Cryo-Correlative Light and X-ray Microscopies: Expanding the Intracellular Chemical Map

Area of Science:

• Cellular Ultrastructure and Composition Analysis
• Nanoscale Imaging Technologies
• Biophysical Characterization

Background:

• Understanding cellular ultrastructure and composition at the nanoscale is crucial for biological research.
• Existing imaging techniques often have limitations in resolution, elemental sensitivity, or require complex sample preparation.
• Synchrotron-based X-ray fluorescence nanoimaging offers high elemental sensitivity but can be limited by sample preparation and throughput.

Purpose of the Study:

• To develop and validate an integrated workflow combining cryo-optical fluorescence microscopy and cryogenic synchrotron radiation X-ray fluorescence nanoimaging.
• To achieve high-resolution 2D and 3D visualizations of cellular ultrastructure and elemental composition.
• To demonstrate the capability for simultaneous elemental mapping, nanoparticle tracking, and organelle-specific imaging.

Main Methods:

• Fusion of cryo-optical fluorescence microscopy with cryogenic synchrotron radiation X-ray fluorescence nanoimaging.
• Utilization of luminescent cyclometalated iridium complexes for mitochondrial imaging.
• Employing molecular probes with different heavy elements (Re, Ir, Br, I) for multiplex elemental mapping.
• Development of a streamlined workflow to minimize sample preparation and maximize synchrotron beamtime.

Main Results:

• Achieved sharp 2D and 3D visualizations of cellular ultrastructure.
• Enabled simultaneous elemental mapping and tracking of nanoparticles within cells.
• Demonstrated multiplex elemental 'painting' of organelles using heavy element probes.
• Successfully imaged mitochondrial features using an iridium complex.

Conclusions:

• The integrated workflow provides powerful nanoscale insights into cellular ultrastructure and composition.
• This streamlined approach accelerates data acquisition and maximizes the use of synchrotron resources.
• The method serves as a practical reference for advanced cryo-nanoscale imaging studies, enabling detailed cellular analysis.