Fixative Induced Effects in Labeled and Unlabeled Fluorescence: Implications for Biomedical Imaging Studies

Eliana H. Stilson, Natzem Lima, Julianne Setiadi, Ricky Sontz, Suzann Duan, Juanita Merchant, Travis Sawyer

Research output: Chapter in Book/Report/Conference proceedingConference contribution


Paraformaldehyde (PFA) is one of the most common fixatives in biological and biomedical research. It is used to preserve tissue or cell morphology while preventing contamination by crosslinking proteins and other biological molecules. Although fixation is required for histology, it has been documented that chemical fixation can cause alterations in the fluorescence properties of exogenous and endogenous fluorophores, which are valuable markers for understanding biological processes, ultimately reducing the accuracy and reliability of quantitative fluorescence measurements. Therefore, there is a need for understanding the behavior of tissue fluorescence during PFA fixation. Multispectral fluorescence imaging (MFSI) is an imaging technique used in biological and biomedical research to visualize and quantify the fluorescence properties of tissue over several wavelength bands, enabling measurement of several fluorophores simultaneously. To evaluate the effects of PFA on tissue fluorescence, we imaged brain tissue samples using MSFI from two cohorts of mice: the SOX10 Cre; R26R-Brainbow 2.1/Confetti mice (expressing four exogenous fluorophores), and wild type Cre-negative controls. Specimens from each were immersed in 10 ml of PFA or phosphate buffer saline (PBS) as a control. The fluorescence intensity was captured using MFSI every 15 minutes over three hours. Analysis was performed on the resulting images to produce quantitative metrics of the resulting fluorescence signal. The results show that exogenous fluorophores are dramatically quenched within the first half hour when fixed in PFA, whereas endogenous fluorescence increased slightly in the same time period. These results are valuable to understand how fixation can influence fluorescence properties and can inform optimal fixation protocols.

Original languageEnglish (US)
Title of host publicationMultiscale Imaging and Spectroscopy V
EditorsPaul J. Campagnola, Darren M. Roblyer, Alex J. Walsh
ISBN (Electronic)9781510669130
StatePublished - 2024
EventMultiscale Imaging and Spectroscopy V 2024 - San Francisco, United States
Duration: Jan 27 2024Jan 28 2024

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
ISSN (Print)1605-7422


ConferenceMultiscale Imaging and Spectroscopy V 2024
Country/TerritoryUnited States
CitySan Francisco


  • endogenous fluorophores
  • exogenous fluorophores
  • fluorescence quenching
  • Multispectral fluorescence imaging
  • tissue fixation
  • tissue fluorescence

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics
  • Biomaterials
  • Radiology Nuclear Medicine and imaging


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