Total Internal Reflection Fluorescence Microscopy for High‐Resolution Imaging of Cell‐Surface Events

Jaiswal, Jyoti K.; Simon, Sanford M.

doi:10.1002/0471143030.cb0412s20

Cited by 12 publications

(11 citation statements)

References 14 publications

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“…There are several commercially available TIRF systems and any of these systems compatible with live cell imaging can be used. Additionally, we have described approaches for setting up homemade TIRF microscopes elsewhere 17,18 . Independent of the TIRF setup there is a need to establish approaches that allow monitoring various fates of subcellular vesicles prior to and following injury.…”

Section: Discussionmentioning

confidence: 99%

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

Defour

Sreetama

Jaiswal

2014

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The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

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Section: Discussionmentioning

confidence: 99%

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

Defour

Sreetama

Jaiswal

2014

JoVE

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“…The TIR‐FM illumination was set up as described previously (42). Total Internal Reflection was achieved using the objective setup (44) on an inverted fluorescence microscope (IX‐70; Olympus) equipped with high numerical aperture lens (Apo 60× NA 1.45; Olympus). A custom‐built temperature‐controlled enclosure was used to maintain the microscope at 37°C during imaging.…”

Section: Methodsmentioning

confidence: 99%

“…The depth of the evanescent field was typically 70–120 nm for the Apo 60× N.A. 1.45 lens (42,44). The emission was collected through emission band pass filter (HQ525/50M; Chroma Technologies Corp., Rockingham, VT, USA).…”

Section: Methodsmentioning

confidence: 99%

Patients with a Non‐dysferlin Miyoshi Myopathy have a Novel Membrane Repair Defect

Jaiswal

Marlow

Summerill

et al. 2006

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Two autosomal recessive muscle diseases, limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), are caused by mutations in the dysferlin gene. These mutations result in poor ability to repair cell membrane damage, which is suggested to be the cause for this disease. However, many patients who share clinical features with MM‐type muscular dystrophy do not carry mutations in dysferlin gene. To understand the basis of MM that is not due to mutations in dysferlin gene, we analyzed cells from patients in one such family. In these patients, we found no defects in several potential candidates – annexin A2, caveolin‐3, myoferlin and the MMD2 locus on chromosome 10p. Similar to dysferlinopathy, these cells also exhibit membrane repair defects and the severity of the defect correlated with severity of their disease. However, unlike dysferlinopathy, none of the conventional membrane repair pathways are defective in these patient cells. These results add to the existing evidence that cell membrane repair defect may be responsible for MM‐type muscular dystrophy and indicate that a previously unsuspected genetic lesion that affects cell membrane repair pathway is responsible for the disease in the non‐dysferlin MM patients.

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“…Special TIRF lenses are commercially available from Zeiss (NA = 1.45) and Olympus (NA = 1.45 or, with special expensive coverglass and toxic immersion media, NA = 1.65). See reviews by Oheim (), Axelrod (), or Jaiswal and Simon () for additional details on TIRF.…”

Section: Types Of Microscopymentioning

confidence: 99%