Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

K Höhn,J Fuchs,C Dietrich,B Glass,M Anders‐Össwein,R Kirmse,P Walther,A Fröber,H.‐G Kräusslich

doi:10.1111/jmi.12230

Abstract

SummaryIn this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV‐pulsed mature human dendritic cells.

Highlights

Life science research increasingly demands multimodal imaging methods to obtain comprehensive data about dynamic events
Its foundation is the preservation of fluorescent signals in the resin after electron microscopy (EM) preparation, which allows identification and precise localization of target structures, i.e. volume(s) of interest (VOI)
In our case the fluorescence signal was used to identify the distribution pattern of mCherry labelled virus-like particles captured by human mature dendritic cell (mDC)

Summary

Introduction

Life science research increasingly demands multimodal imaging methods to obtain comprehensive data about dynamic events. A novel imaging technique for acquiring high resolution 3D data of bulky biological tissue is focused ion beam scanning electron microscopy (FIB-SEM). This method utilizes a SEM equipped with a gallium ion source to produce a second FIB. Both beams coincide at one point in the microscope where the sample is located and can be used to image the same area without moving the microscope stage under the two beams (Heymann et al, 2006; Hekking et al, 2009 and others). The ion beam can be used to remove a few nanometres of the sample surface exposing the freshly generated block-face that is imaged with the

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Results

Conclusion