HIGH-THROUGHPUT IMAGING PLATFORM

• US 20170336394A1
• Filed: 10/27/2015
• Published: 11/23/2017
• Est. Priority Date: 10/27/2014
• Status: Active Grant

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Ben-Yaker, Adela, Hegarty, Evan, Mondal, Sudip, Ghorashian, Navid, Gke, Sertan Kutal, Martin, Christopher
• Granted Patent: US 10,539,554 B2
• Time in Patent Office: Days
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/023 Sending and receiving of information, e.g. using bluetooth
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/14 Means for pressure control
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502723 characterised by venting arrangements
• CPC Class Codes: B01L 3/502746 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: G01N 2021/6482 Sample cells, cuvettes
• CPC Class Codes: G01N 2035/00148 Test cards, e.g. Biomerieux or McDonnel multiwell test cards
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 2201/12 Circuits of general importance; Signal processing
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5085 of invertebrates
• 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: G06V 20/693 : Acquisition
• CPC Class Codes: G06V 20/695 : Preprocessing, e.g. image segmentation

Abstract

A microfluidic device capable of trapping contents in a manner suitable for high-throughput imaging is described herein. The microfluidic device may include one or more trapping devices, with each trapping device having a plurality of trapping channels. The trapping channels may be configured to receive contents via an inlet channel that connects a sample reservoir to the trapping channels via fluid communication. The trapping channels are shaped such that contents within the trapping channels are positioned for optimal imaging purposes. The trapping channels are also connect to at least one exit channel via fluid communication. The fluid, and contents within the fluid, may be controlled via hydraulic pressure.

Claims

1. A microfluidic device comprising:

a substrate; and

a trapping device, comprising;

a sample reservoir;

an inlet channel in fluid communication with the sample reservoir;

a plurality of trapping channels in fluid communication with the inlet channel, each trapping channel having a length and a height, wherein the length of each trapping channel is measured in a direction substantially parallel to a surface of the substrate and the height of each trapping channel is measured in a direction substantially perpendicular to the surface of the substrate;

an exit channel in fluid communication with the plurality of trapping channels; and

wherein the length of each trapping channel comprises a plurality of sections, and wherein for at least one trapping channel, the heights of at least two of the plurality of sections differ from one another.

2. The microfluidic device of claim 1, wherein a first section of the trapping channel is proximate to the inlet channel, a third section of the trapping channel is distal to the inlet channel, and a second section is positioned between the first and third sections;

and wherein the height of the first section is greater than the height of the second section, and wherein the height of the second section is greater than the height of the third section.

3. The microfluidic device of claim 1, wherein at least one trapping channel comprises gradually changing height along at least a portion of the length of the channel.

4. The microfluidic device of claim 1, comprising a plurality of trapping devices in fluid communication with one another.

5. The microfluidic device of claim 4, wherein at least two of the plurality of trapping devices comprise exit channels in fluid communication with one another via an intermediate exit channel.

6. The microfluidic device of claim 5, wherein a plurality of exit channels join in fluid communication with a final exit channel.

7. The microfluidic device of claim 5, wherein the respective exit channels of the at least two trapping devices are sized such that a flow through each exit channel experiences similar hydraulic resistance.

8. The microfluidic device of claim 1, wherein the trapping device further comprises a micro-well having a substantially frustoconical shape and configured such that the well deposits samples and reagents into the sample reservoir.

9. The microfluidic device of claim 4, wherein the plurality of trapping devices are pressurized via a shared gasket system.

10. The microfluidic device of claim 9, wherein the shared gasket system applies pressure in cycles between about 0 and 30 psi.

11. The microfluidic device of claim 10, wherein the shared gasket system comprises a vent to release gas.

12. The microfluidic device of claim 10, wherein each pressure cycle is maintained for a time period of 0 to 600 seconds.

13. The microfluidic device of claim 1, wherein the inlet channel comprises a height of about 1 μ

m to about 500 μ

m and a width of about 1 μ

m to about 750 mm.

14. The microfluidic device of claim 1, wherein the length of each of the plurality of trapping channels is between about 200 μ

m to about 6 mm.

15. The microfluidic device of claim 1, wherein the trapping channel comprises a width, and wherein the width varies such that the aspect ratio of the width to the height remains within a range of about 0.2 to about 2.0.

16. The microfluidic device of claim 1, wherein the geometry of the trapping channel is configured to position a single worm in a specific lateral orientation.

17. The microfluidic device of claim 1, further comprising an imaging apparatus comprising a camera that images the contents of the trapping channel.

