US 12,417,538 B2
Validation methods for multiplexed imaging method
Douglas Wood, Hingham, MA (US)
Assigned to Ultivue, Inc., Cambridge, MA (US)
Filed by Ultivue, Inc., Cambridge, MA (US)
Filed on Feb. 7, 2024, as Appl. No. 18/435,336.
Application 18/435,336 is a division of application No. 16/213,391, filed on Dec. 7, 2018, granted, now 11,935,240.
Claims priority of provisional application 62/630,405, filed on Feb. 14, 2018.
Claims priority of provisional application 62/596,587, filed on Dec. 8, 2017.
Prior Publication US 2024/0242353 A1, Jul. 18, 2024
Int. Cl. G06T 7/00 (2017.01); C12Q 1/6804 (2018.01); G01N 21/64 (2006.01); G01N 33/569 (2006.01); G06T 3/14 (2024.01)
CPC G06T 7/0014 (2013.01) [G01N 21/64 (2013.01); G01N 21/6458 (2013.01); G01N 33/56966 (2013.01); G06T 3/14 (2024.01); C12Q 1/6804 (2013.01); G01N 2333/7051 (2013.01); G01N 2458/10 (2013.01); G06T 2207/10056 (2013.01); G06T 2207/10064 (2013.01); G06T 2207/10081 (2013.01); G06T 2207/30024 (2013.01)] 47 Claims
OG exemplary drawing
 
1. A quantitative method of validating at least one candidate imaging method or candidate imaging reagent for use in evaluating a biological sample for the presence of one or more targets comprising:
a. obtaining a first imaging signal using a first imaging method and/or first imaging reagents comprising:
i. contacting a biological sample with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a nucleic acid strand and wherein target-specific binding partners of different specificity, if present, are linked to different nucleic acid strands, wherein the nucleic acid strand is either a docking strand or a primer strand for amplification of docking strands;
ii. contacting the biological sample with labeled imager strands for a first imaging method, wherein the labeled imager strands are capable of binding a docking strand, directly or indirectly,
iii. generating a first imaging signal;
b. optionally removing the bound labeled imager strands from the docking strands;
c. obtaining a second imaging signal using a second imaging method and/or second imaging reagents comprising contacting the biological sample with either (1) labeled imager strands, wherein the labeled imager strands are capable of binding a docking strand, directly or indirectly, or (2) a secondary binding partner for the target-specific binding partner,
wherein the second imaging method and/or the second imaging reagent is different from the first imaging method and/or first imaging reagent;
d. optionally aligning the first imaging signal and the second imaging signal to adjust for signal orientation, image parity, scale, rotation, and/or translation mismatch,
e. identifying a first imaging zone in both the first imaging signal and second imaging signal, wherein the first imaging zone in both imaging signals represents the same location in the biological sample;
f. comparing the first imaging zone in the first imaging signal and the first imaging zone in the second imaging signal by performing a cross-correlation.