Multiphoton Microscopy of FITC-labelled Fusobacterium nucleatum in a Mouse in vivo Model of Breast Cancer

Abstract

Over the past decades, the main techniques used to visualize bacteria in tissue have improved but are still mainly based on indirect recognition of bacteria. Both microscopy and molecular recognition are being improved, but most procedures for bacteria detection in tissue involve extensive damage. Here, we describe a method to visualize bacteria in tissue slices from an in vivo model of breast cancer. This method allows examining trafficking and colonization of fluorescein-5-isothiocyanate (FITC)-stained bacteria in various tissues. The protocol provides direct visualization of fusobacterial colonization in breast cancer tissue. Rather than processing the tissue or confirming bacterial colonization by PCR or culture, the tissue is directly imaged using multiphoton microscopy. This direct visualization protocol causes no damage to the tissue; therefore, all structures can be identified. This method can be combined with others to co-visualize bacteria, types of cells, or protein expression in cells.

Keywords: Bacterial colonization; Breast cancer; Fusobacterium nucleatum; Multiphoton microscopy.

Figure 1.. Nikon SMZ25 images of breast cancer tissue and adjacent mammary tissue.

Representative microscopy images of tumor and normal adjacent mammary samples harvested 24 h post intravenous injection of 5 × 10⁷ FITC-labeled F. nucleatum ATCC 23726 or with PBS vehicle into AT3 cancer-bearing mice.

Figure 2.. Nikon multiphoton A1MP microscope images of breast cancer tissue and adjacent mammary tissue.

Representative multiphoton microscopy images of tumor and normal adjacent mammary samples harvested 24 h post intravenous injection of AT3 cancer-bearing mice with 5 × 10⁷ FITC-labeled F. nucleatum ATCC 23726. Images were taken from Parhi et al. (2020). No changes were made.