Photoswitches: Key molecules for subdiffraction‐resolution fluorescence imaging and molecular quantification

Abstract

Optical microscopes, often referred to as 'light microscopes', use visible light and a system of lenses to provide us with magnified images of small samples. Combined with highly sensitive fluorescence detection techniques and efficient fluorescent probes they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to the diffraction barrier of light which imposes an optical resolution limit of approximately 200 nm in the imaging plane. In the recent past new techniques emerged that break the diffraction barrier and enable structural investigations with so far unmatched resolution. They are all based on the selective switching of fluorophores between a fluorescent and a nonfluorescent state and are therefore generalized under the denotation "Photoswitching Microscopy". Here we review recent progress in subdiffraction-resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies. Special emphasis will be placed on the design and development of photoswitches and the requirements photoswitches have to fulfill for successful use in photoswitching microscopy. Moreover, we demonstrate how photoswitches can be used advantageously for molecular quantification, i.e. the determination of densities and absolute numbers of proteins located in specific subcellular compartments and discuss concepts how standard organic fluorophores can be used successfully for photoswitching microscopy.All subdiffraction-resolution fluorescence imaging methods are based on the separation of fluorescence emission in time. Thus, they critically depend on the availability of photoswitches, i.e. molecules that can be switched between a fluorescent and nonfluorescent state upon irradiation with light with high reliability. Here we describe the development and operation methods of diverse photoswitches and their application for superresolution fluorescence imaging and molecular quantification.

Photoactivatable Fluorophores

“…It follows that this convenient imaging technique cannot appreciate the subtle factors that govern biological processes and define biological structures at the molecular level. These stringent limitations can be overcome with the aid of photoactivatable fluorophores [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]. Indeed, the ability to switch fluorescence on under optical control permits the separation of distinct probes in time and the sequential reconstruction of images with subdiffraction resolution.…”

“…Indeed, fluorescence photoactivation permits the monitoring of dynamic processes in real time [13][14][15][16][17][18][19][20][21][22][23][24] as well as the acquisition of images with spatial resolution at the nanometer level [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]. As a result, significant research efforts are presently directed to the development of strategies for fluorescence photoactivation with fluorescent proteins and synthetic dyes together with their implementation in imaging applications.…”

Nanoscopic Visualization of Soft Matter Using Fluorescent Diarylethene Photoswitches

“…In the following, we will demonstrate that the photoswitch meets all the requirements of an ideal dye for super-resolved fluorescence microscopy: [28,29] spectrally well-separated on and off states, high absorption coefficients, high fluorescence quantum yield of …”

Stochastic approach to the molecular counting problem in superresolution microscopy

“…However, several obstacles remain: (i) PA-FP "blinking" leads to severe overcounting biases. Blinking refers to a process by which a PA-FP produces a series of intermittent emission bursts, instead of a single continuous burst (24,25), by transiently transitioning to a "dark" state. Accordingly, counting the number of emission bursts over a region of interest (ROI) will lead to overestimating the number of labeled molecules; and (ii) Unknown blinking statistics.…”