A new technique in single molecule biophysics, called PhotoGate microscopy, allows tracking of single molecules in highly dense environments such as inside cells where traditional single molecule microscopy techniques have limitations and PhotoGate microscopy can yield longer tracking times and produce more accurate measurements using photobleaching to control the number of fluorescent particles.
The physics behind the technique and results are described in this article “PhotoGate microscopy to track single molecules in crowded environments” from the Biophysics Graduate Group, University of California, Berkeley.
Digital Light Sheet Microscopy (DLSM) Selective or Selective Plane Illumination Microscopy (SPIM)
Digital Light Sheet Microscopy (DLSM) / Lattice Light-Sheet Microscopy (LLSM) or Selective Plane Illumination Microscopy (SPIM) is a fast optical-sectioning fluorescence technique allowing for 3D volume imaging under the diffraction limit. Light sheet microscopy reduces photodamage caused by long exposure to high laser intensity to cells (such as is common with confocal microscopy), a significant advantage for research on biological processes in live cells. The illumination in LSM happens 90 degrees to the direction of observation, with only the focal plane (typically <1um thick) of the sample exposed to laser light for the time required for imaging.
Light sheet microscopy reduces image acquisition time and photobleaching because it eliminates out-of-focus excitation and spreads the laser light energy over the entire field compared to a single point as common in traditional fluorescence microscopies. The speed of LSM is a great advantage for acquiring 3D images and in some cases has enabled real time video imaging – a key to better understanding the biological development process over time.
Important areas to consider for light sheet systems is the spatial resolution, temporal resolution, scanning area, and long term imaging stability. Longer data collection time requires good mechanical design with low-drift.
There are several designs of light sheet microscopes: isotropic multi-view light-sheet microscopy, lattice light-sheet microscopy, dual-view light-sheet microscopy, etc.