Large Stokes-shift bioorthogonal probes for STED, 2P-STED and multi-color STED nanoscopy

Abstract

Synthesis and multiple STED imaging applications of four, red-emitting (610–670 nm), tetrazine-functionalized fluorescent probes (CBRD = Chemical Biology Research group Dye 1–4) with large Stokes-shift is presented. Present studies revealed the super-resolution microscopy applicability of the probes as demonstrated through bioorthogonal labeling scheme of cytoskeletal proteins actin and keratin-19, and mitochondrial protein TOMM20. Furthermore, super-resolved images of insulin receptors in live-cell bioorthogonal labeling schemes through a genetically encoded cyclooctynylated non-canonical amino acid are also presented. The large Stokes-shifts and the wide spectral bands of the probes enabled the use of two common depletion lasers (660 nm and 775 nm). The probes were also found suitable for super-resolution microscopy in combination with two-photon excitation (2P-STED) resulting in improved spatial resolution. One of the dyes was also used together with two commercial dyes in the three-color STED imaging of intracellular structures.

Beyond the Bioorthogonal Inverse-Electron-Demand Diels–Alder Reactions of Tetrazines: 2-Pyrone-Functionalized Fluorogenic Probes

“…Finally, we added an azide-bearing probe, CBRD. 17b The reaction mixture was then analyzed by LC-MS. To our delight, only the three expected products could be detected; namely, 4 -BCN, PheCou-BCN, and CBRD-DBCO (see the Supporting Information).…”

Fluorescent Probes for STED Optical Nanoscopy

“…Hence, a probe must have small adverse excitability, i.e., a small excitation cross-section in the red tail of the emission spectrum, to be used for high-quality STED imaging [ 38 ]. A probe with a large Stokes shift between excitation and the emission spectrum is favorable [ 39 ].…”

Super‐Resolution Fluorescence Microscopy Methods for Assessing Mouse Biology

“…It is, however, possible and even beneficial to deplete multiple fluorophores with a single STED laser, as long as these fluorophores can be sufficiently separated by their excitation and emission properties. For example, Török et al (2021) report using large Stokes shift tetrazine-functionalized bio-orthogonal probes for multicolor STED; also see Leica Microsystems and Abberior Instruments guides for appropriate dye selection. As the area of fluorescence emission is determined by the STED torus, imaging multiple fluorophores with a single depletion laser ensures intrinsic alignment of the different channels (Leica Microsystems, 2019).…”

“…STED and localization microscopy make huge demands on fluorophores, so it is crucial to use the latest, most robust dyes (e.g., see Current Protocols article by Kasuboski, Sigal, Joens, Lillemeier, & Fitzpatrick, 2012; Török et al., 2021). You can get away with a lower labeling density with diffraction‐limited microscopy, where the diffraction limit “fills the gap” in the structure, but this is not possible with super‐resolution microscopy.…”

“…For example, Török et al. (2021) report using large Stokes shift tetrazine‐functionalized bio‐orthogonal probes for multicolor STED; also see Leica Microsystems and Abberior Instruments guides for appropriate dye selection. As the area of fluorescence emission is determined by the STED torus, imaging multiple fluorophores with a single depletion laser ensures intrinsic alignment of the different channels (Leica Microsystems, 2019).…”