CELS-3D-Cutting edge light source for exciting fluorescence in microtome-based 3D microscopy and targeted correlative...

Area of Science:

• Microscopy and Imaging Technologies
• Biotechnology
• Materials Science

Background:

• Traditional microscopy techniques struggle with imaging large, non-transparent biological samples due to light penetration limitations.
• Confocal microscopy, for instance, can only image depths up to 50 μm in dense samples like liver spheroids.
• There is a need for advanced imaging methods capable of deep tissue visualization and targeted feature identification.

Purpose of the Study:

• To introduce a novel imaging system, CELS-3D (Cutting Edge Light Source, Three-Dimensional), for 3D imaging of biological samples.
• To demonstrate the utility of CELS-3D for deep imaging of large, non-transparent specimens, overcoming limitations of existing technologies.
• To showcase CELS-3D's capability for targeted correlative microscopy by visualizing fluorescence during serial sectioning.

Main Methods:

• Developed a prototype microscope (CELS-3D) that utilizes light exiting an ultramicrotome's glass knife as an oblique light sheet for sample illumination.
• Mounted the CELS-3D system on an ultramicrotome for serial sectioning and 3D imaging of biological samples.
• Characterized the CELS-3D system and applied it to 3D imaging of human liver spheroids and targeted correlative microscopy of C. elegans.

Main Results:

• Successfully achieved 3D imaging and reconstruction of nuclei and tight junctions throughout 500 μm human liver spheroids.
• Demonstrated that CELS-3D can image samples to depths far exceeding the capabilities of confocal microscopy.
• Successfully applied CELS-3D for targeted correlative microscopy of human liver spheroids and C. elegans, identifying fluorescent features during sectioning.

Conclusions:

• CELS-3D provides a breakthrough in 3D imaging of less- and non-transparent samples, including organoids, biopsies, cartilage, and bone.
• The system's ability to provide online fluorescence visualization during sectioning facilitates targeted correlative microscopy.
• CELS-3D is easily mountable on commercial ultramicrotomes and offers an intuitive operation for advanced biological imaging.