Table 1.

Summary of Ex Vivo Imaging Techniques Presented

Technique	Description of principle	Examples of applications	Investigators
In situ hybridization	Radioactive	Detection of HIV/SIV RNA and DNA on tissue sections	Haase
	Chromogenic		Haase
	RNAscope	Detection of HIV/SIV RNA on tissue sections, allows for simultaneous signal amplification and background suppression	Haase, Estes, Connick
	DNAscope	Detection of HIV/SIV DNA in situ, technique similar to RNAscope, but for DNA	Estes, Yu, Germain
	BASEscope	Detection of multiple spliced RNA in situ, technique similar to RNA scope, but for multiple spliced RNA	Estes
Tyramide signal amplification	Novel technique using a biotinylated tyramine to detect specific proteins or nucleic acid sequences in situ, where tyramide, a phenolic compound, has the ability to bind to the electron-rich surface of targets	Visualization of virions with the light microscope	Haase
In situ tetramer staining	Fluorescent detection of epitope-specific T cell receptors	Identification and quantification of antigen-specific CTL in situ	Haase, Skinner
Visualizing the virus	Gp160-GFP, mCherry-Vpr, integrase-Ruby, tetracysteine-p24	Detection of fluorescent or luciferase-labeled reporter viruses	Hope, Connick, Young
	GFP or ELuc reporter virus
EM tomography	Correlated Light and EM microscopy (CLEM)	Identification of intracellular HIV reverse transcribing complexes	Hope
Antibody labeling	VRC01, VRC01-LS labeling with Cy3, Cy5, and 64Cu	Tracking of labeled antibodies, which reveal antibody distribution and new mechanisms of antibody transport	Hope
Multiplexed confocal imaging assays	Combined with RNAscope	Visualization of vaccination or infection-induced germinal centers	Petrovas
Histocytometry	For visualizing and quantifying phenotypically complex cell populations directly in tissue sections	In situ multiplex cell phenotyping, quantification, and spatial analysis	Gerner, Yu, Germain, Petrovas
	Technology is based on multiplexed antibody staining, tiled high-resolution confocal microscopy, voxel gating, volumetric cell rendering, and quantitative analysis	Cell distance mapping
		Analysis of local cellular interactions
		Monitoring immune responses to vaccines
	Expanded to 3D volume imaging	Quantifying cellular distributions of SIV provirus in tissues
High-throughput imaging and analysis	(1) Automated liquid handling	Nuclear features of Hutchinson–Gilford Progeria syndrome cells	Shachar (Misteli)
	(2) High-throughput microscopy
	(3) High-content image and data analysis	Measure cellular phenotype of aging
	Immunofluorescence or fluorescence in situ hybridization (FISH)-based	Identification and characterization of novel cellular factors and molecular mechanisms
		Study nuclear organization
		Detect interactions between genes or loci and nuclear bodies
		Measure distances between loci, such as enhancer-promoter