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Imaging Techniques

LLSM Applications

1 week ago

1011  0
Posted on Jun 11, 2018, 4 p.m.

Live cell lattice light sheet microscopy is an imaging technique used to gain images of cells rapidly. Live cell lattice light sheet microscopy technique was developed by Eric Betzig, making use of lattice patterned thin light beams, reducing total amount of light delivered to the sample minimizing phototoxicity.

It is possible to make continuous fluorescence movies of developing organisms without having a phototoxic effect on them by using live cell lattice light sheet microscopy, as patterns are adjustable and provide images that exceed resolution of confocal microscopy.

 

Studying cells poses challenges one of them being observing them without affecting their behavior, live cell lattice light sheet microscopy overcomes the issue as cell exposure to light is minimized, and use of plane illumination light rather than a point which allows data to be acquired faster than standard confocal microscopes; enabling more detailed accurate studies.

 

Live cell lattice light sheet microscopy has been used in human cancer cells to visualize membrane nanotubes which provide means of communication between cells using soluble messengers such as in paracrine, exosome, of endocrine signaling, which have been investigated for possible role in cancer progression and therapy resistance. Lack of marker or imaging methodology has prevented nanotubes from being studied in intact live tissues. Live cell lattice light sheet microscopy created a thin sheet of light to put over the specimen rapidly to acquire a 3D image, human breast cancer cell line expressing bright fluorescent genetically encoded GFP membranes markers were used. Cells were shown to form multiple nanotubes in culture to propagate Ca2+ signals between cells and traffic GFP tagged membrane aggregates using Live cell lattice light sheet microscopy techniques. 

 

Live cell lattice light sheet microscopy can also be used to image membrane dynamics. Membranes divide cells into compartments, remodeling the membranes is important in changes of cell size or transfer of components to and from the surface. In mitosis changes in the shape of cells lead to birth of two cells. Live cell lattice light sheet microscopy was used to study dynamics of pit formation in plasma membrane on cell surface, no difference was found in lifetimes or intensities of coated pits on top or bottom surfaces of cells at interphase.

 

Potentials applications for use of live cell lattice light sheet microscopy are arguable limitless. Researchers have reported that the technique had been used on 20 different biological processes spanning across four orders of magnitude of space and time including: neutrophil motility in a collagen mesh; subcellular protein localization and dynamics during embryogenesis; binding kinetics of a Sox2 transcription factor molecule; dynamic organelle rearrangements and tracking of microtubules during mitosis; and 3D resolution photoactivated microscopy of nuclear lamins.

 

Photobleaching and phototoxicity were found to be reduced by 2 times magnitude. Live cell lattice light sheet microscopy use has been favorably established by these experiments with previous techniques and revealed potential opportunity for further use in future studies to characterize previously unknown biological phenomena.

 

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