1：Experimental Design and Method Selection:

The study involved designing a CLEM system using a silicon nitride film as a substrate. Optical properties (transmittance and autofluorescence) of SiN films of different thicknesses were measured to determine suitability for microscopy. A novel linkage system with exchangeable components was developed for switching between OM and TEM observations.

2：Sample Selection and Data Sources:

Silicon nitride films (30 nm and 50 nm thick) fabricated using chemical vapor deposition and lithography. A blue-color toner sample was used for application testing, embedded in Epon 812 resin and sliced to 70 nm sections.

3：List of Experimental Equipment and Materials:

Microscopic spectrophotometer (MSV-370, JASCO Corporation), confocal laser microscopy system (ECLIPSE Ti2 and confocal system A1+, Nikon), optical microscope (ECLIPSE LV100ND, Nikon), transmission electron microscope (JEM-1400Flash, JEOL), ultramicrotome (Leica UC6, Leica), silicon nitride films, cover slips (MATSUNAMI Micro cover glass), Texas Red filter cube, imaging software (Picture Overlay, JEOL Ltd.), and custom-developed CLEM retainer, TEM holder, and OM adapter.

4：Experimental Procedures and Operational Workflow:

- Measure light transmittance of SiN films using spectrophotometer. - Measure autofluorescence using confocal microscopy. - Fabricate SiN window chips. - Develop and assemble CLEM linkage system components. - Mount toner sections on SiN window chips. - Observe samples sequentially with OM (bright field and fluorescence) and TEM, using the exchangeable retainer system. - Merge images using software for correlative analysis.

5：Data Analysis Methods:

Quantitative analysis of transmittance and fluorescence intensity from spectrophotometer and microscopy data. Image merging and comparison using Picture Overlay software to correlate OM and TEM observations.