From the Ultramikroskop to 4Pi Microscopy to Theta Microscopy to Tetrahedral Microscopy to Light sheet-based fluorescence microscopy (LSFM)

Specimens scatter and absorb light, thus, the delivery of the probing light and the collection of the signal light become inefficient; many endogenous biochemical compounds also absorb light and suffer degradation of some sort (photo-toxicity), which induces malfunction of a specimen.  In conventional and confocal fluorescence microscopy, whenever a single plane is observed, the entire specimen is illuminated (Verveer 2007).  Recording stacks of images along the optical z-axis thus illuminates the entire specimen once for each plane.  Hence, cells are illuminated 10-20 and fish embryos 100-300 times more often than they are observed (Keller 2008).  This can be avoided by using light sheets, which are fed into the specimen from the side and overlap with the focal plane of a wide-field fluorescence microscope.  In contrast to an epi-fluorescence arrangement, such an azimuthal fluorescence arrangement uses two independently operated lenses for illumination and detection (Stelzer 1994; Huisken 2004).

Optical sectioning and no photo-toxic damage or photo-bleaching outside a small volume close to the focal plane are intrinsic properties.  Light sheet-based fluorescence microscopy (LSFM) takes advantage of modern camera technologies.  LSFM can be operated with laser cutters (e.g. Colombelli 2009) and in fluorescence correlation spectroscopy (FCS, Wohland 2010).  We have also successfully evaluated the application of structured illumination in a SPIM (Breuninger et al., 2007).  Christoph Engelbrecht investigated the performance of our SPIMs (Engelbrecht & Stelzer, 2006) both theoretically and practically.  We also designed and implemented a wide field frequency domain Fluorescence Lifetime Imaging (FLIM) setup, which is based on a SPIM.  This arrangement provides an inherent optical sectioning capability and reduces photo bleaching compared to conventional wide field and confocal fluorescence microscopes.  Most recently, we implemented incoherent structured illumination in our DSLM (Keller 2010).  The intensity modulated light sheets can be generated with dynamic frequencies and allow us to estimate the effect of the specimen on the image formation process at various depths in objects of different age.

The single plane illumination microscope (SPIM) employs a cylindrical lens to generate a light sheet.  A collimated laser beam is focused into the plane of the detection lens along one direction while the other direction remains collimated (Greger et al., 2007).  While this approach is relatively simple and straightforward it suffered from the low quality of the cylindrical lens and the inefficiency of the illumination system (Breuninger et al., 2007).  The major advantage of the digital scanned laser light sheet-based fluorescence microscope (DSLM; Keller et al., 2008) is that it relies entirely on cylindrically symmetric optics and hence provides a very good optical quality.  In addition, the DSLM employs a minimal number of optical components and does not suffer from excessive wave front aberrations.

The development of LSFM at EMBL draws on many previous developments.  In particular confocal theta fluorescence microscopy, which was originally developed together with Steffen Lindek, played a very important role.  About a dozen papers on theta microscopy describe its properties and that of LSFM (single & two-photon, annular/Bessel beams, (a)symmetric arrangements, …) theoretically as well as practically.
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For further information, please contact Prof. Dr. Ernst H.K. Stelzer