Construction of an instant structured illumination microscope

Curd, Alistair; Cleasby, Alexa J.; Makowska, Katarzyna; York, Andrew G.; Shroff, Hari; Peckham, Michelle

doi:10.1016/j.ymeth.2015.07.012

Cited by 36 publications

(40 citation statements)

References 15 publications

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“…To visualize the excitation illumination of the iSIM, we used a concentrated dye solution 28 , and imaged the emitted light onto the camera without scanning ( Supplemental information, Supplemental Figure 4a ). Using an approximately Gaussian beam (M 2 <1.1, FWHM diameter ∼12 mm) to create the excitation pattern, as used in the initial iSIM design 9,29 , results in excitation points of spatially varying intensity closely following a Gaussian distribution ( Figure 2a ), as expected from the simulation ( Figure 1a ). Scanning the Gaussian excitation points partially homogenizes the excitation along the scan direction; however, the resulting illumination features a bright central region and strong roll-off away from the central optical axis ( Supplemental information, Supplemental Figure 5a,b ).…”

Section: Resultsmentioning

confidence: 56%

“…The iSIM setup was partly based on previously described implementations 9,29 . Two lasers with wavelengths of 488 nm (Sapphire 488-300 CW CDRH, Coherent) and 561 nm (gem 561, Laser Quantum GmbH) were combined using a dichroic mirror (F48-486, Analysentechnik) and controlled through an acousto-optic tunable filter (AOTFnC-400.650-TN, AA Optoelectronic).…”

Section: Methodsmentioning

confidence: 99%

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Homogeneous multifocal excitation for high-throughput super-resolution imaging

Mahecic

Gambarotto

Douglass

et al. 2020

Preprint

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19Super-resolution microscopies, which allow features below the diffraction limit to be 20 resolved, have become an established tool in biological research. However, imaging 21 throughput remains a major bottleneck in using them for quantitative biology, which requires 22 large datasets to overcome the noise of the imaging itself and to capture the variability 23 inherent to biological processes. Here, we develop a multi-focal flat illumination for field 24 independent imaging (mfFIFI) module, and integrate it into an instant structured illumination 25 microscope (iSIM). Our instrument extends the field of view (FOV) to >100x100 µm 2 without 26 compromising image quality, and maintains high-speed (100 Hz), multi-color, volumetric 27 imaging at double the diffraction-limited resolution. We further extend the effective FOV by 28 stitching multiple adjacent images together to perform fast live-cell super-resolution imaging 29 of dozens of cells. Finally, we combine our flat-fielded iSIM setup with ultrastructure 30 expansion microscopy (U-ExM) to collect 3D images of hundreds of centrioles in human 31 cells, as well as of thousands of purified Chlamydomonas reinhardtii centrioles per hour at 32 an effective resolution of ~35 nm. We apply classification and particle averaging to these 33 large datasets, allowing us to map the 3D organization of post-translational modifications of 34 centriolar microtubules, revealing differences in their coverage and positioning. 35 (STED) 5 . Initial implementations of these methods were relatively slow, in part due to the 42 use of the time domain to separate single molecules (SMLM), the need to collect multiple 43 images to cover Fourier space (SIM), or the scanning of a reduced excitation volume 44 3 (STED). In both wide-field and patterned illumination, the imaging throughput -defined as 45 the area imaged per time -can be increased by parallelizing the acquisition, as long as the 46 necessary illumination can be maintained over a larger surface in the sample plane. 47Extending the array of patterned excitation has been used to increase the speed or FOV of 48 confocal 6 , STED 7,8 and SIM imaging 9 , but ensuring the quality of the extended pattern 49 across a larger FOV remains a limiting factor. For SMLM, flat-fielding approaches made it 50 possible to extend super-resolution imaging over ~100x100 µm 2 FOVs 10,11 , but their transfer 51 to patterned illumination remains limited. 52An entirely different approach to effectively achieve super-resolution modifies 53 sample preparation rather than image acquisition. By physically increasing the size of the 54 specimen in an isotropic manner via expansion microscopy 12 (ExM), overall imaging 55 throughput can be increased by opting for faster diffraction-limited microscopes 12-14 . 56 Alternatively, combining ExM with existing super-resolution microscopies allows even 57 further improvement in resolution [14][15][16][17][18] , since the resolvable scale is reduced by the 58 expansion factor. However, expansion exacerbates limita...

