Super‐resolution imaging prompts re‐thinking of cell biology mechanisms

Saka, Sinem K.; Rizzoli, Silvio O.

doi:10.1002/bies.201100080

Cited by 22 publications

(5 citation statements)

References 57 publications

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“…For preservation of cytoskeletal elements, such as tubulin, two alternatives are useful: either 100% (wt/wt) methanol at −20 °C for 20 min, or 1 min of 0.3% (vol/vol) glutaraldehyde in extraction buffer at 37 °C, followed by 10 min of 2% (vol/vol) glutaraldehyde in cytoskeleton buffer at room temperature 19,20 Target structures are labeled discontinuously This can be due to poor probe coverage of the target structures during immunostaining (Step 4) Coverage of dense and continuous target structures with imaging probes can be incomplete due to steric hindrance, limited epitope accessibility, low affinity of probes, denaturation of epitopes, and so on. This is a problem not specific to expansion microscopy but that is encountered in all super-resolution methods 14,52 . This problem can sometimes be addressed by increasing probe concentration or extending labeling time.…”

Section: The Polymerization Reaction Proceeded Too Quicklymentioning

confidence: 99%

“…Super-resolution imaging has revolutionized biological research in a surprisingly short period of time [13][14][15][16][17] . It has not only enabled direct visualization of the molecular organization of biological systems and the discovery of biological phenomena that went hitherto unnoticed [18][19][20][21][22][23][24][25][26][27] , but it has also resulted in a rethinking of how to obtain and interpret imaging data.…”

Section: Introductionmentioning

confidence: 99%

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A practical guide to optimization in X10 expansion microscopy

et al. 2019

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Expansion microscopy is a relatively new approach to super-resolution imaging that uses expandable hydrogels to isotropically increase the physical distance between fluorophores in biological samples such as cell cultures or tissue slices. The classic gel recipe results in an expansion factor of ~4×, with a resolution of 60-80 nm. We have recently developed X10 microscopy, which uses a gel that achieves an expansion factor of ~10×, with a resolution of ~25 nm. Here, we provide a step-by-step protocol for X10 expansion microscopy. A typical experiment consists of seven sequential stages: (i) immunostaining, (ii) anchoring, (iii) polymerization, (iv) homogenization, (v) expansion, (vi) imaging, and (vii) validation. The protocol presented here includes recommendations for optimization, pitfalls and their solutions, and detailed guidelines that should increase reproducibility. Although our protocol focuses on X10 expansion microscopy, we detail which of these suggestions are also applicable to classic fourfold expansion microscopy. We exemplify our protocol using primary hippocampal neurons from rats, but our approach can be used with other primary cells or cultured cell lines of interest. This protocol will enable any researcher with basic experience in immunostainings and access to an epifluorescence microscope to perform super-resolution microscopy with X10. The procedure takes 3 d and requires ~5 h of actively handling the sample for labeling and expansion, and another ~3 h for imaging and analysis.

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Section: The Polymerization Reaction Proceeded Too Quicklymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A practical guide to optimization in X10 expansion microscopy

et al. 2019

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“…Super resolution (SR) microscopy unlocked new opportunities for cell biologists to investigate cells and cellular functions at unprecedented resolution up to few nanometers, which require re-thinking of biologists about new and previous discoveries [1]. SR microscopy visualizing single molecules clusters at nanometer resolution has made image analysis a more complicated practice.…”

Section: Introductionmentioning

confidence: 99%

CellProfiler: Novel Automated Image Segmentation Procedure for Super-Resolution Microscopy

Soliman

2015

Biol Proced Online

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BackgroundSuper resolution (SR) microscopy enabled cell biologists to visualize subcellular details up to 20 nm in resolution. This breakthrough in spatial resolution made image analysis a challenging procedure. Direct and automated segmentation of SR images remains largely unsolved, especially when it comes to providing meaningful biological interpretations.ResultsHere, we introduce a novel automated imaging analysis routine, based on Gaussian, followed by a segmentation procedure using CellProfiler software (www.cellprofiler.org). We tested this method and succeeded to segment individual nuclear pore complexes stained with gp210 and pan-FG proteins and captured by two-color STED microscopy. Test results confirmed accuracy and robustness of the method even in noisy STED images of gp210.ConclusionsOur pipeline and novel segmentation procedure may benefit end-users of SR microscopy to analyze their images and extract biologically significant quantitative data about them in user-friendly and fully-automated settings.

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“…This diffraction barrier, however, has been overcome by the invention and development of super-resolution or diffraction-unlimited fluorescence imaging techniques within the last two decades [ 4 – 6 ]. The manner in which the higher precision of nanoscopic information helps in understanding biological processes has been reviewed recently [ 7 – 9 ].…”

Section: Introductionmentioning

confidence: 99%

Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope

2015

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By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as “easy-STED”, achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating.

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Super‐resolution imaging prompts re‐thinking of cell biology mechanisms

Cited by 22 publications

References 57 publications

A practical guide to optimization in X10 expansion microscopy

A practical guide to optimization in X10 expansion microscopy

CellProfiler: Novel Automated Image Segmentation Procedure for Super-Resolution Microscopy

Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope

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