Fluorescence microscopy coupled to electrochemistry: a powerful tool for the controlled electrochemical switch of fluorescent molecules

2008 ◽  
pp. 1913 ◽  
Author(s):  
Fabien Miomandre ◽  
Rachel Meallet-Renault ◽  
Jean-Jacques Vachon ◽  
Robert Bernard Pansu ◽  
Pierre Audebert
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Molecules

scholarly journals Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent

2017 ◽  
Vol 114 (9) ◽  
pp. 2125-2130 ◽  
Author(s):  
Fabian Göttfert ◽  
Tino Pleiner ◽  
Jörn Heine ◽  
Volker Westphal ◽  
Dirk Görlich ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Stimulated Emission ◽  
Limiting Factor ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Initial State ◽  
Intensity Pattern ◽  
Optical Fluorescence ◽  
Pore Complexes ◽  
Fluorescent Molecules

Photobleaching remains a limiting factor in superresolution fluorescence microscopy. This is particularly true for stimulated emission depletion (STED) and reversible saturable/switchable optical fluorescence transitions (RESOLFT) microscopy, where adjacent fluorescent molecules are distinguished by sequentially turning them off (or on) using a pattern of light formed as a doughnut or a standing wave. In sample regions where the pattern intensity reaches or exceeds a certain threshold, the molecules are essentially off (or on), whereas in areas where the intensity is lower, that is, around the intensity minima, the molecules remain in the initial state. Unfortunately, the creation of on/off state differences on subdiffraction scales requires the maxima of the intensity pattern to exceed the threshold intensity by a large factor that scales with the resolution. Hence, when recording an image by scanning the pattern across the sample, each molecule in the sample is repeatedly exposed to the maxima, which exacerbates bleaching. Here, we introduce MINFIELD, a strategy for fundamentally reducing bleaching in STED/RESOLFT nanoscopy through restricting the scanning to subdiffraction-sized regions. By safeguarding the molecules from the intensity of the maxima and exposing them only to the lower intensities (around the minima) needed for the off-switching (on-switching), MINFIELD largely avoids detrimental transitions to higher molecular states. A bleaching reduction by up to 100-fold is demonstrated. Recording nanobody-labeled nuclear pore complexes in Xenopus laevis cells showed that MINFIELD-STED microscopy resolved details separated by <25 nm where conventional scanning failed to acquire sufficient signal.

scholarly journals Accuracy and precision in quantitative fluorescence microscopy

2009 ◽  
Vol 185 (7) ◽  
pp. 1135-1148 ◽  
Author(s):  
Jennifer C. Waters
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Imaging System ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Digital Cameras ◽  
Accuracy And Precision ◽  
Fluorescent Molecules ◽  
Quantitative Fluorescence ◽  
Biology Research

The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.

scholarly journals Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter

2020 ◽  
pp. 202001849 ◽  
Author(s):  
Peter D. Dahlberg ◽  
Saumya Saurabh ◽  
Annina M. Sartor ◽  
Jiarui Wang ◽  
Patrick G. Mitchell ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Asymmetric Cell Division ◽  
Caulobacter Crescentus ◽  
Electron Tomography ◽  
Integral Membrane Protein ◽  
Single Molecule Fluorescence ◽  
Cellular Context ◽  
Specific Proteins ◽  
Fluorescent Molecules

Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ∼30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.

scholarly journals Solving the boundary artifact for the enhanced deconvolution algorithm SUPPOSe applied to fluorescence microscopy

2021 ◽  
Vol 45 (3) ◽  
pp. 418-426
Author(s):  
M. Toscani ◽  
S. Martínez
Keyword(s):  
Fluorescence Microscopy ◽  
Point Sources ◽  
Total Flux ◽  
Image Frame ◽  
Image Source ◽  
Recent Advances ◽  
Deconvolution Algorithm ◽  
Virtual Point ◽  
Fluorescent Molecules

The SUPPOSe enhanced deconvolution algorithm relies in assuming that the image source can be described by an incoherent superposition of virtual point sources of equal intensity and finding the number and position of such virtual sources. In this work we describe the recent advances in the implementation of the method to gain resolution and remove artifacts due to the presence of fluorescent molecules close enough to the image frame boundary. The method was modified removing the invariant used before given by the product of the flux of the virtual sources times the number of virtual sources, and replacing it by a new invariant given by the total flux within the frame, thus allowing the location of virtual sources outside the frame but contributing to the signal inside the frame.

scholarly journals Three Dimensional Orientation Measurements of Single Fluorescent Molecules by Newly Developed Polarized Fluorescence Microscopy

2011 ◽  
Vol 100 (3) ◽  
pp. 116a
Author(s):  
Masashi Ohmachi ◽  
Yasunori Komori ◽  
Atsuko Iwane ◽  
Fumihiko Fujii ◽  
Toshio Yanagida
Keyword(s):  
Fluorescence Microscopy ◽  
Three Dimensional ◽  
Polarized Fluorescence ◽  
Fluorescent Molecules

scholarly journals 3D Adaptive Optical Nanoscopy for Thick Specimen Imaging at sub-50 nm Resolution

2020 ◽  
Author(s):  
Xiang Hao ◽  
Edward S. Allgeyer ◽  
Jacopo Antonello ◽  
Katherine Watters ◽  
Julianne A. Gerdes ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Adaptive Optics ◽  
Diffraction Limit ◽  
Three Dimensions ◽  
Cellular Organization ◽  
Optical Aberrations ◽  
Isotropic Resolution ◽  
Neuronal Synapses ◽  
Ring Canals ◽  
Fluorescent Molecules

Understanding cellular organization demands the best possible spatial resolution in all three dimensions (3D). In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here, we have developed a nanoscope that, by utilizing an advanced adaptive optics strategy, achieves sub-50 nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue.

Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

1988 ◽  
Vol 46 ◽  
pp. 118-119
Author(s):  
K. Jacobson ◽  
A. Ishihara ◽  
B. Holifield ◽  
F. Zhang
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Extended Period ◽  
Dynamic Structure ◽  
Living Cells ◽  
Membrane Dynamics ◽  
Molecular Cell Biology ◽  
Image Averaging ◽  
Single Living Cells ◽  
Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

Scanning x-ray fluorescence microscopy and principal component analysis

1992 ◽  
Vol 50 (2) ◽  
pp. 1752-1753
Author(s):  
Brian Cross
Keyword(s):  
Principal Component Analysis ◽  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
High Speed ◽  
Image Acquisition ◽  
Principal Component ◽  
Fluorescence Microscope ◽  
Acquisition Rate ◽  
X Ray ◽  
Speed Up

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.

Two-photon excitation fluorescence microscopy in living systems

1993 ◽  
Vol 51 ◽  
pp. 154-155 ◽  
Author(s):  
David W. Piston
Keyword(s):  
Confocal Microscopy ◽  
Fluorescence Microscopy ◽  
Singlet State ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In our fluorescence experiments, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

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