Doxorubicin is a commonly used chemotherapeutic employed to treat multiple human cancers, including numerous sarcomas and carcinomas. Furthermore, doxorubicin possesses strong fluorescent properties that make it an ideal reagent for modeling drug delivery by examining its distribution in cells and tissues. However, while doxorubicin fluorescence and lifetime have been imaged in live tissue, its behavior in archival samples that frequently result from drug and treatment studies in human and animal patients, and murine models of human cancer, has to date been largely unexplored. Here, we demonstrate imaging of doxorubicin intensity and lifetimes in archival formalin-fixed paraffin-embedded sections from mouse models of human cancer with multiphoton excitation and multiphoton fluorescence lifetime imaging microscopy (FLIM). Multiphoton excitation imaging reveals robust doxorubicin emission in tissue sections and captures spatial heterogeneity in cells and tissues. However, quantifying the amount of doxorubicin signal in distinct cell compartments, particularly the nucleus, often remains challenging due to strong signals in multiple compartments. The addition of FLIM analysis to display the spatial distribution of excited state lifetimes clearly distinguishes between signals in distinct compartments such as the cell nuclei versus cytoplasm and allows for quantification of doxorubicin signal in each compartment. Furthermore, we observed a shift in lifetime values in the nuclei of transformed cells versus nontransformed cells, suggesting a possible diagnostic role for doxorubicin lifetime imaging to distinguish normal versus transformed cells. Thus, data here demonstrate that multiphoton FLIM is a highly sensitive platform for imaging doxorubicin distribution in normal and diseased archival tissues.
Bibliographical noteFunding Information:
This work was supported by the CDMRP-NFRP (W81XWH-15-1-0114 to DL and PPP), the Children’s Tumor Foundation (2014A-05-020 to DL and PPP), and grants from the NIH (R01CA181385 to PPP; U54CA210190 University of Minnesota Physical Sciences in Oncology Center Project 2 to PPP; P50CA101955 SPORE career development award to PPP), the American Cancer Society (Research Scholar Grant, RSG-14-171-01-CSM to PPP, Research Professor Award #123939 to DL), the Randy Shaver Research and Community Fund (PPP), and the UMN Institute for Engineering in Medicine. ALW was supported by the Children’s Tumor Foundation Young Investigator’s Award Grant 2011-01-018. We thank Kianna Elahi Gedwillo and Rachel Edwards for assistance with sample preparation and helpful comments. The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
- fluorescence lifetime imaging microscopy
- multiphoton microscopy