Multi-well fourier ptychographic and fluorescence imaging

• US 10,168,525 B2
• Filed: 01/26/2016
• Issued: 01/01/2019
• Est. Priority Date: 01/26/2015
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Kim, Jinho, Yang, Changhuei
• Primary Examiner(s): Taningco, Marcus
• Assistant Examiner(s): Guitierrez, Gisselle
• Application Number:
  US15/007,159
• Publication Number:
  US 20160216208A1
• Time in Patent Office: 1,071 Days
• Field of Search: 2504591, 2504581
• CPC Class Codes: G01N 2021/6478 Special lenses
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 2201/0446 Multicell plate, sequential
• CPC Class Codes: G02B 13/0095 Relay lenses or rod lenses in instruments for viewing the inside of hollow bodies G02B23/2446
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/365 Control or image processing arrangements for digital or video microscopes G02B21/361, G02B21/362 take precedence
• CPC Class Codes: G02B 21/367 providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• CPC Class Codes: H04N 23/45 for generating image signals from two or more image sensors being of different type or operating in different modes, e.g. with a CMOS sensor for moving images in combination with a charge-coupled device [CCD] for still images
• CPC Class Codes: H04N 23/56 provided with illuminating means
• CPC Class Codes: H04N 23/957 Light-field or plenoptic cameras or camera modules

Abstract

Certain aspects pertain to multi-well systems, devices, and methods of Fourier ptychographic and fluorescence imaging.

Claims

1. An imaging device for bright field Fourier ptychographic imaging and fluorescence imaging, the imaging device comprising:

a first fluorescence illumination source configured to provide excitation light of a first range of wavelengths to a transparent well;

an optical system having a pair of objectives, the pair of objectives comprising a first objective and a second objective positioned oppositely; and

an image sensor configured to receive light propagated through the optical system from the transparent well, the image sensor further configured to acquire a sequence of uniquely illuminated intensity measurements of light passing through a sample being imaged during operation in the well based on sequential illumination at different illumination angles from a variable illumination source, the image sensor further configured to acquire a first fluorescence image of the sample based on light emitted by the sample in response to excitation light of the first range of wavelengths, wherein the first objective is configured to collect light from the transparent well and the second objective is configured to focus light to the image sensor.

2. The imaging device of claim 1, further comprising a processor configured to iteratively determine an improved resolution bright field image of the sample that is self-consistent with the sequence of uniquely illuminated intensity measurements.

3. The imaging device of claim 2, wherein the improved resolution bright field image of the sample and the first fluorescence image are determined on a periodic basis for time lapse imaging.

4. The imaging device of claim 1, wherein the excitation light of the first range of wavelengths and the illumination sequentially at different illumination angles are provided at different image acquisition times.

5. The imaging device of claim 1, wherein the optical system has an emission filter between the first and second objectives, the emission filter configured to block excitation light of the first range of wavelengths.

6. The imaging device of claim 5, wherein illumination from the variable illumination source is of a range of wavelengths within a passband of the emission filter.

7. The imaging device of claim 1,

further comprising a second fluorescence illumination source configured to provide excitation light of a second range of wavelengths to the transparent well;

wherein the image sensor is further configured to acquire a second fluorescence image of the sample based on light emitted by the sample in response to excitation light of the second range of wavelengths;

wherein the processor is further configured to combine the first fluorescence image and the second fluorescence image to generate a multi-color fluorescence image of the sample.

8. The imaging device of claim 7, wherein the optical system has a dual band emission filter between the objectives, and wherein the dual band emission filter is configured to block excitation light of the first range of wavelengths and block excitation light of the second range of wavelengths.

9. The imaging device of claim 8, wherein illumination from the variable illumination source is of a range of wavelengths within a passband of the dual band emission filter.

10. The imaging device of claim 1, wherein wherein the first objective and the second objective have the same numerical aperture.

11. The imaging device of claim 1, wherein each objective has a numerical aperture of less than 0.55.

12. The imaging device of claim 1, wherein the first fluorescence illumination source directs excitation light of the first range of wavelengths at a small angle to a plane at a bottom surface of the transparent well and to a center of the transparent well.

13. The imaging device of claim 1, wherein the transparent well is in a multi-well plate.

14. The imaging device of claim 1, wherein the transparent well is in a 6-well plate or a 12-well plate.

15. The imaging device of claim 1, wherein the transparent well is at least 20 mm in width.

34. The imaging device of claim 1, wherein the first fluorescence illumination source is side mounted.

16. A system for Fourier ptychographic imaging and fluorescence imaging, the system comprising:

a body configured to receive a multi-well plate;

imaging devices arranged for one to one correspondence with transparent wells in the multi-well plate, each imaging device comprising;

a first fluorescence illumination source configured to provide excitation light of a first range of wavelengths to the corresponding well;

an optical system having a pair of objectives, the pair of objectives comprising a first objective and a second objective positioned oppositely; and

an image sensor for capturing intensity measurements based on light received from the corresponding well, wherein the first objective is configured to collect light from the corresponding well and the second objective is configured to focus light to the image sensor; and

a processor configured to generate an improved resolution brightfield image of each sample being imaged in a respective well during operation, the processor configured to generate the improved brightfield image using Fourier ptychographic reconstruction based on a sequence of uniquely illumination intensity measurements acquired during sequential illumination at different illumination angles by a variable illumination source; and

wherein the system is also configured to generate a first fluorescence image of each sample being imaged based on light emitted by the sample in response to receiving excitation light of the first range of wavelengths.

