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Photon Counting - Fundamentals and Applications || Detectors for Super-Resolution & Single-Molecule Fluorescence Microscopies
摘要： The resolution of light microscopy was thought to be limited to 250–300 nanometers based on the work of Ernest Abbe. This Abbe diffraction limit was believed to be insurmountable until the invention of Super-resolution microscopic techniques in the late 20th century. These techniques remove this limit and have provided unprecedented detail of cellular structures and dynamics down to several nanometers. An emerging goal in this field is to quantitatively measure individual molecules. Measurement of single-molecule dynamics, such as diffusion coefficients and complex stoichiometries, can be accomplished using fluorescence fluctuation techniques to reveal nanosecond-to-microsecond temporal reactions. These powerful complimentary experimental approaches are made possible by sensitive low-light photodetectors. In this chapter, an overview of the principles of super-resolution and single-molecule microscopies are provided. The different types of photodetectors employed in these techniques are explained. In addition, the advantages and disadvantages for these detectors are discussed, as well as the development of next generation detectors. Finally, example super-resolution and single-molecule cellular studies that take advantage of these detector technologies are presented.
关键词： biophysical techniques,STORM,nanoscopy,STED,protein dynamics,palm,spectroscopy,molecular brightness,fluorescence fluctuation
更新于2025-09-23 15:23:52
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Specialty probes give super-res imaging that special blink
摘要： Fluorescent probes light the way to cellular detail, but light can also get in the way. Because of the diffraction limit, structures closer to one another than 200 nanometers (nm) or so cannot be discerned. Unless you use probes with super-resolution imaging. These techniques, such as reversible saturable optical linear fluorescence transitions (RESOLFT) or photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM), use specialty probes, dyes and fluorescent proteins (FPs) that can switch from dark to light and from one color to another. 'We need the labels in combination with the microscope to overcome the diffraction barrier,' says Stefan Jakobs, who develops probes at University Medical Center G?ttingen and the Max Planck Institute for Biophysical Chemistry. In structured illumination microscopy (SIM), labs routinely achieve 100-nm resolution, he says. Scientists using stimulated emission depletion microscopy (STED), RESOLFT, PALM or STORM reach beyond 50-nm resolution. In principle, he says, the methods are diffraction unlimited.
关键词： STORM,STED,RESOLFT,super-resolution imaging,SIM,fluorescent probes,diffraction limit,PALM
更新于2025-09-23 15:21:21
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Reference Module in Materials Science and Materials Engineering || Super Resolution Fluorescence Microscopy
摘要： Superresolution in microscopy has intrigued researchers for many years. There is always a challenge in overcoming what seems to be a fundamental limit. Even since the award of the Nobel prize in Chemistry in 2014 for stimulated emission depletion (STED) microscopy and localization microscopy, there has been continuing activity to improve these techniques, by reducing specimen radiation exposure for example. The term superresolution refers to overcoming the classical limit to resolution, usually defined in terms of either the Abbe resolution limit, or the Rayleigh two-point resolution criterion. These two measures are robust, the Abbe limit in particular providing a hard limit that can only be beaten by a fundamentally new approach, almost like a ‘trick’. Rayleigh recognized that his criterion was arbitrary, but considered it robust enough. Originally defined for incoherent imaging of self-luminous objects, its applicability has been extended to coherent and partially coherent systems, and later to confocal microscopy and other developments. This is usually achieved by the so-called ‘generalized Rayleigh resolution criterion’, according to which two points are taken as being just resolved when the intensity of the image midway between the points is 0.735 times that at the points themselves. It is found that around this value the image contrast changes quickly for a small change in the separation of the points.
关键词： Abbe resolution limit,Rayleigh criterion,Localization microscopy,STED,Superresolution,Microscopy
更新于2025-09-23 15:21:01
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Microscope laser assisted photooxidative activation of bioorthogonal ClickOx probes
摘要： A photoactivatable fluorogenic tetrazine-rhodaphenothiazine probe was synthesized and studied in light-assisted, bioorthogonal labeling schemes. Experimental results revealed that the bioorthogonally conjugated probe efficiently sensitizes 1O2 generation upon illumination with green or orange light and undergoes self-oxidation leading to an intensely fluorescent sulfoxide product. An added value of the present probe is that it is also suitable for STED super-resolution microscopy using a 660 nm depletion laser.
