Features of endogenous cardiomyocyte chromatin revealed by super‐resolution STED microscopy

AbstractStimulated emission depletion (STED) microscopy is routinely used to resolve the ultra-structure of cells with a ∼10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides sub-diffraction resolution by physically enlarging the sample before microscopy. Expansion of fixed cells by crosslinking and swelling of hydrogels easily enlarges the sample ∼4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complimentary approaches, we here combined ExM with STED (ExSTED) and demonstrate an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ∼50 nm isotropic). While the increase in resolution is straight forward, we found that high fidelity labelling via multi-epitopes is required to obtain emitter densities that allow to resolve ultra-structural details with ExSTED. Our work provides a robust template for super resolution microscopy of entire cells in the ten nanometer range.

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Super-Resolution Imaging of the A- and B-Type Lamin Networks: A Comparative Study of Different Fluorescence Labeling Procedures

International Journal of Molecular Sciences ◽

10.3390/ijms221910194 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10194

Author(s):

Merel Stiekema ◽

Frans C. S. Ramaekers ◽

Dimitrios Kapsokalyvas ◽

Marc A. M. J. van Zandvoort ◽

Rogier J. A. Veltrop ◽

...

Keyword(s):

Layer Thickness ◽

Super Resolution ◽

Dermal Fibroblasts ◽

Stimulated Emission ◽

Immunofluorescence Staining ◽

Fluorescence Labeling ◽

Confocal Scanning Laser Microscopy ◽

Lamin A ◽

Intermediate Filament Proteins ◽

Sted Microscopy

A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G>T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.

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STED super-resolution imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes

10.1101/2021.06.05.446432 ◽

2021 ◽

Author(s):

Pablo Carravilla ◽

Anindita Dasgupta ◽

Gaukhar Zhurgenbayeva ◽

Dmytro I. Danylchuk ◽

Andrey S. Klymchenko ◽

...

Keyword(s):

Plasma Membrane ◽

Real Time ◽

Spatial Resolution ◽

Super Resolution ◽

Membrane Dynamics ◽

Live Cell ◽

Stimulated Emission ◽

Sted Microscopy ◽

Temporal And Spatial ◽

Polarity Sensitive

Understanding the plasma membrane nano-scale organisation and dynamics in living cells requires microscopy techniques with high temporal and spatial resolution and long acquisition times, that also allow for the quantification of membrane biophysical properties such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolution, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile Red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dyes is showcased by live-cell real-time 3D-STED recordings of bleb formation and lipid exchange during membrane fusion, as well as by STED-fluorescence correlation spectroscopy (STED-FCS) experiments for the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live-cell membranes.

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Polarity sensitive probes for super resolution STED microscopy

10.1101/107334 ◽

2017 ◽

Author(s):

E Sezgin ◽

F Schneider ◽

V Zilles ◽

E Garcia ◽

D Waithe ◽

...

Keyword(s):

Plasma Membrane ◽

Lipid Membrane ◽

Membrane Lipids ◽

Super Resolution ◽

Membrane Vesicles ◽

Molecular Organization ◽

Live Cells ◽

Sted Microscopy ◽

Two Photon Microscopy ◽

Polarity Sensitive

AbstractThe lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed (or ordered). Polarity sensitive dyes are powerful tools to characterize such lipid membrane order, employing for example confocal and two-photon microscopy. The investigation of potential lipid nanodomains, however, requires the use of super resolution microscopy. Here, we test the performance of the polarity sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA and NR12S in super resolution STED microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles show the high potential of these dyes for probing nanoscale membrane heterogeneity.

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Photobleaching Reduction in Modulated Super Resolution Microscopy

Microscopy ◽

10.1093/jmicro/dfaa062 ◽

2020 ◽

Author(s):

Jafar H Ghithan ◽

Jennifer M Noel ◽

Thomas J Roussel ◽

Maureen A McCall ◽

Bruce W Alphenaar ◽

...

Keyword(s):

Continuous Wave ◽

Imaging Techniques ◽

Fluorescence Emission ◽

Super Resolution ◽

Optical Power ◽

Stimulated Emission ◽

Laser Beams ◽

Synchronous Detection ◽

Major Drawback ◽

Sted Microscopy

Abstract Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases of photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive-index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.

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