A general method to fine-tune fluorophores for live-cell and<i>in vivo</i>imaging

Grimm, Jonathan B.; Muthusamy, Anand K.; Liang, Yajie; Brown, Timothy A.; Lemon, William C.; Patel, Ronak; Lu, Rongwen; Keller, Phillip J.; Ji, Na; Lavis, Luke D.

doi:10.1101/127613

Cited by 27 publications

(32 citation statements)

References 48 publications

(71 reference statements)

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“…We used cryo-SIM to visualize transiently expressed junctional adhesion molecule (JAM)-C (64,66,69), a tight-junction component, fused to JF549i-conjugated SNAP- (35,70), and 2x-mVenus-drebrin, a cytoplasmic actin-microtubule crosslinker protein (71), in cryofixed mouse cerebellar granule neurons (CGNs) ( Fig. 6A , Movie 5).…”

Section: Molecular Underpinnings Of Ultrastructural Specialization Inmentioning

confidence: 99%

Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

Hoffman

Shtengel

et al. 2019

Preprint

119

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words):Living cells function through the spatial compartmentalization of thousands of distinct proteins serving a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as a pathway to directly view nanoscale protein relationships to the underlying global ultrastructure, but has traditionally suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional correlative cryogenic SR and focused ion beam milled block-face EM across entire vitreously frozen cells that addresses these issues by preserving native ultrastructure and enabling independent SR and EM workflow optimization. Application to a variety of biological systems revealed a number of unexpected protein-ultrastructure relationships and underscored the value of a comprehensive multimodal view of ultrastructural variability across whole cells. modalities (supplementary note 1, table S1), allowing specific molecular components to be visualized at nanoscale resolution in the context of the crowded intracellular environment.However, SR/EM correlation often involves tradeoffs in sample preparation between the retention of fluorescent labels, sufficiently dense heavy metal staining for high contrast EM, and faithful preservation of ultrastructure, particularly when chemical fixation is used (19)(20)(21)(22).Here we describe a pipeline ( fig. S1) for correlative cryo-SR/FIB-SEM imaging of whole cells designed to address these issues. Specifically, cryogenic, as opposed to room temperature, SR performed after high pressure freezing (HPF), allowed us to use a standard EM sample preparation protocol without compromise. We used cryogenic 3D structured illumination (SIM) and single molecule localization (SMLM) microscopy for SR protein specific contrast with 3D FIB-SEM for global contrast of subcellular ultrastructure. The SR modality highlights features not readily apparent from the EM data alone, such as exceptionally long or convoluted endosomes, and permits unique classification of vesicles of like morphology, such as lysosomes, peroxisomes, and mitochondrial-derived vesicles. Cell-wide 3D correlation also reveals unexpected localization patterns of proteins, including intranuclear vesicles positive for an ER marker, intricate web-like structures of adhesion proteins at cell-cell junctions, and heterogeneity in euchromatin or heterochromatin recruitment of transcriptionally-associated histone H3.3 and heterochromatin protein 1α (HP1α) in the nuclei of neural progenitor cells as they transition into differentiated neurons). More generally, whole cell cryo-SR/FIB-SEM can reveal compartmentalized proteins within known subcellular components, help discover new subcellular components, and classify unknown EM morphologies and their roles in cell biology. Cryogenic SR below 10K: motivations and photophysical characterization

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Section: Molecular Underpinnings Of Ultrastructural Specialization Inmentioning

confidence: 99%

Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

Hoffman

Shtengel

et al. 2019

Preprint

119

View full text Add to dashboard Cite

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“…To simplify future experiments, transgenic mouse lines that express SNAPand/or Halo-tags constitutively [36] offer a user-friendly alternative. Moreover, instead of topically applying the cell-permeable dye to the exposed brain surface, one may consider intravenously injecting dyes that are able to cross the blood-brain barrier [41]. Either or both modifications to the labelling protocol will minimize the exposure and manipulation of the imaged tissue.…”

Section: Discussionmentioning

confidence: 99%

3D super-resolution deep-tissue imaging in living mice

Velasco

Zhang

Antonello

et al. 2019

Preprint

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Stimulated emission depletion (STED) microscopy enables the three-dimensional (3D) visualization of dynamic nanoscale structures in living cells, offering unique insights into their organization. However, 3D-STED imaging deep inside biological tissue is obstructed by optical aberrations and light scattering.We present a STED system that overcomes these challenges. Through the combination of 2-photon excitation, adaptive optics, far-red emitting organic dyes, and a long-working distance water-immersion objective lens, our system achieves aberration-corrected 3D super-resolution imaging, which we demonstrate 164 µm deep in fixed mouse brain tissue and 76 µm deep in the brain of a living mouse.

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“…Lavis and his team started to play around a little more with their new rhodamine synthesis strategy, eventually developing a way to incorporate 3-substituted azetidine rings into rhodamine (2). The impact of being able to incorporate these azetidine rings into the structure of rhodamine was profound.…”

Section: Finding a New Routementioning

confidence: 99%