Ultra-thin fluorocarbon foils optimise multiscale imaging of three-dimensional native and optically cleared specimens

Area of Science:

• Biophysics
• Microscopy
• Materials Science

Background:

• Optical density heterogeneity limits 3D light microscopy penetration depth and resolution.
• Refractive index changes along the optical path cause aberrations and reduce image quality.
• Current light sheet fluorescence microscopy setups often involve immersion media and separate specimen containers, leading to optical imperfections.

Purpose of the Study:

• To develop and evaluate ultra-thin fluorocarbon (FEP) foil cuvettes for minimizing refractive index mismatch in 3D light microscopy.
• To assess the performance of FEP cuvettes in improving image quality, resolution, and sample handling.
• To demonstrate the suitability of FEP cuvettes for various biological samples and experimental workflows.

Main Methods:

• Fabrication of multi-faceted cuvettes from vacuum-formed ultra-thin FEP foils (10-12 µm wall thickness).
• Utilizing FEP cuvettes in light sheet fluorescence microscopy for imaging diverse specimens.
• Assessing cuvette properties: impermeability, inertness, durability, flexibility, and gas permeability.
• Evaluating sample mounting, fixation, clearing, and organoid culture within the cuvettes.

Main Results:

• FEP cuvettes effectively minimize refractive index variations, enhancing optical quality and resolution.
• High-quality 3D images were obtained from whole organs (mouse oocytes), thick tissue sections (brain, kidney), and organoid clusters (pancreas, liver).
• The cuvettes allow specimens to remain in place from seeding to observation, simplifying workflows and preserving delicate structures.
• FEP cuvettes support organoid seeding and growth for at least ten days, with fixation and staining possible within the holder.

Conclusions:

• Ultra-thin FEP foil cuvettes offer a significant advancement for 3D light microscopy by overcoming limitations of refractive index heterogeneity.
• These cuvettes provide superior sample mounting, compatibility with various media, and enable high-fidelity imaging of fragile biological structures.
• The FEP cuvettes facilitate simplified workflows, including organoid culture and multi-view imaging, making them a versatile tool for advanced microscopy applications.