Retention of fluorescent probes during aldehyde-free anhydrous freeze-substitution

Area of Science:

• Cell biology
• Microscopy techniques
• Biochemistry

Background:

• Fluorescent probes are essential for live-cell microscopy.
• Few probes are suitable for fixed samples without aldehyde fixation.
• Aldehyde fixation can cause artifacts in structure and probe localization.

Purpose of the Study:

• To evaluate chloromethyl aminocoumarin (CMAC) for fixed-cell microscopy without aldehyde fixation.
• To assess probe retention and imaging quality in immobilized fungal hyphae.
• To determine if freeze-substitution is a viable alternative to aldehyde fixation for fluorescent probe microscopy.

Main Methods:

• Fungal hyphae were loaded with chloromethyl aminocoumarin (CMAC).
• Samples underwent anhydrous freeze-substitution without aldehyde fixation.
• Imaging was performed using fluorescence microscopy.
• Probe retention during Spurr's resin embedding was assessed.

Main Results:

• Fluorescence patterns in freeze-substituted hyphae resembled those in live hyphae.
• CMAC and other tested probes showed some loss during embedding.
• Vacuoles were successfully imaged due to probe retention.

Conclusions:

• Anhydrous freeze-substitution enables fluorescent probe microscopy of fixed fungal hyphae without aldehyde fixation.
• CMAC is a viable fluorescent probe for fixed samples, despite some probe loss during embedding.
• This method offers an alternative to aldehyde fixation, reducing potential artifacts.