Highly resolved intravital striped-illumination microscopy of germinal centers

J Vis Exp. 2014 Apr 9:(86):51135. doi: 10.3791/51135.

Abstract

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 µm tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells - on the level of a few protein molecules in germinal centers.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Animals
  • Antigen-Antibody Complex / analysis
  • Antigen-Antibody Complex / immunology
  • B-Lymphocytes / cytology
  • B-Lymphocytes / immunology
  • Dendritic Cells / cytology
  • Dendritic Cells / immunology
  • Germinal Center / cytology*
  • Germinal Center / immunology
  • Green Fluorescent Proteins / chemistry
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods*

Substances

  • Antigen-Antibody Complex
  • Green Fluorescent Proteins