Table 3.

Fluorescence labeling technologies and their benefits and drawbacks

Label	Target biomolecule	Description	Benefits	Drawbacks
DNA Binding Dyes (DAPI, Hoechst, SiR-DNA, and SPY650)	DNA	These dyes fluoresce when they intercalate into the minor groove of DNA [262–264]	• Requires minimal sample preparation • Labels all DNA indiscriminately	• Cannot label specific genes
FiSH	DNA/RNA	Fluorescence in-situ hybridization (FiSH) labels gene loci or RNA specifically with fluorescently labeled single stranded probes [265,266]	• Labels DNA gene loci or RNA specifically • Multiple gene loci labeled at one time	• Cannot be used for live cell imaging • Requires specific probe design
LacR & TetR	DNA	LacR and TetR specifically label chromatin locus in living cells with a GFP-fusion protein [267,268]	• Results in stable cell line that can be used over and over • Specific gene loci and individual gene loci can be imaged in live cells over multiple generation without the addition of probes	• Requires integration of prokaryotic operon sequences into the DNA • The gene editing may result in abnormal gene expression profiles
dCas9	DNA	dCas9 uses the CRISPR gene editing system for DNA labeling with a fluorescently tagged nuclease dead Cas9 in combination with specifically engineered guide RNAs [221–228].	• Live cell imaging without laborious or disruptive gene editing • Multiple gene loci labeled at one time • Ideal for studying chromatin dynamics	• Requires multiple CRISPR/Cas9 to produce a bright enough signal for imaging • The binding affinity of CRISPR/Cas9 is highly dependent upon the gRNA sequence
MS2/PP7	RNA	Fluorescent molecules bind to repetitive stem loops that have been introduced into the gene of interest. Each stem loop, of which there are often up to 24 copies, binds to a dimer of a chimeric protein composed of the phage protein, a nuclear localization signal and a fluorescent protein [197,269].	• Actively transcribing RNA can be imaged in real-time within a cell • Since MS2-RNA and PP7-RNA are sequence specific, both can be used simultaneously within a given cell, allowing for multiple RNAs to be visualized at the same time.	• Can only be used to label two distinct RNAs at a time • The multimerization of the stem loops results in a bulky label that can alter RNA kinetics
dCas13	RNA	dCas13 uses the CRISPR gene editing system for RNA labeling with a nuclease dead Cas13 in combination with specifically engineered guide RNAs [222,229] . Either the gRNA or the Cas13 can be fluorescently tagged.	• Versatile method for labeling RNA’s which have not been modified through the insertion of an RNA hairpin or other sequence • Sequence specific • Ideal for studying RNA dynamics	• Requires multiple copies of the RNA of interest and multiple CRISPR/Cas13 to produce a bright enough signal for imaging • The binding affinity of CRISPR/Cas13 is highly dependent upon the gRNA sequence
RNA Aptamers	RNA	RNA aptamers, like RNA Mango [230], are sequences designed as molecular beacons and selected through SELEX [233,234]. The resulting aptamer is capable of binding specific fluorophore derivatives with nanomolar affinity.	• Provides a fluorescence enhancement upon binding (up to 1000×), lowering the considerable fluorescence background that is typically present	• Requires binding to a target molecule to fluoresce • Requires specific environmental parameters to perform optimally (magnesium concentration, temperature, ect.)
Fluorescent Protein Tags (ex. GFP)	Protein	Fluorescent proteins can be inserted into a cell line so that as a protein is expressed it fluoresces [270].	• Proteins are produced directly by the cell • 100% labeling efficiency	• These protein labels are bulky and can change protein dynamics and function.
HaloTag and SNAP-tag	Protein	Self-labeling protein tags such as HaloTag and SNAP-tag [237,238] are organic protein tags that can be inserted into cloning vectors [237], allowing for a specific binding site for fluorophores.	• Can be used with a wide range of fluorophores • Improved brightness and photostability • Self-labeling	• Does not have 100% labeling efficiency, therefore “dark” or unlabeled proteins sometimes occur • Requires gene editing
Fluorescent Antibody Fragments (Fabs)	Protein	This is a technique that uses monoclonal antibodies which lack the Fc component to specifically tag proteins of interest [271]. The fluorophore is conjugated to a single chain antibody specific to the protein of interest [272].	• Ideal method of quantifying the timing of post-translational modifications and their effects in living cells	• Challenging to design probes • Low yield when designing Fabs