Resolution in the ApoTome and the confocal laser scanning microscope: comparison.

The essential feature of the confocal laser scanning microscope (cLSM) is the generation of optical sections by the removal of out-of-focus light. About ten years ago, structured illumination microscopy (SIM) was introduced as an alternative method for obtaining optical sections from biological specimens. Here we compare the resolution of the ApoTome (commercial SIM by Zeiss) to that achieved by a cLSM (Zeiss LSM 510). If fluorescent beads are used as test objects, then the ApoTome will achieve a lower axial resolution than the cLSM. In contrast to that, its lateral resolution scores slightly better. If subresolution homogeneous fluorescent layers are used as test objects, then the ApoTome will achieve a higher axial resolution than the cLSM. The ApoTome's axial resolution is homogeneous over the field-of-view while that of the cLSM changes markedly. Finally, the anisotropy of the ApoTome's resolution was found to be negligible for standard applications while its capability to resolve fine structures within stained tissue slices is limited to one or two cell layers and thus worse than in the cLSM.

[1]  J. Faber,et al.  Normal table of Xenopus laevis. , 1994 .

[2]  D. Taylor,et al.  Cytomechanics applications of optical sectioning microscopy. , 2003, Methods in enzymology.

[3]  M. Gustafsson,et al.  S: Widefield Light Microscopy with 100-nm-scale Resolution in Three Dimensions , 2007 .

[4]  Murray.  Evaluating the performance of fluorescence microscopes , 1998, Journal of microscopy.

[5]  T Wilson,et al.  Whole-field optically sectioned fluorescence lifetime imaging. , 2000, Optics letters.

[6]  S. Hell,et al.  Comparison of I5M and 4Pi‐microscopy , 2006, Journal of microscopy.

[7]  A. Boccara,et al.  Optimization and characterization of a structured illumination microscope. , 2007, Optics express.

[8]  L H Schaefer,et al.  Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach , 2004, Journal of microscopy.

[9]  T. Wilson,et al.  Image formation in structured illumination wide-field fluorescence microscopy. , 2008, Micron.

[10]  T. Wilson,et al.  Real time 3D fluorescence microscopy by two beam interference illumination , 1998 .

[11]  H. Rahn,et al.  Acid-base balance in cold-blooded vertebrates as a function of body temperature. , 1970, The American journal of physiology.

[12]  K. Jalink,et al.  Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts , 2005, Journal of microscopy.

[13]  J. Gurdon,et al.  Normal table of Xenopus laevis (Daudin) , 1995 .

[14]  D. Schild,et al.  Anisotropic diffusion in mitral cell dendrites revealed by fluorescence correlation spectroscopy. , 2002, Biophysical journal.

[15]  W. Decraemer,et al.  A simple method for checking the illumination profile in a laser scanning microscope and the dependence of resolution on this profile. , 2006, Scanning.

[16]  T. Wilson,et al.  Method of obtaining optical sectioning by using structured light in a conventional microscope. , 1997, Optics letters.

[17]  P. French,et al.  Optical sectioning microscopes with no moving parts using a micro-stripe array light emitting diode. , 2007, Optics express.

[18]  E. H. Linfoot,et al.  Phase Distribution near Focus in an Aberration-free Diffraction Image , 1956 .

[19]  Neil,et al.  Wide‐field optically sectioning fluorescence microscopy with laser illumination , 2000, Journal of microscopy.

[20]  Jan Siegel,et al.  A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning , 2002 .

[21]  M. Gustafsson.  Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[22]  R. Heintzmann,et al.  Saturated patterned excitation microscopy--a concept for optical resolution improvement. , 2002, Journal of the Optical Society of America. A, Optics, image science, and vision.

[23]  J. Siegel,et al.  Time‐domain whole‐field fluorescence lifetime imaging with optical sectioning , 2001, Journal of microscopy.

[24]  J. Pawley,et al.  Handbook of Biological Confocal Microscopy , 1990, Springer US.

[25]  M. Gustafsson,et al.  Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. , 2008, Biophysical journal.