High accuracy, fiducial marker-based image registration of correlative microscopy images

Elly Van Donselaar,Judith Klumperman,Sajjad Mohammadian,Gerhard A Blab,Hans C Gerritsen,Alexandra V Agronskaia,Nalan Liv,Cecilia De Heus,Jantina Fokkema

doi:10.1038/s41598-019-40098-4

Elly Van Donselaar, Judith Klumperman + Show 7 more

Open Access

https://doi.org/10.1038/s41598-019-40098-4

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Abstract

Fluorescence microscopy (FM) and electron microscopy (EM) are complementary techniques. FM affords examination of large fields of view and identifying regions of interest but has a low resolution. EM exhibits excellent resolution over a limited field of view. The combination of these two techniques, correlative microscopy, received considerable interest in the past years and has proven its potential in biology and material science. Accurate correlation of FM and EM images is, however, challenging due to the differences in contrast mechanism, size of field of view and resolution. We report an accurate, fast and robust method to correlate FM and EM images using low densities of fiducial markers. Here, 120 nm diameter fiducial markers consisting of fluorescently labelled silica coated gold nanoparticles are used. The method relies on recording FM, low magnification EM and high magnification EM images. Two linear transformation matrices are constructed, FM to low magnification EM and low magnification EM to high magnification EM. Combination of these matrices results in a high accuracy transformation of FM to high magnification EM coordinates. The method was tested using two different transmission electron microscopes and different Tokuyasu and Lowicryl sections. The overall accuracy of the correlation method is high, 5–30 nm.

Highlights

Correlative light and electron microscopy (CLEM) combines the high sensitivity and large field of view of fluorescence microscopy with the high resolution of electron microscopy
In Fluorescence microscopy (FM), only parts of the specimen that are fluorescently labelled with e.g. a fluorescent antibody are visible while in EM the contrast relies on electron density variations usually generated by heavy metal staining and visible throughout the specimen
The low magnification EM image is correlated with the high magnification TEM image[6,7]

Summary

Introduction

Correlative light and electron microscopy (CLEM) combines the high sensitivity and large field of view of fluorescence microscopy with the high resolution of electron microscopy. In FM, only parts of the specimen that are fluorescently labelled with e.g. a fluorescent antibody are visible while in EM the contrast relies on electron density variations usually generated by heavy metal staining and visible throughout the specimen This results in similar features having very different appearances in the FM and EM images. A straight-forward method is to manually identify and overlay regions in FM and high magnification EM images[1,2,3,4,5] This method is strongly user dependent and its accuracy cannot be determined. The low magnification EM image is correlated with the high magnification TEM image[6,7] This method relies on (auto)-fluorescence in an ultra-thin section of the sample[7] or the signal from Uranyl acetate under cryo conditions[6].

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