Live Cell Imaging describes methods which allow time-lapse observations of living samples for many minutes, hours, or days. While any imaging modality can record time lapse images, most biological samples are ultimately limited by photo-damage to the sample. This makes modalities like point-scanning laser confocal microscopes poor choices for most live cell imaging due to the high photon flux in the laser beam. Two main approaches have been developed to minimize the laser excitation light delivered to the sample are:
- Spinning disk confocal Here, laser light is delivered to the sample simultaneously through thousands of pin holes in a revolving disk, greatly diluting how much laser light is delivered to each point in the sample.
- Light sheet microscopy uses separate lenses for excitation and observation so that only a small region of the sample is exposed to excitation light at a time. Lattice light sheet microscopy is a further refinement of light sheet microscopy in which the sheet of excitation light is thinner than the depth of focus of the excitation lens. This reduces the sample exposure 100 to 1000-fold from that observed with a spinning disk confocal.
In our imaging facility, we have a spinning disk confocal and a lattice light sheet microscope, both of which were manufactured by 3i, Incorporated.
Benefits of SDC and LLSM approaches:
- Relatively simple sample preparation
- Sample preps are simple modifications of existing immunofluorescence protocols
- Can label three or four objects simultaneously
- Images are acquired on familiar inverted microscope stands