Filter-Dense Multicolor Microscopy

PLoS One. 2015 Mar 4;10(3):e0119499. doi: 10.1371/journal.pone.0119499. eCollection 2015.

Abstract

Immunofluorescence microscopy is a unique method to reveal the spatial location of proteins in tissues and cells. By combining antibodies that are labeled with different fluorochromes, the location of several proteins can simultaneously be visualized in one sample. However, because of the risk of bleed-through signals between fluorochromes, standard multicolor microscopy is restricted to a maximum of four fluorescence channels, including one for nuclei staining. This is not always enough to address common scientific questions. In particular, the use of a rapidly increasing number of marker proteins to classify functionally distinct cell populations and diseased tissues emphasizes the need for more complex multistainings. Hence, multicolor microscopy should ideally offer more channels to meet the current needs in biomedical science. Here we present an enhanced multi-fluorescence setup, which we call Filter-Dense Multicolor Microscopy (FDMM). FDMM is based on condensed filter sets that are more specific for each fluorochrome and allow a more economic use of the light spectrum. FDMM allows at least six independent fluorescence channels and can be applied to any standard fluorescence microscope without changing any operative procedures for the user. In the present study, we demonstrate an FDMM setup of six channels that includes the most commonly used fluorochromes for histology. We show that the FDMM setup is specific and robust, and we apply the technique on typical biological questions that require more than four fluorescence microscope channels.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Atherosclerosis / pathology
  • Biomarkers
  • Carotid Arteries / pathology
  • Color
  • Fluorescent Dyes / chemistry
  • Mice
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods*

Substances

  • Biomarkers
  • Fluorescent Dyes

Grants and funding

The work was supported by grants from the Swedish Research Council (A0282301 and A0282302), the Swedish Heart and Lung Foundation (20120262), and the Sahlgrenska University Hospital ALF research grants (LUA 70270). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.