222 research outputs found
Confocal microscopy with an increased detection aperture: type-B 4Pi confocal microscopy
Two-color STED microscopy reveals different degrees of colocalization between hexokinase-I and the three human VDAC isoforms
The voltage-dependent anion channel (VDAC, also known as mitochondrial porin) is the major transport channel mediating the transport of metabolites, including ATP, across the mitochondrial outer membrane. Biochemical data demonstrate the binding of the cytosolic protein hexokinase-I to VDAC, facilitating the direct access of hexokinase-I to the transported ATP. In human cells, three hVDAC isoforms have been identified. However, little is known on the distribution of these isoforms within the outer membrane of mitochondria and to what extent they colocalize with hexokinase-I. In this study we show that whereas hVDAC1 and hVDAC2 are localized predominantly within the same distinct domains in the outer membrane, hVDAC3 is mostly uniformly distributed over the surface of the mitochondrion. We used two-color stimulated emission depletion (STED) microscopy enabling a lateral resolution of ~40 nm to determine the detailed sub-mitochondrial distribution of the three hVDAC isoforms and hexokinase-I. Individual hVDAC and hexokinase-I clusters could thus be resolved which were concealed in the confocal images. Quantitative colocalization analysis of two-color STED images demonstrates that within the attained resolution, hexokinase-I and hVDAC3 exhibit a higher degree of colocalization than hexokinase-I with either hVDAC1 or hVDAC2. Furthermore, a substantial fraction of the mitochondria-bound hexokinase-I pool does not colocalize with any of the three hVDAC isoforms, suggesting a more complex interplay of these proteins than previously anticipated. This study demonstrates that two-color STED microscopy in conjunction with quantitative colocalization analysis is a powerful tool to study the complex distribution of membrane proteins in organelles such as mitochondria
Switchable Fluorescent and Solvatochromic Molecular Probes Based on 4-Amino-N-methylphthalimide and a Photochromic Diarylethene
New fluorescent photochromic compounds (1-H and 1-Boc)have been synthesized and characterized in different solvents.The fluorescence emission can be switched âonâ andâoffâ with visible light and UV, respectively, by means of thephotochromic reaction. The emission wavelength and efficiencystrongly depend on the polarity of the solvent. Thecompounds show a positive solvatochromic effect in theemission maxima, and their fluorescence quantum yield decreasesas the solventâs polarity increases (from cyclohexaneto dioxane). In solvents more polar than dioxane the emissionis too weak and therefore undetectable, and thus 1-H and 1-Boc behave as ânormalâ photochromic compounds. The photochromic reaction is also sensitive to the environment. A decreaseof more than an order of magnitude was found for thequantum yield of the colouring reaction (ΊOFCF) for 1-H inethanol compared with cyclohexane, and an about threefolddecrease in ΊOFCF was observed for the compound 1-Bocin polar solvents (compared with apolar solvents). For bothcompounds the ring-opening reaction was found not to dependenton the solvent. The novel fluorescent molecularswitches 1-H and 1-Boc are able to probe the polarity of theirmicroenvironment.Fil: Yan, Sergey F.. Max Planck Institute for Biophysical Chemistry; AlemaniaFil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry; AlemaniaFil: Bossi, Mariano Luis. Max Planck Institute for Biophysical Chemistry; Alemania. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de QuĂmica, FĂsica de los Materiales, Medioambiente y EnergĂa. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de QuĂmica, FĂsica de los Materiales, Medioambiente y EnergĂa; ArgentinaFil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemani
Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters
We demonstrate nanoscale resolution in far-field fluorescence microscopy using reversible photoswitching and localization of individual fluorophores at comparatively fast recording speeds and from the interior of intact cells. These advancements have become possible by asynchronously recording the photon bursts of individual molecular switching cycles. We present images from the microtubular network of an intact mammalian cell with a resolution of 40 nm
Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane â a minimally invasive investigation by STED-FCS
Photoactivatable Fluorophore for Stimulated Emission Depletion (STED) Microscopy and Bioconjugation Technique for Hydrophobic Labels
The use of photoactivatable dyes in STED microscopy has so far been limited by twoâphoton activation through the STED beam and by the fact that photoactivatable dyes are poorly solvable in water. Here we report ONBâ2SiR, a fluorophore that can be both photoactivated in the UV and specifically deâexcited by STED at 775 nm. Likewise, we introduce a conjugation and purification protocol to effectively label primary and secondary antibodies with moderately waterâsoluble dyes. Greatly reducing dye aggregation, our technique provides a defined and tunable degree of labeling, and improves the imaging performance of dye conjugates in general
STED Nanoscopy Combined with Optical Tweezers Reveals Spatial Dynamics of Proteins on Densely Covered DNA
STED nanoscopy of the centrosome linker reveals a CEP68-organized, periodic rootletin network anchored to a C-Nap1 ring at centrioles
The centrosome linker proteins C-Nap1, rootletin, and CEP68 connect the two centrosomes of a cell during interphase into one microtubule-organizing center. This coupling is important for cell migration, cilia formation, and timing of mitotic spindle formation. Very little is known about the structure of the centrosome linker. Here, we used stimulated emission depletion (STED) microscopy to show that each C-Nap1 ring at the proximal end of the two centrioles organizes a rootletin ring and, in addition, multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers originating from the two centrosomes form a web-like, interdigitating network, explaining the flexible nature of the centrosome linker. The rootletin/CEP68 filaments are repetitive and highly ordered. Staggered rootletin molecules (N-to-N and C-to-C) within the filaments are 75 nm apart. Rootletin binds CEP68 via its C-terminal spectrin repeat-containing region in 75-nm intervals. The N-to-C distance of two rootletin molecules is âŒ35 to 40 nm, leading to an estimated minimal rootletin length of âŒ110 nm. CEP68 is important in forming rootletin filaments that branch off centrioles and to modulate the thickness of rootletin fibers. Thus, the centrosome linker consists of a vast network of repeating rootletin units with C-Nap1 as ring organizer and CEP68 as filament modulator
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