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Scanning Angle Interference Microscopy (SAIM)
Published in Qiu-Xing Jiang, New Techniques for Studying Biomembranes, 2020
Cristina Bertocchi, Timothy J. Rudge, Andrea Ravasio
In total internal reflection fluorescence microscopy (TIRFM),30 an evanescent field of excitation light is generated when a propagated wave strikes the boundary between two media with different refractive indices at an angle larger than a particular critical angle with respect to the normal to the surface (Figure 4.1b). The evanescent field decays exponentially from the total internal reflection (TIR) interface with a characteristic 1/e distance. Only fluorophores within approximately 200 nm of the TIR interface are excited to an appreciable extent, the objects outside of the evanescent field are cut out and are not excited. In VA-TIRFM, also referred to as multiple angle-TIRFM, the characteristic 1/e distance of the evanescent field is modulated by varying the incidence angle at the TIR interface (Figure 4.1b). The incident light is delivered via a monomode fiber and focused onto a sample that is optically coupled to a hemi-cylindrical glass prism, under different angles of total internal reflection. Thus, fluorophores in close proximity to a cell–substrate interface are excited by an evanescent wave with variable penetration depth and localized with high (nanometer) axial z-resolution. Although VA-TIRFM could be used to image isolated objects, its applicability has been hampered by its experimental complexity, especially due to the need for a through-prism TIRFM geometry, to achieve the range of incidence angles required.
Enabling drug discovery and development through single-cell imaging
Published in Expert Opinion on Drug Discovery, 2019
Andrea K. Pomerantz, Farid Sari-Sarraf, Kerri J. Grove, Liliana Pedro, Patrick J. Rudewicz, John W. Fathman, Thomas Krucker
High-resolution single-cell imaging techniques employ state-of-the-art chemistry, optical hardware, and data analysis algorithms to push the limits of spatial resolution and multiplexing. Reporters and stains used for target protein or compound detection in single cells must be bright and specific, and in recent years, chemists have been able to synthesize brighter and more photostable dyes that are also compatible with live cell imaging [66]. These high-performance fluorophores have made it possible to carry out single-molecule imaging in cells for analysis of potent binders beyond the detection range of conventional instrumentation, such as SPR/Biacore [67]. Paired with imaging methods such as total internal reflection fluorescence microscopy, membrane-bound targets [68,69] like G-protein-coupled receptors (GPCRs) can be visualized. Single-molecule super-resolution (SR) imaging that transcends the conventional diffraction limit (~0.2 microns) for optical microscopy is also making inroads in drug discovery. With single-molecule localization methods such as STORM (stochastic optical reconstruction microscopy) and PALM (photoactivated localization microscopy), or structured illumination microscopy (SIM), scientists can interrogate structures that were previously incapable of being visualized in single cells. These include sub-diffraction-limited (~tens of nanometers) protein aggregates, mitochondrial cristae, and synapse architecture in neuronal and immune cells [70–73]. Many SMLM methods still suffer from low time resolution and low throughput, as it can take tens of minutes to acquire a STORM image for one cell. This is being addressed by the application of deep learning algorithms to accelerate SR imaging to a point where 96-well plate-based assays are now feasible [10].
From proteomic landscape to single-cell oncoproteomics
Published in Expert Review of Proteomics, 2021
Vivian Weiwen Xue, Sze Chuen Cesar Wong, William Chi Cho
Nowadays, new single-peptide identification methods such as protein sequencing, nanopore technology, and fluorescent protein fingerprint are emerging to replace MS-based proteomic profiling. By combining these platforms with effective molecular tags, it can realize parallel peptide detection at the zeptomole level [1]. Among them, protein sequencing is a fusion strategy merging Edman degradation with the next-generation sequencing. During sequencing, proteins are fragmented and labeled with fluorescent tags on specific amino acids (AAs). The C-terminal of fragments was immobilized to flow cells. Edman degradation is applied to degrade an AA in each cycle from the N-terminal of fragments, and fluorescence microscopy captures images after each degradation cycle. Although only specific residues with fluorescence labels can be detected, the identified sequence patterns with unknown AAs will be sufficient to match with peptide sequences in databases to identify its parent protein. These sequence blocks with recognized AAs and unknown AAs are also known as peptide fingerprinting [1]. Besides, Ginkel et al. developed a single-peptide identification based on ClpX6P14-mediated protein degradation. ClpX6P14 protein complex is an enzymatic motor that unfolds and degrades proteins. Through immobilizing an array of ClpX6P14 proteases on a PEG-coated surface, ClpX6 subunit recognizes peptides with 11-AA C-terminal ssrA tag and translocate them to ClpP14 for degradation. Fluorescence-labeled AA residues are detected by total internal reflection fluorescence microscopy and alternating laser excitation imaging [32]. Two methods as stated are based on peptide fingerprinting and fluorescence imaging therefore they are limited by imaging accuracy. Differently, aerolysin nanopore-based peptide sequencing aims to realize electrical recognition of single AAs and it was used to successfully identify 13 natural AAs based on the different ionic current patterns in a recent study [2]. This platform is shown to be promising for the direct and de novo sequencing with long reading length in the future.