Red Blood Cell and Heme-Related Autofluorescence

Symptom

Strong autofluorescence across multiple channels in blood-rich tissues such as spleen, liver, brain, bone marrow, and vascularized tumors. Heme and porphyrins dominate signal especially when tissue is not thoroughly perfused.

1. 1 Residual red blood cells containing heme and porphyrins
2. 2 Insufficient perfusion prior to fixation
3. 3 High tissue vascularity
4. 4 Blood clotting during perfusion

1. 1 Perfuse with PBS prior to fixation when possible
2. 2 Include approximately 10 U/mL heparin in perfusion solution to reduce clotting
3. 3 For human/postmortem or non-perfused tissues, wash sections longer
4. 4 Consider pigment quenchers such as Sudan Black B or cupric sulfate
5. 5 Assign low-abundance targets to far-red channels where RBC background is lower

Related Video (3)

Current Protocols ★ 85

Deciding on an Approach for Mitigating Autofluorescence

"Directly addresses autofluorescence mitigation strategies and decision-making, core to understanding and preventing heme-related autofluorescence artifacts"

Current Protocols ★ 82

Heterogeneous Autofluorescence

"Explicitly covers heterogeneous autofluorescence sources and their characterization, directly applicable to identifying and managing heme/porphyrin-driven background"

JoVE (Open Access) ★ 72

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

"Demonstrates native autofluorescence measurement and management in tissue imaging using sFLIM, relevant for distinguishing tissue autofluorescence from specific signals"