Multiplexed imaging with nanobody probes
Methods include (a) contacting a biological sample with a composition featuring a plurality of different types of probes, where each type of probe of the plurality of different types of probes includes a detection moiety that selectively binds to a different type of protein target in the sample, a nanobody bound to the detection moiety, and an oligonucleotide linked to the nanobody and featuring an oligonucleotide sequence, where the oligonucleotide sequence of each type of probe is different from the oligonucleotide sequences of each of the other types of probes of the plurality of probes; and (b) contacting the sample with a set of one or more different types of optical labels, where each different type of optical label of the set of optical labels includes an oligonucleotide that selectively hybridizes to only one type of probe.
1 . A method, comprising:
(a) contacting a biological sample with a composition comprising a plurality of different probes, wherein each different probe of the plurality of different probes comprises:
a detection moiety that selectively binds to a different protein in the sample;
a nanobody bound to the detection moiety; and
an oligonucleotide linked to the nanobody and comprising an oligonucleotide sequence, wherein the oligonucleotide sequence of each different probe of the plurality of different probes is different from the oligonucleotide sequences of each of the other different probes of the plurality of different probes;
(b) contacting the biological sample with a set of one or more different optical labels, wherein each different optical label of the set of one or more different optical labels comprises an oligonucleotide that selectively hybridizes to only one probe among the plurality of different probes, and an optical moiety; and
(c) obtaining at least one image of the biological sample, wherein the at least one image comprises optical signals generated by at least one of the optical moieties of the set of one or more different optical labels.
2 . The method of claim 1 , further comprising identifying one or more protein targets in the sample based on the optical signals in the at least one image of the sample.
3 . The method of claim 2 , further comprising determining an amount of at least one of the one or more protein targets in the sample based on the optical signals in the at least one image of the sample.
4 . The method of claim 1 , wherein the set of one or more different optical labels comprises multiple different optical labels.
5 . The method of claim 4 , wherein the set of one or more different optical labels comprises at least three different optical labels.
6 . The method of claim 1 , wherein the detection moiety of at least one probe of the plurality of different probes comprises an antibody or antibody fragment.
7 . The method of claim 1 , wherein the composition comprises at least 10 different probes.
8 . The method of claim 7 , wherein the composition comprises at least 30 different probes.
9 . The method of claim 1 , wherein the at least one of the optical moieties of the set of one or more different optical labels comprises a fluorescent dye.
10 . The method of claim 1 , wherein for one or more of the different probes among the plurality of different types, the nanobody is bound to the detection moiety with a dissociation constant K d that is 1.0×10 −9 mol/L or less.
11 . The method of claim 1 , wherein the composition comprises multiple different probes comprising antibody binding moieties of a common species and isotype.
12 . The method of claim 1 , wherein the composition further comprises at least one fragment of a binding target of at least one of the nanobodies among the plurality of different probes.
13 . The method of claim 12 , wherein the composition further comprises multiple different fragments of binding targets of multiple nanobodies among the plurality of different probes.
14 . The method of claim 1 , further comprising, prior to contacting the sample with the composition, forming each of the different probes of the plurality of different probes, wherein each different probe of the plurality of different probes is formed by:
contacting the detection moiety for the different probe of the plurality of different probes with a labeling moiety comprising the nanobody for the different probe of the plurality of different probes linked to the oligonucleotide for the different probe of the plurality of different probes; and
incubating the binding moiety and the labeling moiety to bind the nanobody to the binding moiety.
15 . The method of claim 14 , wherein the nanobody undergoes site-specific binding to the binding moiety.
16 . The method of claim 1 , further comprising, following step (a), contacting the sample with at least one fixative.
17 . The method of claim 1 , further comprising, following step (a), washing the sample to remove unbound probes from the sample.
18 . The method of claim 1 , wherein at least one optical label of the set of one or more different optical labels comprises the oligonucleotide of the at least one optical label of the set of one or more different optical labels linked to the optical moiety of the at least one optical label of the set of one or more different optical labels through a streptavidin-biotin linkage.
19 . The method of claim 1 , wherein the at least one image comprises at least one fluorescence image of the sample.
20 . The method of claim 1 , further comprising repeating steps (b)-(c) with at least one additional set of one or more different optical labels, to obtain at least one additional image of the sample comprising optical signals generated by at least one of the optical moieties of the at least one additional set of one or more different optical labels.
21 . The method of claim 20 , further comprising identifying one or more additional protein targets in the sample based on the optical signals in the at least one additional image of the sample.
22 . The method of claim 20 , further comprising, for each sequence of steps (b)-(c), removing the set of one or more different optical labels from the sample prior to repeating step (b) with at least one additional set of one or more different optical labels.
23 . The method of claim 22 , wherein removing the set of one or more different optical labels comprises dehybridizing the set of one or more different optical labels from the plurality of different probes.