SYSTEMS, METHODS, AND APPARATUS FOR IN VITRO SINGLE-CELL IDENTIFICATION AND RECOVERY

• US 20160209319A1
• Filed: 01/15/2016
• Published: 07/21/2016
• Est. Priority Date: 01/15/2015
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Adalsteinsson, Viktor A., Stockdale, Alan, Loginov, Denis, Love, J. Christopher, Gierahn, Todd
• Granted Patent: US 10,078,778 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0851 Bottom walls
• CPC Class Codes: B01L 2300/0896 Nanoscaled
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 3/50 Containers for the purpose of retaining a material to be analysed, e.g. test tubes devices for taking samples of blood A61B5/15
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: G01B 21/08 for measuring thickness
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1425 using an analyser being characterised by its control arrangement
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2035/00148 Test cards, e.g. Biomerieux or McDonnel multiwell test cards
• CPC Class Codes: G01N 33/4833 of solid biological material, e.g. tissue samples, cell cultures tissue in vivo A61B5/00; cell suspensions G01N33/48735
• CPC Class Codes: G01N 35/00029 provided with flat sample substrates, e.g. slides G01N35/028 takes precedence
• CPC Class Codes: G01N 35/00871 Communications between instruments or with remote terminals
• CPC Class Codes: G02B 21/006 focusing arrangements; selection of the plane to be imaged
• CPC Class Codes: G02B 21/008 Details of detection or image processing, including general computer control
• CPC Class Codes: G02B 21/06 Means for illuminating specimens
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/26 : Stages; Adjusting means therefor
• CPC Class Codes: G06V 20/695 : Preprocessing, e.g. image segmentation
• CPC Class Codes: G06V 20/698 : Matching; Classification
• CPC Class Codes: H04N 23/56 : provided with illuminating means

Abstract

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.

Claims

1. A multiscale deposition-well plate comprising:

one or more sample wells; and

a plurality of macro-scale wells, wherein each macro-scale well with any one or more of length, width, and/or depth of at least 1 mm,

wherein each of the macro-scale wells comprise a plurality of micro-scale and/or nano-scale wells, wherein each micro-scale well with any one or more of length, width, and/or depth of at least 1 μ

m,

wherein each nano-scale well with any one or more of length, width, and/or depth of at least 1 nm, and

wherein each micro-scale well contains no greater than about 1000 pL of sample volume.

3. The multiscale deposition-well plate of claim 1, wherein each of the wells has a sample-contacting surface compatible with live cells, the sample-contacting surface comprising one or more members selected from the group consisting of glass, silicon, polymer, elastomer, a thermoplastic, and a medical grade plastic.

49. The multiscale deposition-well plate of claim 1, wherein each macro-scale well has a dimension (e.g., length, width, and/or depth) of at least 3 mm, and wherein each micro-scale well has a dimension (e.g., length, width, and/or depth) of no greater than about 100 μ

m.

50. The multiscale deposition-well plate of claim 1, wherein each micro-scale well contains no greater than about 500 pL of sample volume.

51. The multiscale deposition-well plate of claim 1, wherein the one or more sample wells comprise from three to twenty sample wells.

52. The multiscale deposition-well plate of claim 1, further comprising a plurality of recovery wells.

2. (canceled)

4. A deposition well-plate comprising one or more sample wells and one or more recovery wells, wherein the sample well(s) and recovery well(s) are positioned in proximity to each other.

5. The deposition well-plate of claim 4, wherein at least one of the sample well(s) and/or at least one of the recovery well(s) is a multiscale deposition well.

6. The deposition well-plate of claim 4, further comprising one or more wash stations.

7. The deposition well-plate of claim 4, wherein at least one of the sample wells comprises tapered walls.

53. The deposition well-plate of claim 4, wherein each sample well is within 50 mm, of the nearest recovery well.

8-10. -10. (canceled)

11. 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 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 the light source(s) to turn it/them on, then signals a detector to begin integration of light, and upon completion of exposure time, the microcontroller is operable to signal the light source(s) to turn them 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.

12. The system of claim 11, the system comprising an optical train comprising a demagnification lens.

17. The system of claim 11, 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 mm².

54. The system of claim 11, wherein the system is a multicolor cytometry system.

55. The system of claim 11, 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.

56. The system of claim 11, wherein the microcontroller is operable to start a next cycle of image acquisition by signaling movement of the stage to the next position.

57. The system of claim 12, wherein the demagnification lens optimizes resolution for cytometry and increase imaging speed.

13. A system for automated identification and/or recovery of individual cells of interest, the system comprising a microscope comprising a light source, an optical train, and a detector capable of imaging a deposition-well plate positioned on a motorized stage.

16. The system of claim 13, further comprising elements for hardware triggering of light sources.

58. The system of claim 13, wherein the system images in one or more fluorescent channels.

59. The system of claim 58, wherein the system images in from 10 to 40 fluorescent channels.

14-15. -15. (canceled)

18-20. -20. (canceled)

21. 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 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, the light source(s) to turn it/them on, then signaling a detector to begin integration of light, and upon completion of exposure time, signaling, by the microcontroller, the light source(s) to turn them 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.

60. The method of claim 21, 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.

22-48. -48. (canceled)