17. The system of claim 16, wherein the processor is configured to iteratively determine the improved resolution bright field image that is self-consistent with the sequence of uniquely illuminated intensity measurements.

18. The system of claim 16, wherein the improved resolution bright field image of the sample and the first fluorescence image are determined on a periodic basis for time lapse imaging.

19. The system of claim 16, wherein the excitation light of the first range of wavelengths and the illumination sequentially at different illumination angles are provided at different image acquisition times.

20. The system of claim 16, wherein the optical system has an emission filter between the first and second objectives, the emission filter configured to block excitation light of the first range of wavelengths.

21. The system of claim 20, wherein illumination from the variable illumination source is of a range of wavelengths within a passband of the emission filter.

22. The system of claim 16,

wherein each imaging device further comprises a second fluorescence illumination source configured to provide excitation light of a second range of wavelengths to the corresponding transparent well;

wherein the image sensor is further configured to acquire a second fluorescence image of the sample being imaged based on light emitted by the sample in response to receiving excitation light of the second range of wavelengths; and

wherein the processor is further configured to overlay the first fluorescence image and the second fluorescence image to generate a multi-color fluorescence image of the sample.

23. The system of claim 22, wherein the optical system has a dual band emission filter between the first and second objectives, and wherein the dual band emission filter is configured to block excitation light of the first range of wavelengths and block excitation light of the second range of wavelengths.

24. The system of claim 23, wherein illumination from the variable illumination source is of a range of wavelengths within a passband of the dual band emission filter.

25. The system of claim 16, wherein the first objective and the second objective have the same numerical aperture.

26. The system of claim 16, wherein each of the first and second objectives has a numerical aperture of less than about 0.55.

27. The system of claim 16, wherein the first fluorescence illumination source directs excitation light of the first range of wavelengths at a small angle to a plane at a bottom surface of the corresponding transparent well and to a center of the corresponding transparent well.

28. The system of claim 16, wherein the first fluorescence illumination source is side mounted.

29. The system of claim 16, wherein the multi-well plate is a 6-well plate or a 12-well plate.

30. The system of claim 16, wherein each of the transparent wells is at least 20 mm in diameter.

31. An imaging method, comprising:

calibrating positions of light sources of a variable illumination source to locations of image sensors receiving light from wells in a multi-well plate;

illuminating the multi-well plate with excitation light of a first range of wavelengths;

for each well in the multi-well plate, acquiring a first fluorescence image of a sample being imaged during operation in the well;

sequentially illuminating the multi-well plate with plane wave illumination at a plurality of illumination angles using a variable illumination source;

for each well in the multi-well plate, acquiring, using an image sensor, a sequence of uniquely illuminated intensity measurements from light passing through the sample being imaged in the corresponding well based on the sequential illumination at the plurality of illumination angles; and

for each well in the multi-well plate, reconstructing with a Fourier ptychography reconstruction process an improved resolution brightfield image of the sample being imaged based on the sequence of uniquely illuminated intensity measurements.

32. The method of claim 31, wherein calibrating the positions of the light sources comprises:

illuminating a central light element of the variable illumination source providing the sequential illumination;

acquiring an intensity image from light passing through the sample being imaged in a respective well of the multi-well plate;

determining the center of the intensity image;

measuring a shift in the intensity image;

finding the central light element displacement using a lookup data; and

determining the plurality of illumination angles based on the central light displacement.

33. An imaging method, comprising:

sequentially illuminating a multi-well plate with plane wave illumination at a plurality of illumination angles using a variable illumination source;

for each well in the multi-well plate, acquiring, using an image sensor, a sequence of intensity measurements from light passing through the well based on sequential illumination at the plurality of illumination angles;

for each well in the multi-well plate, reconstructing, using a Fourier ptychography reconstruction process, an improved resolution brightfield image using the acquired sequence of intensity measurements;

illuminating the multi-well plate with excitation light of a first range of wavelengths;

for each well in the multi-well plate, acquiring, using the image sensor, a first fluorescence image based on emissions resulting from the illumination by excitation light of the first range of wavelengths;

illuminating the multi-well plate with excitation light of a second range of wavelengths; and

for each well in the multi-well plate, acquiring, using the image sensor, a second fluorescence image based on emissions resulting from the illumination by excitation light of the second range of wavelengths and overlaying the first and second fluorescence images to generate a multicolor fluorescence image.

35. An imaging method, comprising:

sequentially illuminating a multi-well plate with plane wave illumination at a plurality of illumination angles using a variable illumination source;

for each well in the multi-well plate, collecting light using a first objective and focusing the collected light to an imaging sensor using a second objective, the first objective and the second objective positioned oppositely;

for each well in the multi-well plate, acquiring, using the image sensor, a sequence of intensity measurements from light passing through the well based on the sequential illumination at the plurality of illumination angles;

reconstructing an improved resolution brightfield image using a Fourier ptychography reconstruction process and based on the acquired sequence of intensity measurements;

illuminating the multi-well plate with excitation light; and

for each well in the multi-well plate, acquiring, using the image sensor, a fluorescence image.

36. The imaging method of claim 35, wherein the first objective and the second objective have the same numerical aperture.