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Section: Resultsmentioning

confidence: 56%

Section: Methodsmentioning

confidence: 99%

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Mahecic

Gambarotto

Douglass

et al. 2020

Preprint

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“…For iSIM experiments, imaging was performed on a custom-built microscope setup as previously described [60,61]. The microscope was equipped with a 1.49 NA oil immersion objective (APONXOTIRF;…”

Section: Mitochondrial Fission Differs From Other Fission Processes Smentioning

confidence: 99%

Mitochondrial membrane tension governs fission

Mahecic

Carlini

Kleele

et al. 2018

Preprint

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2Mitochondria rely on cellular machinery for their division, which is an essential component of metabolic response of the cell. Many division factors have been identified; however, a framework accounting for the energetic requirements of the mitochondrial fission process is lacking. We report that the presence of an active constriction does not ensure fission. Instead, by measuring constrictions down to ~100 nm with time-lapse super-resolution microscopy, we found that 34% of constrictions failed to divide and 'reversed' to an unconstricted state. Higher local curvaturesreflecting an increased bending energy -made constriction sites more likely to divide, but often with a significant residual energy barrier to fission. Our data suggest that membrane tension, largely arising from pulling forces, could account for this missing energy. These results lead us to propose that mitochondrial fission is probabilistic, and can be modeled as arising from bending energy complemented by a fluctuating membrane tension.Mitochondria are highly dynamic organelles, transported through the cytoplasm along cytoskeletal networks as they change in size and shape. Mitochondrial morphologies can range from a filamentous, connected network to a fragmented collection of individuals. Underlying such morphological changes are altered equilibria between fusion and division 1,2 . These transformations have been linked to an adaptive response to cellular energy requirements, for example in response to stress [3][4][5] or the cell cycle 6 . As a vestige of their bacterial origins, mitochondria cannot be generated de novo, but must multiply by division, or fission, of existing mitochondria 7 . Division has also been suggested to act as part of a quality control mechanism 8,9 and an intracellular signal for mitophagy 10,11 .All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/255356 doi: bioRxiv preprint first posted online 3 In bacterial division systems, internal assembly of the fission machinery is tightly regulated and a series of cell cycle checkpoints ensure daughter cell viability. In contrast, the mitochondrial division machinery is external to the organelle, allowing cells to flexibly regulate fission. Initially, the division site is marked by a pre-constriction defined by contact with ER tubules 12 and deformed by targeted actin polymerization [13][14][15] Live-cell Structured Illumination Microscopy (SIM) of Drp1-mediated mitochondrial constriction events enabled us to measure dynamic changes in membrane geometry leading up to and following division. We discovered that a fraction of mitochondria constrict dramatically before relaxing to an unconstricted state, termed 'reversals'. Using a custom-written image analysis package and classical elasticity theory, we calculated the energy differences between fission and reversal events, which al...

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“…To investigate the effect of CCG-1423 treatment on glioma cells at higher resolution and in 3D, we used instant structured illuminated microscopy (iSIM), which captures images at high spatio-temporal resolution (approx. 150 nm, greater than 100 fps), allowing rapid capture of high-resolution volumes (Z-stacks) [6,7]. Having labelled spheroids encased in collagen (see Methods/electronic supplementary material), we acquired 3D images of single migratory cells.…”

Section: Introductionmentioning

confidence: 99%

A novel workflow for three-dimensional analysis of tumour cell migration

et al. 2020

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One contribution of 7 to a theme issue '3D biological cultures and organoids'.The limitations of two-dimensional analysis in three-dimensional (3D) cellular imaging impair the accuracy of research findings in biological studies. Here, we report a novel 3D approach to acquisition, analysis and interpretation of tumour spheroid images. Our research interest in mesenchymal-amoeboid transition led to the development of a workflow incorporating the generation and analysis of 3D data with instant structured illumination microscopy and a new ImageJ plugin.

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Construction of an instant structured illumination microscope

Abstract: Graphical abstract

Cited by 36 publications

References 15 publications

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Mitochondrial membrane tension governs fission

A novel workflow for three-dimensional analysis of tumour cell migration

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