Granted Patent B2

US 12,618,099 · App. 18/936,042 · Granted May 5, 2026

Inventors: George M. Church (Brookline, MA); Jehyuk Lee (Allston, MA); Daniel Levner (Boston, MA); Michael Super (Lexington, MA)

Assignee: President and Fellows of Harvard College

C12Q1/6806C12P19/34C12Q1/6804C12Q1/6816C12Q1/6837C12Q1/6844C12Q1/6869C12Q1/6874G01N33/53G01N2458/10

Abstract

Claims (35)

1 . A system, comprising:

(a) a cell or tissue sample immobilized on a solid support, wherein said cell or tissue sample comprises an analyte at a spatial location of said cell or tissue sample;

(b) a detection reagent comprising: (i) a probe configured to bind to said analyte, and (ii) a plurality of predetermined subsequences;

(c) a plurality of decoder probes, wherein a decoder probe of said plurality of decoder probes comprises a sequence complementary to a predetermined subsequence of said plurality of predetermined subsequences, and wherein said decoder probe is associated with an optically detectable label;

(d) an optical detection subsystem configured to detect an optical signal signature from said optically detectable label at said spatial location in said cell or tissue sample; and

(e) a computer having software or an algorithm that:

(i) performs a comparison of temporal readouts of optical signal signatures detected by said optical detection subsystem in a plurality of temporally sequential readout cycles with an optical code assigned to said plurality of predetermined subsequences, and

(ii) identifies said analyte based on agreement between a temporal order of optical signal signatures of said optical signal signatures and said optical code.

2 . The system of claim 1 , further comprising a wash buffer.

3 . The system of claim 2 , wherein said wash buffer comprises a salt, a detergent, or a denaturant.

4 . The system of claim 2 , wherein said wash buffer comprises formamide or dimethyl sulfoxide (DMSO).

5 . The system of claim 1 , further comprising a signal removal buffer.

6 . The system of claim 5 , wherein said signal removal buffer comprises a cleavage reagent, a quenching agent, a bleaching agent, a reducing agent, an oxidation agent, or any combination thereof.

7 . The system of claim 1 , further comprising a photo-bleaching subsystem configured to photo-bleach said optically detectable label.

8 . The system of claim 1 , further comprising a buffer for performing a hybridization reaction.

9 . The system of claim 1 , further comprising a nucleic acid ligase.

10 . The system of claim 1 , wherein said software or said algorithm determines a location of said optical signal signatures detected in said plurality of temporally sequential readout cycles and corrects a spatial shift in said location during said plurality of temporally sequential readout cycles.

11 . The system of claim 10 , wherein said software or said algorithm aligns a distinguishable feature detected in readout cycles of said plurality of temporally sequential readout cycles to correct said spatial shift in said location.

12 . The system of claim 1 , wherein said optical detection subsystem comprises a laser or a light emitting diode.

13 . The system of claim 1 , wherein said optical detection subsystem comprises a microscope.

14 . The system of claim 13 , wherein said microscope comprises an inverted fluorescent microscope, an epi-fluorescent microscope, or a confocal microscope.

15 . The system of claim 1 , wherein said analyte is a nucleic acid analyte and wherein said detection reagent comprises nucleic acid.

16 . The system of claim 1 , wherein said analyte is a protein analyte and said probe comprises an antibody.

17 . The system of claim 1 , wherein said optically detectable label comprises a fluorescent label.

18 . The system of claim 17 , wherein said optically detectable label is directly attached to said decoder probe via a covalent bond.

19 . The system of claim 17 , wherein said optically detectable label is indirectly attached to said decoder probe via a non-covalent bond.

20 . The system of claim 17 , wherein said optically detectable label is indirectly attached to said decoder probe via an intermediary molecule.

21 . The system of claim 17 , wherein said optically detectable label is indirectly attached to said decoder probe via a binding molecule that hybridizes to said decoder probe.

22 . The system of claim 1 , wherein predetermined subsequences of said plurality of predetermined subsequences are each about 5 to about 30 nucleotides in length.

23 . The system of claim 22 , wherein said plurality of predetermined subsequences comprises at least 4 predetermined subsequences.

24 . The system of claim 23 , wherein each decoder probe of said plurality of decoder probes is at least about 14 nucleotides in length.

25 . The system of claim 1 , wherein said cell or tissue sample comprises a plurality of fixed cells.

26 . The system of claim 1 , wherein said cell or tissue sample is a fixed tissue section.

27 . The system of claim 1 , wherein said decoder probe is associated with a plurality of optically detectable labels.

28 . The system of claim 1 , wherein said decoder probe is associated with optically detectable labels at a ratio of optically detectable labels to said decoder probe from about 1:1 to about 100:1.

Assignments (1)

Continuity (13)

References Cited (400)