An aldehyde-crosslinking mitochondrial probe for STED imaging in fixed cells

Area of Science:

• Cellular and Molecular Imaging
• Microscopy Techniques
• Biophysics

Background:

• Fluorescence labeling is crucial for super-resolution microscopy, enabling detailed visualization of cellular structures.
• Existing probes for mitochondrial super-resolution imaging often fail to retain fluorescence after chemical fixation.
• This limitation restricts mitochondrial super-resolution imaging primarily to live cells.

Purpose of the Study:

• To develop a mitochondria-specific probe for super-resolution imaging of fixed specimens.
• To overcome the challenge of probe retention in mitochondria after chemical fixation.
• To enable correlative super-resolution light and electron microscopy of mitochondria.

Main Methods:

• Introduction of PK Mito Orange FX (PKMO FX), a probe with a fixation-driven cross-linking motif.
• Assessment of PKMO FX fluorescence retention after chemical fixation.
• Evaluation of PKMO FX for stimulated emission depletion (STED) imaging at 775 nm.
• Demonstration of compatibility with immunolabeling and correlative light and electron microscopy.

Main Results:

• PKMO FX exhibits high fluorescence retention in mitochondria post-fixation.
• The probe enables nanoscopic imaging before and after aldehyde fixation.
• PKMO FX is compatible with standard immunolabeling protocols.
• The probe facilitates correlative super-resolution light and electron microscopy by leveraging mitochondrial morphology.

Conclusions:

• PKMO FX is a novel mitochondria-specific probe suitable for super-resolution imaging of fixed samples.
• This probe expands the available toolkit for multimodal microscopy of mitochondria at nanometer resolution.
• PKMO FX enables advanced imaging of cellular structures previously limited to live-cell studies.