PROJECT SUMMARY/ABSTRACT
Correlative light and electron microscopy (CLEM) combines dynamic information from florescence light
microscopy (LM) with ultrastructural information from electron microscopy (EM) of the same biological specimen,
addressing fundamental questions neither LM nor EM alone can answer. However, performing CLEM is
technically challenging even for many experienced scientists. In particular, CLEM study is currently impossible
or impractical for cells cultured on permeable substrates (e.g., Transwell inserts, thin 3D substrates) that are
required by a broad range of biomedical research. This discrepancy is due to the lack of a convenient method
or tools to target, track and locate the regions of interest in EM specimens of the cells previously examined by
LM. Our long-term goal is to develop convenient tools and methods to simplify CLEM studies for cells, tissues
and organoids. Our objective is to develop a working prototype for CLEM technology, including a new type of
substrate and associated protocols that will enable CLEM studies allowing functional cells cultured on
permeable/porous substrates to regain their functional state/morphology, polarity or desired orientations. Our
strategy is to use microfabrication to develop and produce a designable polymer-based porous substrate with
easy-to-recognize indexing patterns for cells to culture on, enabling fluorescence LM imaging and resin
embedding, followed by EM of serial sections of the same object. The substrate will also be developed and
validated for CLEM studies using focused ion beam (FIB) milling for FIB-scanning electron microscopic (SEM)
tomography. We will develop the substrates and CLEM protocols to address two typical needs for functional cell
culture, confluent cell culture and thin 3D cell culture. We will accomplish the research through three specific
aims: (1) Develop a method for routine production of polymer substrates with a cutting property matching that of
resin-embedded cell samples; (2) Develop accessible substrates/protocols for CLEM of functional cells on
porous substrates; and (3) Develop accessible substrates/protocols for CLEM of cells cultured on desired thin
3D substrates. This innovative research will develop a novel CLEM tool by combining techniques in cellular EM
specimen preparation with a unique method of microfabrication to produce a previously unavailable, polymer-
based, indexing patterned substrate, and enable, the first time, CLEM studies for functional cells that must be
cultured on porous substrates. The newly developed substrate will also facilitate convenient CLEM of EM
specimens prepared by a high pressure freezing/freeze substitution method. The anticipated contribution will be
significant because it will not only enable CLEM applications for a broad range of cell types that must be cultured
on porous substrates for their polarity and functional status, but also facilitate EM specimen preparation by using
a high pressure freezing/freeze substitution for these cells. Additionally, it opens the gate for further development
of substrates and methods for 3D-CLEM that correlate information from 3D-LM live cell imaging with the
ultrastructure of 3D cell cultures on 3D substrates.
