Systems, methods, and workflows for optogenetics analysis

• US 9,702,874 B2
• Filed: 11/16/2015
• Issued: 07/11/2017
• Est. Priority Date: 08/23/2010
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Kralj, Joel, Hochbaum, Daniel, Cohen, Adam E., Douglass, Adam D.
• Primary Examiner(s): NAVARRO, ALBERT MARK
• Application Number:
  US14/942,992
• Publication Number:
  US 20160069876A1
• Time in Patent Office: 603 Days
• CPC Class Codes: A61K 49/0017 Fluorescence in vivo
• CPC Class Codes: A61K 49/0097 Cells, viruses, ghosts, red blood cells, viral vectors, used for imaging or diagnosis in vivo
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

The invention provides methods for characterizing cellular physiology by incorporating into an electrically excitable cell an optical reporter of, and an optical actuator of, electrical activity. A signal is obtained from the optical reporter in response to a stimulation of the cell. Either or both of the optical reporter and actuator may be based on genetically-encoded rhodopsins incorporated into the cell. The invention provides all optical methods that may be used instead of, or as a complement to, traditional patch clamp technologies and that can provide rapid, accurate, and flexible assays of cellular physiology.

Claims

1. A method for evaluating the function of a neuron, the method comprising:

providing a first neuron that includes an optical reporter of electrical activity and a second neuron that is in communication with the first neuron via at least one synapse;

obtaining a signal from the optical reporter in response to a stimulation of the second neuron; and

evaluating the signal in order to determine a functional property of the neuron.

2. The method of claim 1, wherein the optical reporter of electrical activity comprises a microbial rhodopsin with one or more mutations.

3. The method of claim 1, wherein the second neuron includes an actuator of electrical activity.

4. The method of claim 3, wherein the actuator of electrical activity is a protein.

5. The method of claim 4, wherein the protein is a channelrhodopsin.

6. The method of claim 4, wherein characterizing the neuron comprises evaluating a response to exposure to a compound.

7. The method of claim 4, wherein evaluating the signal comprises using a computer system to characterize an AP waveform of the first neuron.

8. The method of claim 4, wherein the optical reporter of electrical activity comprises a microbial rhodopsin with one or more mutations.

9. The method of claim 4 wherein the optical reporter of electrical activity is part of a fusion protein that also includes a fluorescent Ca++ indicator.

10. The method of claim 9, wherein the fluorescent Ca++ indicator is GCaMP6f.

11. A method for evaluating the function of a cardiomyocyte, the method comprising:

providing a first cardiomyocyte that includes an optical reporter of electrical activity and a second cardiomyocyte that is in communication with the first cardiomyocyte via at least one gap junction;

obtaining a signal from the optical reporter in response to a stimulation of the second cardiomyocyte; and

evaluating the signal in order to determine a functional property of the cardiomyocyte.

12. The method of claim 11, wherein the optical reporter of electrical activity comprises a microbial rhodopsin with one or more mutations.

13. The method of claim 11, wherein the second cardiomyocyte includes an actuator of electrical activity.

14. The method of claim 13, wherein the actuator of electrical activity is a protein.

15. The method of claim 14, wherein the protein is a channelrhodopsin.

16. The method of claim 14, wherein characterizing the cardiomyocyte comprises evaluating a response to exposure to a compound.

17. The method of claim 14, wherein evaluating the signal comprises using a computer system to characterize an AP waveform of the first cardiomyocyte.

18. The method of claim 14, wherein the optical reporter of electrical activity comprises a microbial rhodopsin with one or more mutations.

19. The method of claim 14 wherein the optical reporter of electrical activity is part of a fusion protein that also includes a fluorescent Ca++ indicator.

20. The method of claim 19, wherein the fluorescent Ca++ indicator is GCaMP6f.