Methods for High-Throughput Labelling and Detection of Biological Features In Situ Using Microscopy

Abstract

Methods of labelling one or more subcellular components (e.g., an organelle and/or subcellular region) in vivo are provided. Methods of labelling a protein in vivo are provided. Methods of determining a nucleic acid sequence in situ are also provided.

1 .- 31 . (canceled) 
     
     
         32 . A method for identifying a subcellular component in a cell, comprising:
 (a) in said cell, bringing a nucleic acid molecule comprising a barcode in contact with a subcellular component comprising a binding domain such that said nucleic acid molecule couples to said binding domain of said subcellular component; and   (b) identifying said barcode within said cell to identify said subcellular component.   
     
     
         33 . The method of  claim 32 , wherein said subcellular component is a protein. 
     
     
         34 . The method of  claim 32 , wherein said subcellular component is an organelle. 
     
     
         35 . The method of  claim 34 , wherein said organelle is a nucleus, a nucleolus, a mitochondria, a Golgi apparatus, an endoplasmic reticulum, a ribosome, a lysosome, a vacuole, an endocytic vesicle, an exocytic vesicle, a cytoskeleton or a chloroplast of said cell. 
     
     
         36 . The method of  claim 32 , further comprising, subsequent to (a), subjecting said nucleic acid molecule to nucleic acid amplification to generate one or more amplicons derived from said nucleic acid molecule. 
     
     
         37 . The method of  claim 36 , wherein said nucleic acid molecule comprises a ribonucleic acid (RNA) sequence, and wherein subjecting said nucleic acid molecule to said nucleic acid amplification comprises reverse transcribing said RNA sequence to generate a deoxyribonucleic acid (DNA) sequence. 
     
     
         38 . The method of  claim 37 , further comprising generating one or more copies of said DNA sequence. 
     
     
         39 . The method of  claim 38 , wherein (b) comprises detecting said one or more copies of said DNA sequence. 
     
     
         40 . The method of  claim 38 , wherein said DNA sequence is part of a circular nucleic acid molecule, and wherein said one or more copies of said DNA sequence are generated by performing rolling circle amplification on said circular nucleic acid molecule. 
     
     
         41 . The method of  claim 32 , wherein said subcellular component is a subcellular region. 
     
     
         42 . The method of  claim 41 , wherein said subcellular region is a plasma membrane, a cell wall or a ribosomal subunit. 
     
     
         43 . The method of  claim 32 , wherein said nucleic acid molecule is a ribonucleic acid (RNA) molecule. 
     
     
         44 . The method of  claim 43 , wherein prior to (a), said RNA molecule is expressed in said cell. 
     
     
         45 . The method of  claim 43 , further comprising, prior to (a), delivering said RNA molecule to said cell. 
     
     
         46 . The method of  claim 32 , wherein (b) comprises delivering one or more probes to said cell and using at least a subset of said one or more probes to identify said barcode. 
     
     
         47 . The method of  claim 32 , wherein in (b), said nucleic acid molecule or one or more derivatives thereof are attached to a matrix within said cell. 
     
     
         48 . The method of  claim 47 , wherein, prior to (b), said matrix is formed by contacting said cell with a matrix-forming material. 
     
     
         49 . The method of  claim 47 , wherein in (b) said cell comprises a plurality of nucleic acid molecules attached to said matrix, wherein said plurality of nucleic acid molecules includes said nucleic acid molecule or said one or more derivatives thereof, and wherein said matrix maintains a spatial relationship of said plurality of nucleic acid molecules. 
     
     
         50 . The method of  claim 32 , wherein said barcode comprises a nucleic acid sequence that is specific to said subcellular component, and wherein (b) comprises identifying said nucleic acid sequence to identify said subcellular component. 
     
     
         51 . The method of  claim 50 , wherein identifying said nucleic acid sequence comprises sequencing said nucleic acid sequence.