Photochemical Mechanisms of Fluorophores Employed in Single‐Molecule Localization Microscopy

Abstract

Decoding cellular processes requires visualization of the spatial distribution and dynamic interactions of biomolecules. It is therefore not surprising that innovations in imaging technologies have facilitated advances in biomedical research. The advent of super‐resolution imaging technologies has empowered biomedical researchers with the ability to answer long‐standing questions about cellular processes at an entirely new level. Fluorescent probes greatly enhance the specificity and resolution of super‐resolution imaging experiments. Here, we introduce key super‐resolution imaging technologies, with a brief discussion on single‐molecule localization microscopy (SMLM). We evaluate the chemistry and photochemical mechanisms of fluorescent probes employed in SMLM. This Review provides guidance on the identification and adoption of fluorescent probes in single molecule localization microscopy to inspire the design of next‐generation fluorescent probes amenable to single‐molecule imaging.

Method To Diversify Cyanine Chromophore Functionality Enables Improved Biomolecule Tracking and Intracellular Imaging

“…Finally, these studies illustrate the potential for dynamic ring-open/ring-closed chemistry with heptamethine indocyanines, which may enable the discovery for a range of long-wavelength optical probes. The prospect for such efforts is heightened by prior studies on rhodamines, fluoresceins, and rhodols, where a range of fluorogenic sensors and imaging strategies have been developed by taking advantage of nonfluorescent lactone, fluorescent zwitterion equilibria. − Related cyclization chemistry has also been used for single molecule switching in the context of super-resolution imaging . We also note that the development of cell-permeable targeted imaging probes may be useful for NIR surgical applications seeking to image intracellular targets. , Overall, we anticipate that the ability to easily modify the key chromophore unit will provide access to diverse cyanine chemical space, which, in turn, will enable previously challenging NIR imaging applications.…”

“…85−87 Related cyclization chemistry has also been used for single molecule switching in the context of super-resolution imaging. 88 We also note that the development of cell-permeable targeted imaging probes may be useful for NIR surgical applications seeking to image intra-cellular targets. 68,89 Overall, we anticipate that the ability to easily modify the key chromophore unit will provide access to diverse cyanine chemical space, which, in turn, will enable previously challenging NIR imaging applications.…”

Enhanced fluorescence blinking of AF647 fluorophores in Mowiol via violet and UV light induced recovery for superior localization microscopy

“…In recent years, fluorescent probes have been applied extensively in optical super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy [1] and single-molecule localization microscopy (SMLM) [2,3], to visualize the world up to a separation of a few tens of nano-meters. Such separations were previously restricted by diffraction limit of resolution [4].…”

“…As the fluorophores fail to show adequate amount of photo-switched blinking, it is reported [7,19] that their photophysical properties can be regained through a photon induced recovery mechanism by moving the molecules from the dark state to the reversible ground state. Use of UV or violet radiations for the photon induced recovery has been demonstrated in chemical aqueous mounting buffers [2,10,20,21]. There also exists a report [22] on UV-violet light induced recovery in mounting buffers like Mowiol.…”