The application of CorrSight™ in correlative light and electron microscopy of vitrified biological specimens

Correlative light and electron microscopy is a powerful technique for identification and determination of the structures of interested macromolecules in situ. Combined with sample vitrification, it would be much easier to preserve the native state of macromolecule complexes and distinguish them from the crowded structure environment. In this article, we present a detailed process for the application of the CorrSight system, a light microscope equipped with a cryo module, in combination with a cryo-electron microscope. A relatively long course of up to 7–8 h for cryo module preparation and multichannel light microscopy imaging of vitrified specimen can be sustained. Correlation of light and electron microscopy images at both grid levels to locate squares and square level to locate target particles, and verification of target particles can be performed with the help of AutoEMation software. Cryo-electron tomography is used for obtaining the three-dimensional structure information.


INTRODUCTION
Vitrification of biological specimens in liquid nitrogen (LN2) temperature has been proved to be a powerful technique to preserve the native structures of macromolecules either in vitro or in situ. In combination with the most recent hardware and software breakthroughs, cryo-fixed macromolecule complexes isolated from cells can be determined at near atomic resolution using the single particle reconstruction method (Ma et al. 2017;Peng et al. 2016;Wang 2015;Wang et al. 2017a). However, it is still very challenging to identify and determine the structures of interested macromolecules in situ with cryo-electron microscopy (cryo-EM) (Lucic et al. 2008;Oikonomou and Jensen 2017;Plitzko et al. 2009). The major hurdle lies in the fact that many different kinds of macromolecules are crowded within the cell and therefore are hard to be distinguished from each other simply by their shapes revealed in cryo-EM (Bauerlein et al. 2017;Zhang 2013). Furthermore, the large dimension of a cell and a rather small viewing area of EM at a high magnification render it more difficult to localize relatively sparse molecules in a specific cellular structure environment (Plitzko et al. 2009). Light microscopy (LM), more specifically, fluorescence microscopy (FM) is well developed to label specific molecules and localize them in a large viewing area with a resolution of several hundreds of nanometers. The advantages of specific labeling and localization in LM and high spatial resolution in EM can be combined, leading to the development of a powerful technique as the correlative light and electron microscopy (CLEM) (Anderson et al. 2017;Compera et al. 2015;Faas et al. 2013;Koning et al. 2014;Sjollema et al. 2012).
CLEM technique was first reported in the early 1960s for adenovirus study with conventional chemical fixation EM specimen preparation Morgan et al. 1960). While the chemical fixed specimens are still the majority of targets investigated by CLEM (Hellstrom et al. 2015;Kobayashi et al. 2016;Kong and Loncarek 2015;Loussert Fonta and Humbel 2015;Mourik et al. 2014), vitrified biological specimens become more and more popular to avoid the potential artifacts and damages caused by the chemical fixation. Cryo-CLEM is thus developed in order to visualize vitrified specimens by cryo-EM (Bykov et al. 2016;Mahamid et al. 2016;Wolff et al. 2016). Currently, two types of methods are used for correlation: (1) correlative LM/ EM with freezing after FM imaging (Briegel et al. 2010). FM imaging of samples at room temperature could be done with any typical FM instrument. FM can have better resolution with high-powered, oil-immersion lenses with large numerical apertures. After FM imaging, the sample needs to be frozen for further cryo-EM imaging. Various factors can be introduced to the specimen between the LM imaging and specimen vitrification, causing more difficulties for accurate correlation. Therefore, it works better for specimen that is relatively stable and the fluorescently marked structure would not change in the seconds to minutes before vitrification.
(2) Correlative LM/EM with freezing before FM imaging (Briegel et al. 2010). The sample is plunge frozen or cryo-sectioned first, then the grid is visualized under LM and EM sequentially with little disturbance of its structure, thus making the correlation more consistent. Such a practice needs the cryo-fixed specimen to be maintained always in a humidity-free environment and below *-140°C during the whole CLEM process to prevent ice contamination on the specimen surface or recrystallization in the specimen. Special specimen stages designed for the related instruments are essential to preserve the cryo-fixed specimens (Li et al. 2018;Liu et al. 2015;Plitzko et al. 2009;Rigort et al. 2010;Schorb and Briggs 2014;Schorb et al. 2017;Zhang 2013). While the first type of CLEM method provides convenience of use and better resolution of LM, the second type guarantees more accurate correlation and is more favored (Briegel et al. 2010).
For the second type of CLEM method, two different designs are commercially available. One is the integrated (Agronskaia et al. 2008;Faas et al. 2013) and the other is the separated. The first integrated CLEM system called Tecnai with iCorr (Thermo Fisher Scientific Inc.) allows the stage to be tilted 90°to switch between the LM and EM mode for direct correlation of a specimen in situ, preventing the specimen from potential contamination and damage. The experiments efficiency is greatly increased without repeated grid transfers (Agronskaia et al. 2008). Within the iCorr system, however, only small light objective lens as 159/0.5 NA can be integrated due to the limited space between the pole pieces of a standard Tecnai Spirit TEM. The iCorr system thus can only have fluorescence signals at the green channel with a low resolution of about 500 nm (Wang et al. 2017b). In comparison, separated CLEM systems such as the CorrSight system equipped with a cryo module from Thermo Fisher Scientific Inc. (Arnold et al. 2016) and Leica cryo-CLEM System (Hampton et al. 2017) allow the observation of a cryo-fixed specimen in a more advanced LM at better resolution and in an EM with high acceleration voltage. Different image processing software packages have been developed for high-precision correlation. The study of virus-infected or transfected mammalian cells by using Leica Cryo system has been reported. Here we present a detailed process for the application of the CorrSight system in combination with a cryo-TEM. We used plunge-frozen technique to fix the biological specimen and cryoelectron tomography (cryo-ET) for obtaining the threedimensional (3D) structure information.

