Correlative light and electron microscopy imaging of proteinaceous deposits in cell cultures and brain tissues

Jiang, Peizhou; Dickson, Dennis W.

doi:10.1186/s40478-025-01969-2

Acta Neuropathologica Communications

Table 1 Comparison of the strengths and limitations between our method and other conventional methods

From: Correlative light and electron microscopy imaging of proteinaceous deposits in cell cultures and brain tissues

Comparison for CLEM application	Strategy I	Strategy II	Strategy III
Comparison for CLEM application	Single-section imaging	Z-stack LM imaging/ EM processing & imaging	Serial sections for separately imaging of LM and EM	Our improved method
Quality of LM image 1	+ + +	+ + + + +	+	+ +
Quality of EM image 2	+ + + +	+ + + + +	+ + + + +	+ + + + +
Section alignment and overlay accuracy 3	+ + + + +	+	+ + + +	+ + + +
Technical friendliness 4	+	+ + +	+ + + + +	+ + + + +
Simplicity in sample process 5	+	+ + +	+ + + + +	+ + + + +
Fiducial markers usage 6	+ + + + +	+ + + + +	NA	+ + + + +
Multiplex staining for LM image 7	+ + + + +	+ + + + +	NA	+ + + +
Integration with immuno-EM 8	NA	NA	NA	+ + + + +

1—Strategy II captures the original immunofluorescence signal without additional processing, while Strategy I requires cryo-processing, which theoretically preserves antigenicity better than the antigen retrieval process used in Strategy III. However, our method in Strategy III demonstrates superior antigenicity preservation compared to traditional approaches
2—Strategy I does not use OsO₄ staining, resulting in lower contrast in EM images compared to Strategies II and III
3— Strategy I employs a single section for both LM and EM, ensuring the best overlay accuracy. Strategy II relies on Z-stack imaging for LM, which can be challenging to align with the cutting plane of the EM section. In contrast, Strategy III utilizes immediately adjacent sections for LM and EM, significantly minimizing overlay errors. This approach is effective as long as the EM section has no folded areas or the folded regions (e.g., as shown in image 2B of Fig. 3) are excluded from analysis
4—Strategy I and some experiments in Strategy II require high-pressure freezing and freeze substitution
5—Strategy I Involves delicate manipulation for cryo-processing, and Strategy II requires embedding samples in situ
6—Strategy III (prior to our method) did not utilize fiducial markers, which are now incorporated in our improved approach
7—Strategy III (prior to our method) did not perform multiplex staining on ultrathin sections, a feature now enabled by our method
8—Our method introduces the novel integration of immuno-EM into CLEM, significantly enhancing its capabilities

Back to article page

ISSN: 2051-5960

Contact us

General enquiries: journalsubmissions@springernature.com