Home › Cell Biology › Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence
Cell Biology JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/59925-v
What you'll learn
  • ✓ Set up and calibrate a spectral fluorescence lifetime imaging (sFLIM) system for FRET measurements
  • ✓ Prepare plant tissue sections and apply rhodamine-based fluorescent probes to lignin
  • ✓ Acquire and analyze sFLIM data to quantify Förster resonance energy transfer interactions
  • ✓ Interpret lifetime measurements to assess probe-lignin binding in plant cell walls
Protocol

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate Förster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

Difficulty
advanced
Total time
~4–6 hours per experiment (including sample preparation, system calibration, and multi-point measurements)
Model organism
Plant tissue sections (species not specified in abstract)

Steps

1
Prepare plant tissue sections for imaging

Collect and section plant material, then mount on microscope slides for fluorescence measurements. Ensure sections are sufficiently thin to permit light transmission while preserving lignin structure.

▶ 00:54
2
Calibrate sFLIM system and spectral settings

Establish spectrally-resolved fluorescence lifetime measurement parameters and verify detector alignment. Confirm proper wavelength separation and temporal resolution for accurate FRET detection.

▶ 02:21
3
Acquire sFLIM measurements on stained sections

Apply rhodamine-based fluorescent probes to plant tissue sections and collect spectral and lifetime data across regions of interest. Record photon arrival times and spectral information simultaneously.

▶ 03:09
4
Process and analyze sFLIM lifetime data

Extract fluorescence decay curves, fit lifetime components, and calculate FRET efficiency based on donor lifetime changes. Correlate spectral signatures with lignin autofluorescence.

▶ 04:13
5
Interpret representative sFLIM results

Review sample lifetime images, spectral profiles, and FRET maps to visualize probe-lignin interactions across the plant section. Compare spatial distribution of energy transfer.

▶ 05:26

🚨 Failure Case Library (9) + Submit your own case

critical
Improper Channel and Fluorophore Assignment
Everything glows in green/yellow channels. Dim targets overwhelmed by autofluorescence. Blue and green channels showing high background across tissue types. Spectral crowding and bleed-through between channels.
💡 5 · ✓ 6
severe
Red Blood Cell and Heme-Related Autofluorescence
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.
💡 4 · ✓ 5
severe
Collagen and Elastin Structural Protein Autofluorescence
Strong broad-spectrum autofluorescence in collagen and elastin-rich tissues including skin, lung, vessel walls, and fibrotic tissue. Emission persists through fixation and processing.
💡 4 · ✓ 5
severe
Age-Related Lipofuscin Accumulation
Exceptionally broad excitation and emission spectra affecting multiple channels in aged tissues such as brain, heart, skeletal muscle, and retina. Age-dependent lysosomal pigment cannot be confined to single channel.
💡 4 · ✓ 5
severe
Melanin Pigment Interference
Unpredictable autofluorescence and signal quenching in pigmented tissues such as skin and retina. Melanin both autofluoresces and quenches true signal in unpredictable patterns.
💡 4 · ✓ 5
severe
True Signal Loss After Autofluorescence Quenching
Signal drops or disappears after applying Sudan Black B or other quenching treatments. Target staining reduced along with background. Tissue morphology may be affected.
💡 5 · ✓ 6
moderate
Fixation-Induced Aldehyde Fluorescence
Strong blue-green autofluorescence haze in formalin or glutaraldehyde-fixed tissues. Background increases with fixation duration and disproportionately contaminates lower-wavelength channels. Amplified by paraffin embedding and overfixation.
💡 5 · ✓ 6
moderate
Metabolic Cofactor Autofluorescence
Endogenous fluorescence from NADH, FAD, and other metabolic cofactors in high-metabolism tissues such as kidney, liver, pancreas, and spleen. Complex extracellular matrices contribute additional background.
💡 4 · ✓ 5
moderate
Inconsistent Antigen Retrieval Results
Antigen retrieval (AR) performance varies unpredictably across sections and targets. Harsh AR needed to recover signal suggests overfixation. Epitope accessibility inconsistent even with standardized protocols.
💡 5 · ✓ 6
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