Simple Methods to Correlate Light and Scanning Electron Microscopy
Claudia S. López,1,2,3 *
lopezcl@ohsu.edu
Abstract: Based on technologies capable of data collection between the millimeter and nanometer scales, correlative imaging has been transforming how researchers obtain molecular and spatial informa- tion from specimens. Attempts to combine multidimensional data are often met with the challenge of overcoming suboptimal sample condi- tions such as reduced fluorescence signal, poor specimen preserva- tion, anisotropic specimen deformation, and low specimen contrast. These issues motivated the development and use of enhanced sample preparation procedures, as well as specialized imaging software to overcome such challenges. In this work we present three simple meth- ods to correlate optical and scanning electron microscopy images.
Keywords: scanning electron microscopy, fluorescence microscopy, correlative light and electron microscopy, focused ion beam, cancer biology
Introduction Correlative light and electron microscopy (CLEM) can
be done by means of transmission electron (TEM), scanning electron (SEM), and focused ion beam (FIB-SEM) microsco- pies [1–7]. In recent years, we have optimized and developed new protocols to correlate optical microscopy of fixed and live cells with both SEM and 3D FIB-SEM imaging [3,4,8–10] (unpublished results) and to also preserve fluorescence signal in methacrylate resins [8,10] (unpublished results). In some experimental cases, we have observed that 2D EM imaging will suffice and is enough to answer a specific scientific ques- tion. However, over the past ten years the scientific community has shown advantages of using 3D EM. In fact, 3D imaging technologies have enormously advanced knowledge in several scientific fields, such as developmental biology, cancer biol- ogy, and, particularly, neurobiology [11–13]. In our hands, the ability to correlate both fluorescent and bright-field imaging with 3D FIB-SEM has helped us develop protocols that are currently being used in clinical trials [14–16] (
https://dx.doi. org/10.17504/
protocols.io.36vgre6). Tese
protocols assist
clinical pathologists and cancer biology researchers at Oregon Health and Science University to identify specific areas of interest from human cancer biopsies prepared for large format 2D and 3D EM by combining toluidine blue and SEM images (Figure 1). An advantageous technique, sequential CLEM allows
sample imaging using any optical method of choice, including super-resolution fluorescence microscopy [17], and samples are processed for 2D or 3D EM only if the optical imaging was successful. In this workflow, the introduction of the heavy met- als needed to obtain high-quality images by secondary electron SEM (SE-SEM) or backscattered electron SEM (BSE-SEM) is completed in later steps to ensure that fluorescent signal is not compromised [3]. Currently, a broad range of methodologies
24 doi:10.1017/S1551929520001108
to obtain CLEM results exists, each with their own advantages and limitations [1,2,6,18]. Terefore, researchers must care- fully select the optimal methods for their study based on the nature of their specimens and instrumentation available. Here we describe three different CLEM methods that can be easily reproduced in any laboratory using conventional bench pro- cessing methods.
Materials and Methods Instrumentation. Similar instrumentation and imag-
ing conditions were used for all of the results presented here (Figure 2). Wide-field optical and fluorescence microscopy (FM) was performed using a FEI CorrSight™. A FEI Helios NanoLab 650 DualBeam™ (later upgraded to the 660 version) was used for SEM and FIB-SEM data collection. Both instru- ments used the FEI MAPS soſtware package. Sections were cut using a Leica UC7 ultramicrotome. Sample coating was performed on a Leica ACE600 coater and dehydration using a Leica CPD300. Tere are several soſtware programs, both open-source or
licensed, available for optical and EM correlation that include rigid (MAPS used here from Termo Fisher) and/or non-rigid registrations of the images [19]. Depending on the protocol used to prepare the specimen, non-rigid registration is advan- tageous as the image can be warped and therefore adapted to any deformations that have occurred during the preparation steps (mostly during the chemical fixation and dehydration procedures). However, for samples processed near to native conditions, such as those methods used for cryofixation, or in the case of in-resin fluorescence preservation methods, rigid registration is most appropriate since specimen deformation is minimal. Correlation of optical microscopy with 2D SE-SEM
imaging. MCF7 breast cancer cells (American Type Cul- ture Collection) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 35 mm culture dishes. Cells were transiently cotrans- fected with 500 ng of DNA of HER2-eGFP and AKT2-tagRFP expression plasmids each using X-tremeGENE HP DNA Transfection Reagent (Roche) for 24 h. Cells were grown on indium tin oxide (ITO)-coated cover slips pretreated with 0.01% polylysine at a desired confluency and chemically fixed using 4% (v/v) paraformaldehyde (PFA) and 0.1% (v/v) glutar- aldehyde (Glut) in 1× phosphate-buffered saline (PBS) for 1 h at room temperature. Aſter this step, the cells were washed in 1× PBS and incubated with 1μg/ml DAPI prepared in 1× PBS and incubated for 30 minutes at room temperature. Before acquir- ing the optical images, the ITO cover slips were engraved using
www.microscopy-today.com • 2020 July * Erin Stempinski,1,2 and Jessica L. Riesterer1,2
1Multiscale Microscopy Core, Oregon Health & Science University (OHSU), Portland, OR 2OHSU Center for Spatial Systems Biomedicine (OCSSB), Portland, OR 3Pacific Northwest CryoEM Center, Portland, OR
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