18. The microfluidic device of claim 17, wherein the imaging apparatus is configured to simultaneously image the contents of a plurality of parallel trapping channels.

19. The microfluidic device of claim 17, wherein the imaging apparatus images the contents in multiple z-stacks.

20. The microfluidic device of claim 17, wherein the imaging apparatus automatically images the contents.

21. The microfluidic device of claim 17, wherein the imaging apparatus comprises adjustable features for controlling camera exposure time, number of z-stacks, and z-step size.

22. An image analysis apparatus for analyzing images from the imaging apparatus of claim 1, comprising a processor and computer-implemented instructions for determining the individual position of contents of the trapping channels and choosing a best focal plane for pheonotyping.

23. The image analysis apparatus of claim 22, wherein the steps of determining and choosing are carried out automatically.

24. A high-throughput imaging system, comprising:

a microfluidic device comprising a plurality of trapping devices;

a pressure device operably connected to the microfluidic device via fluidic connections and configured to apply hydraulic pressure to the microfluidic device via the fluidic connections;

a microscope comprising a camera, an objective, and a motorized platform supporting the microfluidic device;

a processor comprising instructions for;

applying hydraulic pressure to the microfluidic device to position contents of the microfluidic device;

translating the motorized platform such that the camera captures the contents of the microfluidic device;

capturing the contents of the microfluidic device as image files; and

storing the image files sequentially.

25. The high-throughput imaging system of claim 24, wherein the microfluidic device is in fluid communication with the gasket, and the gasket is in fluid communication with pressure device.

26. The high-throughput imaging system of claim 24, wherein individual trapping devices in the microfluidic device are in fluid communication with individual tubing couplers, and the individual tubing couplers are in fluid communication with pressure device.

27. The high-throughput imaging system of claim 24, wherein the motorized platform is capable of moving in at least three dimensions.

28. A method of automatically imaging contents of a microfluidic device having a plurality of trapping devices, comprising:

receiving parameter information regarding the microfluidic device, said parameter information comprising a well format, number of wells to be imaged, a desired number of z-stack images, and a z-step size;

measuring, in three dimensions, the orientation of the microfluidic device;

calculating offsets in a focusing plane of the contents in the trapping devices;

calculating a curvature of a substrate of the microfluidic device for a given applied pressure;

moving the motorized stages in three dimensions for focusing on the contents of the microfluidic device based on the measured orientation, calculated offsets, and calculated curvature;

acquiring the images of the contents in the microfluidic device; and

storing the image files.

29. The method of claim 28, wherein measuring further comprises obtaining x, y, and z coordinates of a plurality of predetermined locations of the microfluidic device.

30. The method of claim 28, wherein calculating the curvature of the microfluidic device is carried out while the microfluidic device is under hydraulic pressure.

31. The method of claim 28, wherein imaging the microfluidic device comprises imaging more than one trapping channel at a time.

32. An image analyzer for analyzing images of the contents of a microfluidic device having a plurality of trapping devices, comprising:

loading images of at least one of the plurality of trapping devices;

identifying the trapping channels;

identifying desired contents of said trapping channels;

cropping the image to remove portions of the trapping channels that do not have the desired contents;

comparing a plurality of images of the desired contents, the plurality of images representing different focal planes;

for the desired contents of each trapping channel, choosing an ideal focal plane from the plurality of images; and

displaying the chosen focal planes of the desired contents on a graphical user interface.

33. The image analyzer of claim 32, wherein the different focal planes represent a z-stack image.

34. The image analyzer of claim 32, wherein display settings are adjusted in real-time to aid in scoring phenotypes.

35. The image analyzer of claim 32, wherein a particle size filter is used to remove all foreign particles in the area of interest highlighting the features of interest.

36. The image analyzer of claim 32, wherein scored phenotypes of the contents in the microfluidic device are saved into a multi-dimensional array for graphical representation and statistical analysis.

37. An automated image analyzer for analyzing images of the contents in a microfluidic device having a plurality of trapping devices, comprising:

loading images of at least one of the plurality of trapping devices;

identifying the trapping channels;

identifying desired contents of said trapping channels;

cropping the image to remove portions of the trapping channels that do not have the desired contents;

comparing a plurality of images of the desired contents, the plurality of images representing different focal planes;

for the desired contents of each trapping channel, choosing an ideal focal plane from the plurality of images; and

analyzing the features of interest in the chosen focal planes of the desired contents, categorizing and saving their phenotypes.

38. The image analyzer of claims 37, further comprising displaying scored phenotypes on a graphical user interface.