Sample preparation
In our CLEM study, biological samples were vitrified and assembled into EM Autogrids as below: (1) GiG R3.5/1 200 mesh grids (GiG C200F1, Changshu Zhongke Xinghua Technology) with indexed letters and numbers were glow discharged by means of a basic plasma cleaner PDC-32G-2 (Harrick Plasma).
(2) After biological sample was applied onto the grids, cryo-EM grids were prepared using Vitrobot Mark IV (Thermo Fisher Scientific Inc.) at 20°C and with 100 percent humidity. The blot force is set as -2 and the blot time is set as 2-2.5 s. The vitrified specimen on cryo-EM grids was stored in LN2 dewars for further examination. (3) The assembly of Autogrids is executed inside a regular Cryo Transfer Workstation (Thermo Fisher Scientific Inc.) for a TEM with autoloader. Each grid is placed inside a C-clip ring (Thermo Fisher Scientific Inc.) with the sample-application side facing down and then clipped with a C-clip (Thermo Fisher Scientific Inc.) from the top to form a sandwich-type Autogrid. (4) In the cryo shuttle and Transfer Box Assembly Workstation (Thermo Fisher Scientific Inc.) of the CorrSight system, the Autogrids are mounted on a cryo shuttle with the C-clip ring side facing up (Fig. 1). The metal spacing cylinder is then put on top of the Autogrids, and the Plexiglas lid is closed immediately. Two Autogrids can be loaded into one cryo shuttle. The cryo shuttle can be stored in the transfer box.
The steps (2)-(4) should all be performed at LN2 temperature. The Autogrids or transfer boxes are all stored in LN2 dewars for further cryo-LM or cryo-EM imaging.

Fluorescence imaging with the CorrSight system
The flowchart of fluorescence imaging with the Corr-Sight system is illustrated as shown in Fig. 2. Before LM imaging of cryo samples, purging of the objective (chamber) with dry nitrogen gas and cooling down of the cryo module with liquid nitrogen are processed for a total time of 2-3 h (Fig. 3). Because of the continuous objective purging demand during the whole process, a pipeline supply of dry nitrogen gas is preferred or a selfpressurized LN2 dewar is the second choice. In this experiment, a few dry nitrogen gas cylinders were used, which can sustain as long as 8 h, from the objective purging to system heating. High-pressure gas purging on the cover glass must be done for de-icing, after the temperature of sample and chamber reaches a range from -185 to -195°C (Fig. 4C). Live imaging mode of the system can be used to check whether the cover glass is clean enough for light source to penetrate, especially at the location of the grids. When the system is stable, the transfer box can be transferred into the cryo module chamber in a far end, and the cryo shuttle is placed in the center of the bottom, with the screw facing toward the operator (Fig. 3B). The fluorescence imaging steps are detailed as follows: (1) Check the intactness of carbon film, flatness of grid, and thickness of vitreous ice for the Autogrids (Fig. 4A  (3) Multichannel images of interested squares can be acquired at a magnification of 409/0.9 NA as exampled in Fig. 6   imaging by focusing on the cover glass, the crystal ice would be blown away gradually. The temperature changes of the samples and specimen chambers should be closely monitored, especially after continuous imaging for more than 2 h, to ensure that all liquid nitrogen in the cryo module dewar is not used up (Fig. 3A). Note: Turn on the air compressor only at the beginning of the multichannel image acquisition and turn it off before the cryo sample transfer to prevent severe vibration of CorrSight. Once the imaging is completed, the cryo samples should be unloaded and put back in the LN2 tank immediately. (4) After the image acquisition, put the cryo shuttle back to the LN2 storage tank. The cryo module should be heated continuously at 65°C for 2 h and purged at 0.5 bar with pressurized LN2 tank at the same time (Fig. 3C). (5) Overlaid multichannel images can be adjusted and saved manually for EM correlation. Within a good grid, more than 60 squares could be imaged and marked (Figs. 4B, 5A). This is the step for rough and quick screening of squares. Find squares with Fig. 4 The acquisition of multichannel LM images with Maps software. A The samples 1 and 2 correspond to the ones described in Fig. 3B. B Layer of sample 1. Each sample can have a layer which may contain one low magnification grid map and many highmagnification fluorescence square images numbered in sequence. C The temperature of sample and chamber should be kept in the range from -185 to -195°C during imaging process Process for the application of the CorrSight system PROTOCOL interested fluorescence signal and label the locations within the overlaid images (Fig. 5A) for further cryo-EM imaging.

