Multi-Color Imaging

Up to four fluorescence signals can be readily collected by the utilization of fluorophores with distinct excitation/emission spectra. Typically, four distinct laser lines are used to excite fluorophores with emission peaks in the blue, green, red, and far red portions of the visual spectrum. Combinations of fluorescent proteins, DNA stains, conjugated antibodies, etc. are possible within a single imaging protocol.

Suggested Instrument: A1R




Fluorescence Recovery after Photobleaching (FRAP)


Click here for a time-lapse video of the above FRAP experiment.

Often used to determine the rate of diffusion/movement of fluorescently labeled proteins in live cells, a FRAP experiment typically begins with the intentional photobleaching of fluorophores in a designated region of a cell. This region is then monitored over the following seconds and minutes in order to detect the reentry of non-photobleached fluorophores (the "recovery"). The rate of diffusion into the bleached region can be calculated from the change in fluorescence intensity in the region. If the fluorophore is attached to a protein of interest (for example, a GFP-fusion protein), an estimate can be made of the overall diffusion of the native protein. Additionally, the "fluorescence loss in photobleaching" (FLIP) can be determined by monitoring a non-bleached region of the cell following photobleaching of a different region.

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Suggested Instrument: A1R




Live-Cell Imaging


Click here for a time-lapse video of the above image of cells while maintained in a stage-top incubator.

The advantages of imaging living cells and tissues have increased the use of this technique dramatically over the past decade. Cells or tissues expressing fluorescent proteins or stained with membrane-permeant (for intracellular features) or membrane-impermeant (for cell-surface structures) fluorescent dyes can be imaged using either confocal or multiphoton microscopy. The approach is similar to that used for the imaging of fixed samples with the exception that special precautions must be taken if cells are to be maintained out of an incubator for extended periods of time. For such experiments, a stage-top incubator controlling temperature, humidity, and gas concentration is available. Coupled with an autofocus system, the incubator allows for extended imaging (many hours) of live cells or tissues in an environment identical to that of a standard cell culture incubator.

Further Reading:

Suggested Instrument: A1R or Radiance




Intravital (Whole-Animal) Imaging


Click here for a video of labeled cells beneath the surface of a mouse ear.

Endogenous or transplanted cells within 500 microns of the surface of the skin can be imaged using confocal or multiphoton microscopy in anesthetized mice or rats allowing for in vivo observations. The skin of the ear and paw have been successfully imaged in this manner. Potentially, any tissue or organ can be imaged if surgical access is possible.

Further Reading:

Suggested Instrument: A1R or Radiance