Table 3.

Candidate techniques for imaging of HBV nucleic acids: Single cell detection.

Technique	Modality	Target	Possible Finding	Advantages	Drawbacks	References
CRISPR/ Cas	• dCas9-tag: -FP -Sun-tag: -tripartite -Sunspot • sgRNA modification -Aptamers: MS2-MCP, PP7 loops • Cas9 active: -STRIDE	DNA/ RNA dsDNA/ ssDNA	• Single molecule detection • HBV nucleic acids kinetic • Quantification • Distinction between cccDNA and partial DNA	• Fixed and living cells • Allows strategies to improve signal/noise ratio and amplification of signal. • Detection of single molecule and low-abundance of nucleic acids, e.g., Sunspot system • No engineering virus • Specificity • Signal amplification	• Needs signal amplification strategies like Sun-tag system, however large complexes may interfere with nucleic acids kinetic • Requires expression of specific factors in the host cells. • Not applicable in living cells • Antibodies specificity	[52] [53] [23]
Non-CRISPR/ Cas	• Aptamer-protein systems	RNA	• Distinction between transcripts (if combined with other methods)	• Fixed and living cells	• Virus engineering • High background • Sensitive folding conditions • Weak photostability	[54]
	• Light-up aptamer-dye systems	RNA	• Single-molecules detection possible • Distinction between transcripts (if combined with other methods)	• Fixed and living cells	• Virus engineering • Sensitive folding conditions • Weak photostability	[55,56]
	• Molecular beacons	RNA, ssDNA	• Single-molecules detection with advanced microscopes • Distinction between transcripts (if combined with other methods)	• Fixed and living cells	• Prone to nucleases • Difficulties with cell entry • Weak photostability • Prone to non-specific signal	[57,58]
	• Quantum-dots / quantum dots-nanobeacons	RNA, ssDNA	• Single-molecule detection • Distinction between transcripts (if combined with other methods)	• Fixed and living cells • Strong signal • Strong photostability	• Cytotoxicity • Difficulties with cell entry due to size • Single-molecules detection	[59,60]
	• ANCHOR	dsDNA	• Single-molecule detection • HBV nucleic acids kinetics possible due to photostability • Distinction between cccDNA and rcDNA	• High contrast • Signal amplification • Living cells • Photostability due to bleached molecules replenishment	• Virus engineering • Effect of OR protein on cells unknown • High cytosolic background
	• PNA-based probes	RNA, ssDNA/dsDNA	• Distinction between cccDNA and rcDNA (if combined with other methods)	• Fixed and living cells • High sequence specificity and affinity • Resistant to nucleases • Multiple binding modes make them flexible in function	• Difficulties with cell entry • Prone to non-specific signal to hydrophobic surfaces • Binding mode dependent on careful design	[61] [62] [63] [64]
	• Metabolic labeling	dsDNA	• Single-molecules detection • Distinction cccDNA/ rcDNA (if combined with other methods)	• Labelled infectious virus	• Fixed cells only	[65]