Metallic support films reduce optical heating in cryogenic correlative light and electron tomography

Dahlberg, Peter D.; Perez, Davis; Hecksel, Corey W.; Chiu, Wah; Moerner, W. E.

doi:10.1016/j.jsb.2022.107901

Cited by 16 publications

(20 citation statements)

References 49 publications

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“…The second option is to replace the carbon as the material for the support film. Recently, metal support films have proven useful to inhibit devitrification for cryo-light microscopy of bacterial samples 26 . Here we extend this to additional support films for intracellular mammalian proteins.…”

Section: Discussionmentioning

confidence: 99%

Selecting optimal support grids for super-resolution cryogenic correlated light and electron microscopy

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Tuijtel

Voortman

et al. 2022

Preprint

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Cryogenic transmission electron microscopy (cryo-TEM) and super-resolution fluorescence microscopy (FM) are two popular and ever improving methods for high-resolution imaging of biological samples. In recent years, the combination of these two techniques into one correlated workflow has gained attention as a promising route towards contextualizing and enriching cryo-TEM imagery. A problem that is often encountered in the combination of these methods is that of light-induced damage to the sample during fluorescence imaging that renders the sample structure unsuitable for TEM imaging. In this paper, we describe how absorption of light by TEM sample support grids leads to sample damage, and we systematically explore the importance of parameters of grid design. We explain how, by changing the grid geometry and materials, one can increase the maximum illumination power density in fluorescence microscopy by up to an order of magnitude, and demonstrate the significant improvements in super-resolution image quality that are enabled by the selection of support grids that are optimally suited for correlated microscopy.

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Section: Discussionmentioning

confidence: 99%

Selecting optimal support grids for super-resolution cryogenic correlated light and electron microscopy

Last

Tuijtel

Voortman

et al. 2022

Preprint

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“…However, compared to the previously used PAmKate, this photoactivation pathway in mApple appears to be very inefficient−only a small fraction of initially active emitters can be recovered to an "on" state after initial bleach-down, even when using the increased optical intensities recently made possible by advanced metallic sample substrates (Figure S4). 9 The characterization of single emitters in data sets such as those collected with this low-intensity cryogenic imaging technique is often complicated by the overcounting of emitters, which occurs when molecules remain in a bright state and are localized in a large number of frames. This can be addressed by pooling the photons over many frames, but there can be an additional complication arising from overlapping emitters.…”

Section: The Journal Of Physical Chemistrymentioning

confidence: 99%

“…Recently, there has been a growing interest in performing superresolution under cryogenic conditions. Cryogenic fluorescence imaging can be performed in a manner amenable for correlation with cryogenic electron tomography (CryoET), combining the benefits of specific labeling in fluorescence microscopy with the high-resolution cellular context provided by electron microscopy. − However, a key limitation continues to be the dearth of fluorophores with access to efficient active control mechanisms required for SMACM within the limits imposed on the optical intensities to prevent damage of the plunge-frozen sample through melting . Historically, many initial breakthroughs in single-molecule spectroscopy and imaging were achieved through cooling of rigid organic fluorophores in solid hosts to cryogenic temperatures. − Later, when studies of green fluorescent protein (GFP) introduced FPs as candidates for the in situ labeling of proteins in a wide range of biological systems, low-temperature spectroscopy and hole burning studies of GFP mutants provided key insights into the electronic states contributing toward its observed photoswitching behavior. − Understanding of the optical control of this photoswitching was crucial in developing engineered FPs, such as the photoactivatable PA-GFP, establishing the necessary sparsity of active emitters underpinning a wide range of superresolution microscopy techniques relying on single-molecule active control mechanisms. − …”

Section: Introductionmentioning

confidence: 99%

Characterization of mApple as a Red Fluorescent Protein for Cryogenic Single-Molecule Imaging with Turn-Off and Turn-On Active Control Mechanisms

et al. 2023

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Single-molecule superresolution microscopy is a powerful tool for the study of biological structures on size scales smaller than the optical diffraction limit. Imaging samples at cryogenic temperatures (77 K) reduces the quantum yield of photobleaching for many fluorescent labels, yielding localization precisions below 10 nm. Cryogenic imaging further enables correlation with cryogenic electron tomography. A key limitation in applying methods such as PALM and STORM to samples maintained at 77 K is the limited number of fluorophores known to undergo efficient turn-on and turn-off mechanisms necessary to control the sparsity of active emitters. We find that mApple, a redemitting fluorescent protein, undergoes a novel turn-off mechanism in response to simultaneous illumination with two colors of light. This turn-off mechanism enables localization of many individual molecules in initially bright samples, but the final density of localizable emitters is limited by relatively inefficient turn-on (photoactivation). Bulk excitation and emission spectroscopy shows that mApple has access to two distinct emissive states as well as dark states accessible optically or through changes in pH. The bright and stable emission of mApple enables widefield collection of single-molecule emission spectra, which highlight the complex nature and environmental sensitivity of states observed in red fluorescent proteins.

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Section: Introductionmentioning

confidence: 99%

“…Recent reports demonstrate that by avoiding optical heating of samples in correlated fluorescence and transmission electron cryo-microscopy, high illumination power densities can be used that enable better super-resolution fluorescence imaging 1,2 . While this rules out devitrification as a mode of damaging the sample, it is not clear whether fluorescence inspection of a sample prior to cryoEM imaging damages the sample via other possible mechanisms, such as via the illumination of highly absorptive fluorescent proteins (FPs) with intense laser light.…”

Section: Introductionmentioning

confidence: 99%

Super-resolution fluorescence imaging of cryosamples does not limit achievable resolution in cryoEM

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Noteborn

Voortman

et al. 2023

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Correlated super-resolution cryo-fluorescence and cryo-electron microscopy (cryoEM) has been gaining popularity as a method to investigate biological samples with high resolution and specificity. A concern in this combined method (called SR-cryoCLEM), however, is whether and how fluorescence imaging prior to cryoEM acquisition is detrimental to sample integrity. In this report, we investigated the effect of high-dose laser light irradiation on apoferritin samples prepared for cryoEM with excitation wavelengths commonly used in fluorescence microscopy, and comparing these samples to controls that were kept in the dark. We found that laser illumination, of equal duration and intensity as used in super-resolution cryomicroscopy and in the presence of high concentrations of fluorescent protein, did not affect the achievable resolution in cryoEM, with final reconstructions reaching resolutions of ~1.8 Angstrom regardless of the illumination conditions. The finding that super-resolution fluorescence imaging of cryosamples prior to cryoEM data acquisition does not limit the achievable resolution suggests that super-resolution cryo-fluorescence microscopy and in situ structural biology using cryoEM are entirely compatible.

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Metallic support films reduce optical heating in cryogenic correlative light and electron tomography

Cited by 16 publications

References 49 publications

Selecting optimal support grids for super-resolution cryogenic correlated light and electron microscopy

Selecting optimal support grids for super-resolution cryogenic correlated light and electron microscopy

Characterization of mApple as a Red Fluorescent Protein for Cryogenic Single-Molecule Imaging with Turn-Off and Turn-On Active Control Mechanisms

Super-resolution fluorescence imaging of cryosamples does not limit achievable resolution in cryoEM

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