Fluorescent Probes for STED Optical Nanoscopy

Abstract

Progress in developing fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, is inseparable from the advancement in optical fluorescence microscopy. Super-resolution microscopy, or optical nanoscopy, overcame the far-field optical resolution limit, known as Abbe’s diffraction limit, by taking advantage of the photophysical properties of fluorescent probes. Therefore, fluorescent probes for super-resolution microscopy should meet the new requirements in the probes’ photophysical and photochemical properties. STED optical nanoscopy achieves super-resolution by depleting excited fluorophores at the periphery of an excitation laser beam using a depletion beam with a hollow core. An ideal fluorescent probe for STED nanoscopy must meet specific photophysical and photochemical properties, including high photostability, depletability at the depletion wavelength, low adverse excitability, and biocompatibility. This review introduces the requirements of fluorescent probes for STED nanoscopy and discusses the recent progress in the development of fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, for the STED nanoscopy. The strengths and the limitations of the fluorescent probes are analyzed in detail.

Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy

“…These features make xHTLs an important advance for live-cell imaging especially at high resolution. 9 ■ ASSOCIATED CONTENT * sı Supporting Information…”

“…Rhodamines are available in different colors 7 with high brightness and good photostability, required to resist the photobleaching induced by, for example, stimulated emission depletion (STED) 8 microscopy. 9 Furthermore, rhodamine derivatives can be engineered to possess fluorogenicity and cell permeability due to the dynamic equilibrium of rhodamines between a fluorescent zwitterion (i.e., open form) and a nonfluorescent spirocyclic form (i.e., closed form). 10 The nonfluorescent and apolar spirocyclic form increases the membrane permeability of the probes, while binding to HaloTag7 shifts the equilibrium back to the fluorescent zwitterion, resulting in fluorogenicity.…”

Small-molecule fluorogenic probes for mitochondrial nanoscale imaging

“…These fluorogenic probes have excellent photophysical properties including higher brightness ( εΦ ), better photostability, smaller molecular size, lower background fluorescence, and easier chemical modification than fluorescent proteins, quantum dots, and rare earth materials. 83,84 Furthermore, they have good biocompatibility and cell penetration to specifically penetrate the cell membrane. 85–88…”

“…These fluorogenic probes have excellent photophysical properties including higher brightness (eF), better photostability, smaller molecular size, lower background fluorescence, and easier chemical modification than fluorescent proteins, quantum dots, and rare earth materials. 83,84 Furthermore, they have good biocompatibility and cell penetration to specifically penetrate the cell membrane. [85][86][87][88] The protons in the mitochondrial matrix are pumped into the mitochondrial membrane space via the proton pump (PP) in the IMM, resulting in positive charges in the mitochondrial membrane space.…”

Identifying STEDable BF2-Azadipyrromethene Fluorophores

“…The far-red region of the spectrum is useful for biological fluorescence imaging since there is a greater penetration of light at longer wavelengths, a lower phototoxicity, and cellular autofluorescence is minimal [ 1 ]. Advances into super resolution imaging in this region continue to yield a greater understanding of cellular biology with a substantial amount of research continuously ongoing [ 2 , 3 ]. Stimulated emission depletion (STED) nanoscopy represents one super-resolution method in which Abbe’s diffraction limit is overcome by selectively deactivating fluorophores such that the illumination area is minimized [ 4 , 5 ].…”