Home › Microscopy & Imaging › Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi
Microscopy & Imaging JoVE (Open Access) Citable · DOI

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

DOI: 10.3791/60613-v
What you'll learn
  • ✓ Select and apply vital fluorescent dyes for fungal membrane and cell wall imaging
  • ✓ Prepare live filamentous fungal samples for microscopy without fixation
  • ✓ Execute live-cell microscopy protocols to visualize membrane dynamics and exocytosis
  • ✓ Interpret fluorescent imaging results for cell wall morphogenesis analysis
Protocol

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

Difficulty
intermediate
Total time
~1–2 hours per sample set (staining, mounting, and imaging)
Model organism
Filamentous fungi (Aspergillus, Neurospora, or equivalent)
Biosafety
BSL-1

Steps

1
Prepare fungal samples for vital dye staining

Harvest and prepare live filamentous fungal cultures in appropriate growth medium. Ensure cells are viable and at the correct developmental stage for imaging.

▶ 01:06
2
Apply fluorescent dyes and mount specimens

Incubate fungal samples with selected vital fluorescent dyes to label plasma membranes and cell walls. Prepare slides or imaging chambers with mounted samples in aqueous medium.

▶ 01:40
3
Acquire live-cell fluorescence microscopy images

Configure microscope settings (objective, laser/illumination, detector gain) and acquire time-lapse or single-frame images of stained fungal cells while maintaining viability.

▶ 02:57
4
Analyze membrane dynamics and cell wall morphogenesis

Process and interpret acquired images to track plasma membrane movement, endocytic/exocytic events, and cell wall growth patterns in live filamentous fungi.

▶ 04:16
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