Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

Lee, Jun Young; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

doi:10.1016/j.bpj.2012.09.014

Cited by 37 publications

(28 citation statements)

References 31 publications

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“…Given the larger probe volume of confocal microscopy, it seems likely that the mobile population observed in those experiments was not associated with the cell surface. Single-molecule fluorescence measurements have also indicated immobilization of amebal proteins by glass (40) and of human proteins by PLL (41). Despite this, PLL has been a popular choice for studying ''resting'' T cells using TIRFM (9,(11)(12)(13)(14)(15)(16)18,19,23).…”

Section: Discussionmentioning

confidence: 99%

Single-Molecule Light-Sheet Imaging of Suspended T Cells

Ponjavic

Carr

Santos

et al. 2018

Biophysical Journal

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Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose.

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Section: Discussionmentioning

confidence: 99%

Single-Molecule Light-Sheet Imaging of Suspended T Cells

Ponjavic

Carr

Santos

et al. 2018

Biophysical Journal

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“…Actin-based protrusions from cells were imaged using line-scan confocal microscopy (LSCM; Supplementary Fig. 1; Methods) 39 . The colocalization images illustrate two-coloured bridges formed by two filopodia protruding from different cells (FBs, Fig.…”

Section: Dynamic Transition Of Fbs Between Cells To Visualize the Dymentioning

confidence: 99%

F-actin dynamics transform filopodial bridges into intercellular nanotubes capable of distant cell communication

Chang

et al. 2018

Preprint

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A novel actin-based bridge connecting cells has been recognized as a new pathway for the distant transport of cytoplasmic components, viruses, or pathogenic substances between cells. However, it is not yet known how such a fine structure extends over several hundred micrometres and remains robust for several hours. Using optical fluorescence imaging methods, we found that random contact promotes the formation of filopodial bridges through N-cadherin interactions between filopodia, which are slender actin-rich plasma membrane protrusions. These filopodial bridges eventually evolve into a single actin-based bridge (intercellular nanotube) that connects two cells via an intermediate state that involves a helical structure. Surprisingly, the twisting of two filopodia is likely to result from the rotational motion of actin filaments inside the filopodia by myosin V. The accumulated torsion of the filopodia triggers the release of one of the paired filopodia, whose end is attached to the other cell body by an N-cadherin cluster. The resulting retraction of the filopodium by retrograde F-actin flow leaves a single bridge. The N-cadherin/catenin cluster is likely to form a synapse between the intercellular nanotube and the cell body. This study sheds light on the formation mechanism of the filopodial bridge-based intercellular nanotubes for longdistance communication between cells.

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“…For FRET-PAINT to be used for neural tissue imaging, huge background noise problem needs to be solved as well. Recently, we developed a real-time confocal microscopy that may provide superresoltion fluorescence images of thick tissue samples using video-rate confocal microscopy for single-molecule imaging [ 24 ]. We expect that FRET-PAINT combined with our real-time confocal microscopy will finally enable us to reconstruct three-dimensional structures of thick neural tissue samples with both high speed and high resolution.…”

Section: Discussionmentioning

confidence: 99%

Accelerated super-resolution imaging with FRET-PAINT

et al. 2017

Self Cite

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Super-resolution fluorescence microscopy in the current form is hard to be used to image the neural connectivity of thick tissue samples due to problems such as slow imaging speed, severe photobleaching of fluorescent probes, and high background noise. Recently developed DNA-PAINT solved the photobleaching problem, but its imaging speed is extremely low. We report accelerated super-resolution fluorescence microscopy named FRET-PAINT. Compared to conventional DNA-PAINT, the imaging speed of the microscopy increases up to ~30-fold. As demonstrations, we show that 25-50 second imaging time is long enough to provide super-resolution reconstruction of microtubules and mitochondria of COS-7 cells.Electronic supplementary materialThe online version of this article (10.1186/s13041-017-0344-5) contains supplementary material, which is available to authorized users.

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Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

Cited by 37 publications

References 31 publications

Single-Molecule Light-Sheet Imaging of Suspended T Cells

Single-Molecule Light-Sheet Imaging of Suspended T Cells

F-actin dynamics transform filopodial bridges into intercellular nanotubes capable of distant cell communication

Accelerated super-resolution imaging with FRET-PAINT

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