Systems, methods, and apparatus for in vitro single-cell identification and recovery
Described herein are systems, methods, and apparatus for automatically identifying and recovering individual cells of interest from a sample of biological matter, e.g., a biological fluid. Also described are methods of enriching a cell type of interest. These systems, methods, and apparatus allow for coordinated performance of two or more of the following, e.g., all with the same device, thereby enabling high throughput: cell enrichment, cell identification, and individual cell recovery for further analysis (e.g., sequencing) of individual recovered cells.
1. A system for hardware triggering of light sources for image acquisition, the system comprising;
a computer with a processor programmed to:
transmit spatial positions to a memory of a stage, and filter positions to a memory of a filter wheel;
transmit one or other parameters of acquisition to a microcontroller,
wherein the microcontroller is operable to start a cycle of image acquisition without interaction with the computer by signaling the stage to move to a next position stored in its memory,
wherein the stage moves to the stored position and signals the microcontroller upon completion of the move, and
wherein the microcontroller signals the filter wheel to move to the next position stored in its memory, after which the filter wheel moves to the stored position and signals the microcontroller upon completion of the move, upon which the microcontroller signals at least one or more light sources to turn on, then signals a detector to begin integration of light, and upon completion of exposure time, the microcontroller is operable to signal the at least one or more light sources to turn off, after which the microcontroller signals the detector to stop its integration, and the detector automatically transfers an accumulated image to a frame grabber on the computer, after which the microcontroller is operable to start the next cycle of image acquisition.
2. The system of claim 1 , the system comprising an optical train comprising a demagnification lens.
3. The system of claim 2 , wherein the demagnification lens optimizes resolution for cytometry and increase imaging speed.
4. The system of claim 1 , wherein the system is capable of performing an imaging run with at least 12 channels in a total time less than 150 minutes, where the total imaged area is at least 1000 mm 2 .
5. The system of claim 1 , wherein the system is a multicolor cytometry system.
6. The system of claim 1 , wherein the one or more parameters comprises one or more members selected from the group consisting of a number of positions and/or spectral channels to be acquired, one or more light source(s), exposure times, and filter wheel movements set for each channel.
7. The system of claim 1 , wherein the microcontroller is operable to start a next cycle of image acquisition by signaling movement of the stage to the next position.
8. A method for hardware triggering of light sources for image acquisition, the method comprising:
transmitting, by a processor of a computing device, spatial positions to a memory of a stage, and filter positions to a memory of a filter wheel;
transmitting, by the processor, one or other parameters of acquisition to a microcontroller;
starting, by the microcontroller, a cycle of image acquisition without interaction with the computer device by signaling the stage to move to a next position stored in its memory, wherein the stage moves to the stored position and signals the microcontroller upon completion of the move;
signaling, by the microcontroller, the filter wheel to move to the next position stored in its memory, after which the filter wheel moves to the stored position and signals the microcontroller upon completion of the move;
signaling, by the microcontroller, at least one or more light sources to turn on, then signaling a detector to begin integration of light, and upon completion of exposure time, signaling, by the microcontroller, the at least one or more light sources to turn off;
signaling, by the microcontroller, the detector to stop its integration;
automatically transferring an accumulated image from the detector to a frame grabber;
and
beginning, by the microcontroller, a subsequent cycle of image acquisition.
9. The method of claim 8 , wherein the one or more parameters comprises one or more members selected from the group consisting of a number of positions and/or spectral channels to be acquired, one or more light source(s), exposure times, and filter wheel movements set for each channel.