Cell Volume (3D) Correlative Microscopy Facilitated by Intracellular Fluorescent Nanodiamonds as Multi-Modal Probes.

Neeraj Prabhakar,Xavier Heiligenstein,Eija Jokitalo,Jessica M Rosenholm,Markus Peurla,Ilya Belevich,Cecilia Sahlgren,Huan-Cheng Chang

doi:10.3390/nano11010014

Abstract

Three-dimensional correlative light and electron microscopy (3D CLEM) is attaining popularity as a potential technique to explore the functional aspects of a cell together with high-resolution ultrastructural details across the cell volume. To perform such a 3D CLEM experiment, there is an imperative requirement for multi-modal probes that are both fluorescent and electron-dense. These multi-modal probes will serve as landmarks in matching up the large full cell volume datasets acquired by different imaging modalities. Fluorescent nanodiamonds (FNDs) are a unique nanosized, fluorescent, and electron-dense material from the nanocarbon family. We hereby propose a novel and straightforward method for executing 3D CLEM using FNDs as multi-modal landmarks. We demonstrate that FND is biocompatible and is easily identified both in living cell fluorescence imaging and in serial block-face scanning electron microscopy (SB-EM). We illustrate the method by registering multi-modal datasets.

Highlights

Correlative light and electron microscopy (CLEM) combine the strengths of fluorescence and electron microscopy and this allows overcoming their respective limitations for cell imaging [1,2,3]
The majority of developed CLEM methods are based on the correlation of light microscopy (LM) with 2D images of thin cell sections imaged with transmission electron microscopy (TEM) [13,16,17,19,20,21]
Our 3D CLEM workflow begins by seeding Fluorescent nanodiamonds (FNDs) incubated MDA-MB-231 cells over gridded glass-bottom dishes designed for CLEM experiments

Summary

Introduction

Correlative light and electron microscopy (CLEM) combine the strengths of fluorescence and electron microscopy and this allows overcoming their respective limitations for cell imaging [1,2,3]. The majority of developed CLEM methods are based on the correlation of LM with 2D images of thin cell sections imaged with transmission electron microscopy (TEM) [13,16,17,19,20,21]. These CLEM methods provide very limited information on the z-axis direction, as TEM sections are generally licenses/by/4.0/)

Methods

Results

Conclusion