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Review
. 2023 May 16;15(5):1174.
doi: 10.3390/v15051174.

Fluorescence Microscopy in Adeno-Associated Virus Research

Susanne K Golm  1 , Wolfgang Hübner  2 , Kristian M Müller  1
Affiliations
Review

Fluorescence Microscopy in Adeno-Associated Virus Research

Susanne K Golm et al. Viruses. .

Abstract

Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications and new technologies, respectively. The topics converge, since high and super-resolution microscopes facilitate the study of spatial and temporal aspects of cellular virus biology. Labeling methods also evolve and diversify. We review these interdisciplinary developments and provide information on the technologies used and the biological knowledge gained. The emphasis lies on the visualization of AAV proteins by chemical fluorophores, protein fusions and antibodies as well as on methods for the detection of adeno-associated viral DNA. We add a short overview of fluorescent microscope techniques and their advantages and challenges in detecting AAV.

Keywords: AAV; AAV labeling; DNA labeling; adeno-associated virus; laser scanning confocal microscopy (LSCM); microscopy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Scheme of labeling methods for a protein of interest (P.O.I.). Most commonly, proteins are covalently labeled with amine reactive derivatives of established dyes, e.g., from the Cy or AlexaFluor series. More advanced options such as coupling via click chemistry exist. A well-established specific labeling method uses antibodies that are coupled to fluorescent dyes or are recognized by a secondary fluorescently labeled antibody. Furthermore, the fusion of a gene of interest to sequences coding for a fluorescent protein leads to the in vivo expression of a labeled P.O.I. Here, a fusion protein of VP3 of AAV2 as the P.O.I. (PDB 1LP3 [4]) and GFP (PDB 6XZF) is depicted. Green stars represent fluorophores and arrows indicate a chemical coupling reaction.
Figure 2
Figure 2
Overview of techniques for AAV DNA labeling. (A) In vitro labeling techniques. The SABER-FISH relies on repetitive probes generated by primer exchange reaction (PER). RNAscope uses two target probes (z-oligonucleotides) for selective detection and a tree-like assembly for signal amplification. RCAHCR uses a primer -padlock “SNAIL” combination followed by rolling circle amplification (RCA) and hybridization chain reaction (HCR). Furthermore, AAV DNA can be labeled, e.g., during in vivo synthesis with, e.g., BrdU, and this label can be then recognized by fluorescently labeled antibodies. Black or blue lines denote oligonucleotides and stars represent fluorophores, which may be of different colors e.g., green or red. (B) Live cell DNA imaging. Endowing DNA with multiple operator sites and expressing the respective repressor–protein fused to a fluorescent protein enabled live cell detection. Triangles represent operator sites, the partial circle a repressor and the green square a fluorescent protein. In a similar fashion, although not yet reported for AAV, CAS-GFP fusions from the CRISPR system have also been used for DNA painting based on consecutive-binding single-guide RNAs.
Figure 3
Figure 3
Overview of fluorescence microscopy methods. (A) Scheme of methods and their approximate resolution range in three typical categories. Standard and high-resolution fluorescence microscopy methods are all limited in absolute resolution by diffraction. (B) Scheme of light paths in standard and high resolution. High-resolution techniques typically achieve clean optical sectioning by structured illumination with or without further software deconvolution to improve contrast. These methods typically employ lenses with high magnification and numerical aperture. (C) Schematic representation of the resulting point-spread function of microscopy methods and the expected axial and lateral resolution range. Only super-resolution techniques achieve resolutions under the diffraction limit, typically under 100–150 nm in lateral resolution. The resolutions are dependent on the wavelength (red to blue), i.e., lower wavelengths result in higher resolutions (e.g., discussed in [116]). RESOLFT: Saturable Optical Fluorescence Transitions; SR-SIM: super-resolution structured illumination microscopy; STED: stimulated emission depletion; SMLM: single molecule localization microscopy.
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Grants and funding

This research received no external funding. We acknowledge support for the publication costs by the Open Access Publication Fund of Bielefeld University and the Deutsche Forschungsgemeinschaft (DFG).

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