关键词： tetrazine-rhodaphenothiazine,fluorogenic,STED,super-resolution microscopy,photoactivatable,bioorthogonal,labeling
更新于2025-09-23 15:19:57
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Single Molecule Nonlinearity in a Plasmonic Waveguide
摘要： Plasmonic waveguides offer the unique possibility to confine light far below the diffraction limit. Past room temperature experiments focused on efficient generation of single waveguide plasmons by a quantum emitter. However, only the simultaneous interaction of the emitter with multiple plasmonic fields would lead to functionality in a plasmonic circuit. Here, we demonstrate the nonlinear optical interaction of a single molecule and propagating plasmons. An individual terrylene diimide (TDI) molecule is placed in the nanogap between two single-crystalline silver nanowires. A visible wavelength pump pulse and a red-shifted depletion pulse travel along the waveguide, leading to stimulated emission depletion (STED) in the observed fluorescence. The efficiency increases by up to a factor of 50 compared to far-field excitation. Our study thus demonstrates remote nonlinear four-wave mixing at a single molecule with propagating plasmons. It paves the way toward functional quantum plasmonic circuits and improved nonlinear single-molecule spectroscopy.
关键词： quantum emitter,nonlinear optics,stimulated emission depletion STED,two-wire transmission line,single-crystalline silver flake,plasmonic nanocircuit
更新于2025-09-23 15:19:57
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Green emitted CdSe@ZnS quantum dots for FLIM and STED imaging applications
摘要： Inorganic quantum dots (QDs) have excellent optical properties, such as high fluorescence intensity, excellent photostability and tunable emission wavelength, etc., facilitating them to be used as labels and probes for bioimaging. In this study, CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope (FLIM) and stimulated emission depletion (STED) nanoscopy imaging. The emission peak of CdSe@ZnS QDs centered at 526 nm with a narrow width of 19 nm and the photoluminescence quantum yield (PLQY) was 64%. The QDs presented excellent anti-photobleaching property which can be irradiated for 400 min by STED laser with 39.8 mW. The lateral resolution of 42.0 nm is demonstrated for single QDs under STED laser (27.5 mW) irradiation. Furthermore, the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5 nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes. Meanwhile, Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM, and their fluorescence lifetime was around 3.1 ns, consistent with the in vitro value, suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.
关键词： CdSe@ZnS QDs,living cells,STED,FLIM
更新于2025-09-19 17:13:59
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Protein‐specific, multi‐color and 3D STED imaging in cells with DNA‐labeled antibodies
摘要： Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal-to-noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed with exchangeable labels, which transiently bind to and off a target and thereby replenish destroyed labels by intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore-labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies. The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two-color STED imaging of whole cells.
关键词： multicolor imaging,DNA-PAINT,STED microscopy,fluorescence,fluorescent probes
更新于2025-09-19 17:13:59
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STED Direct Laser Writing of 45 nm Width Nanowire
摘要： Controlled fabrication of 45 nm width nanowire using simulated emission depletion (STED) direct laser writing with a rod-shape effective focus spot is presented. In conventional STED direct laser writing, normally a donut-shaped depletion focus is used, and the minimum linewidth is restricted to 55 nm. In this work, we push this limit to sub-50 nm dimension with a rod-shape effective focus spot, which is the combination of a Gaussian excitation focus and twin-oval depletion focus. Effects of photoinitiator type, excitation laser power, and depletion laser power on the width of the nanowire are explored, respectively. Single nanowire with 45 nm width is obtained, which is λ/18 of excitation wavelength and the minimum linewidth in pentaerythritol triacrylate (PETA) photoresist. Our result accelerates the progress of achievable linewidth reduction in STED direct laser writing.
关键词： STED direct laser writing,45 nm width,controlled fabrication,rod-shape effective focus
更新于2025-09-12 10:27:22
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The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy
摘要： Protein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy F?rster resonance energy transfer (fLiM-fRet) and stimulated emission depletion (SteD) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.
关键词： FLIM-FRET,living cells,magnetotactic bacteria,STED microscopy,protein mechanics
更新于2025-09-12 10:27:22
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Protein-specific, multi-color and 3D STED imaging in cells with DNA-labeled antibodies
摘要： Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal-to-noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed with exchangeable labels, which transiently bind to and off a target and thereby replenish destroyed labels by intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore-labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies. The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two-color STED imaging of whole cells.
关键词： multicolor imaging,STED microscopy,fluorescent probes,DNA-PAINT,fluorescence
更新于2025-09-11 14:15:04