of the plurality of containers in position within the reservoir; and
a translating stage configured to translate the tube holder along a translation axis through light from the beam path, wherein the system is configured to scan a set of cross-sections of each sample of the subset of the plurality of containers.
66 . The system of claim 65 , wherein the set of transparent sides comprises a first transparent side and a second transparent side connected to the first transparent side at a right angle.
67 . The system of claim 66 , wherein the translation axis is orientated at a 45° angle relative to the first transparent side and the second transparent side and parallel to the of the subset of the plurality of containers retained by the tube holder.
68 . The system of claim 65 , wherein the plurality of samples comprises at least 96 samples.
69 . The system of claim 65 , wherein the plurality of samples comprises at least 20 samples.
70 . The system of claim 65 , wherein the set of optical elements comprises:
a first cylindrical lens positioned in the beam path and configured to 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 collimated beam along the short axis to a focal plane.
71 . The system of claim 65 , wherein a sample of the plurality of samples comprises a three-dimensional sample containing a distribution of fluorescent molecules.
72 . The system of claim 65 , further comprising a detector configured to detect fluorescent light from each sample.
73 . The system of claim 65 , wherein the reservoir contains an index matching fluid.
74 . The system of claim 65 , wherein a sample of the plurality of samples comprises a distribution of protein molecules.
75 . A method comprising:
transmitting a plurality of containers comprising a plurality of samples through a beam path,
wherein each sample of the plurality of samples comprises a distribution of fluorescent particles and is contained within a respective container of the plurality of containers,
wherein the beam path is generated from a collimated illumination source configured to emit a collimated beam through a set of optical elements configured to elongate the collimated beam along an axis, and
wherein transmitting comprises retaining the plurality of containers within a tube holder positioned within a reservoir, the reservoir comprising a set of transparent sides through which light from the beam path is configured to enter;
producing a dataset upon detecting fluorescent light emitted from each of the plurality of samples; and
from the dataset, quantifying targets of the plurality of samples upon identifying a subset of fluorescent particles conforming to a shape, an intensity threshold condition, and a size range.
76 . The method of claim 75 , wherein a sample of the plurality of samples comprises a distribution of protein molecules.
77 . The method of claim 75 , wherein a sample of the plurality of samples comprises a three-dimensional sample containing a distribution of fluorescent molecules.
78 . The method of claim 75 , wherein identifying a subset of fluorescent particles comprises applying a machine learning algorithm with a set of semi-supervised labels comprising a signal intensity label and a polydispersity label.
79 . The method of claim 75 , wherein the set of optical elements comprises:
a first cylindrical lens positioned in the beam path and configured to 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 collimated beam along the short axis to a focal plane.
80 . The method of claim 75 , wherein the plurality of samples comprises at least 20 samples.
81 . The method of claim 75 , wherein transmitting comprises translating the tube holder along a translation axis through light from the beam, using a translation stage coupled to the tube holder.
82 . The method of claim 75 , wherein quantifying targets of the plurality of samples comprises identifying signal positive particles from the dataset, wherein the signal positive particles have intensities greater than the intensity threshold condition.
83 . The method of claim 75 , wherein quantifying targets comprises removing asymmetric particles from a plurality candidate particles represented in the dataset.
84 . The method of claim 75 , further comprising moving light from the collimated illumination source relative to a sample of the plurality of samples, with movements ranging from 0.1 kHz to 20 kHz.