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

• Published in: Laser & photonics reviews Vol. 3; no. 1-2; pp. 180 - 202
• Main Authors: Heilemann, M., Dedecker, P., Hofkens, J., Sauer, M.
• Format: Journal Article
• Language: English
• Published: Berlin WILEY-VCH Verlag 01.02.2009; WILEY‐VCH Verlag; Wiley-VCH
• Subjects: Barriers; Biological and medical sciences; Density; Diffraction; Exact sciences and technology; Fluorescence; Fundamental and applied biological sciences. Psychology; Fundamental areas of phenomenology (including applications); General aspects; General aspects, investigation technics, apparatus; General equipment and techniques; Imaging; Instrumentation. Materials. Reagents. Research laboratory organization; Instruments, apparatus, components and techniques common to several branches of physics and astronomy; Microscopy; Molecular optical switches and photoswitches; Optical computers, logic elements, interconnects, switches; neural networks; Optical elements, devices, and systems; Optical microscopes; Optics; photoswitching microscopy; Physics; Sensors (chemical, optical, electrical, movement, gas, etc.); remote sensing; subdiffraction- or super-resolution fluorescence imaging; Tissues, organs and organisms biophysics; 87.80.Nj; Resolving power; Optical switches; Superresolution; Biological tissues; 61.05.-a; Fluorescence; Imagery; photoswitching microscopy; Proteins; 87.80.-y; Fluorophore; 87.64.M; Visible radiation; Optical sensors; Optical microscopes; 87.57.cf; Photochromic materials; Fluorescence microscopy; Biomedical imaging; Molecular optical switches and photoswitches; subdiffraction- or super-resolution fluorescence imaging.; 32.50.+d
• ISSN: 1863-8880; 1863-8899
• DOI: 10.1002/lpor.200810043

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. Today, fluorescence microscopes are essential in biological and biomedical sciences for 3D noninvasive imaging of the interior of cells. Optical microscopes are subject to the diffraction barrier of light. 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 can be generalized under the denotation “Photoswitching Microscopy”. Recent progress in subdiffraction‐resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies is presented. Special emphasis is placed on the design and development of photoswitches and the requirements they have to fulfill for successful use in this kind of microscopy.