Correlation of cryo-LM and cryo-EM images
Two functions in the upgraded version 2.0 of AutoEMation software (Lei and Frank 2005) are used for acquisition of montaged EM atlas (Fig. 7A), correlation of LM and EM images (Fig. 7B).
To locate and verify target particles for cryo-ET data collection, there are three sequential steps described as follows: (1) The first step is the correlation of LM and EM grid map to locate the selected squares in EM grid map.
After loading the finder grid from the cryo shuttle into a cryo transmission electron microscope, for example, a Cs-corrected Titan Krios in this work, by using the Cryo Transfer Workstation, a montaged EM atlas of a grid is acquired at a nominal magnification in low magnification range (409 for Gatan Orius SC200 CCD camera, 1009 for Falcon camera, 1759 for Gatan GIF K2 camera) as exampled in Fig. 5B. The acquisition usually starts from the stage center and stage movement is used.
The conversion matrix between the LM and EM grid maps should be determined, which can be derived from two reference positions, i.e., indexed letters and numbers, recognizable in both the LM and EM maps. For each reference position, the coordinate in the LM map is obtained by using any graphic software, while the coordinate in the EM map is obtained and converted to the corresponding EM stage position via a precalibrated matrix between the camera and stage by using AutoEMation.
With the help of the conversion matrix, the coordinates of those marked squares in the LM map (Fig. 5A) can be converted to their corresponding coordinates in   Fig. 7B, and the EM stage positions which can be reached by clicking the ''GoTo'' button near to the ''Mark'' button directly or the ''Go to XYZ'' button ( Fig. 7B) if the marker in the EM map is selected (Fig. 5B).
(2) The second step is the correlation of LM and EM square map to locate target particles in EM square map.
For each marked square, a montaged EM atlas of the square (Fig. 8C) is acquired at the lowest nominal magnification in the SA range (17009 for both Gatan Orius SC200 CCD camera and Falcon camera, 22509 for Gatan GIF K2 camera). The acquisition starts locally after reaching the stage position of the square in the above step and stage movement or a combination of stage movement and image-beam shift is used.
Similarly, the conversion matrix between the LM and EM square maps should be determined, which can be derived from two reference positions, i.e., square corners or any features, recognizable in both the LM and EM maps.
With the help of the conversion matrix, the coordinates of fluorescence target in the LM map (Fig. 8A, B) can be converted to their corresponding coordinates in the EM map (Fig. 8C, D) marked by clicking the ''Mark'' button in Fig. 7B, and the EM stage positions which can be reached by clicking the ''GoTo'' button near to the ''Mark'' button directly or the ''Go to XYZ'' button ( Fig. 7B) if the marker in the EM map is selected. The location of the target particle can be finely tuned with the combination of ''Refresh'' and ''Go to XY'' buttons ( Fig. 7B) as the low-dose alignment between this magnification and a much higher magnification for verification can be set precisely with the inaccuracy of less than 0.02 lm by using AutoEMation.
Sometimes it is useful to verify the target particle visually. In Fig. 8B and D, three vesicles near the fluorescence signal are used as references for verification.
(3) The third step is the verification of the target particles at a much higher magnification to ensure they are suitable for cryo-ET data collection.
The target particles selected from the above two steps may not be suitable for cryo-ET data collection as images acquired at the above magnification cannot provide enough details. Therefore, it is worth imaging the target particles at a much higher magnification (i.e., nominal magnification of 18,0009 for Falcon camera, 2-3 e/Å 2 dose, -8 to -10 lm defocus) for verification as further cryo-ET data collection takes very long time. The imaging for verification can be simply done by clicking the ''Refresh'' button ( Fig. 7B) after switching to the preset high-magnification mode. Cryo-ET data collection and reconstruction is usually performed by using free software packages and/or packages from EM vendors.

Set batch tomography data collection positions for all targets
(1) Set up FEI TEM tomography and build a new specimen session by checking the low-dose and batch options. Select file format and output folder.
(2) Add every target with separate defocus settings, ranging from -5 to -8 lm. Set the focus and tracking areas away from the exposure area. Determine the exposure time of data collection based on the limit of 1-2 e/Å 2 for each tilted image.
(3) As a typical setting, acquisition usually starts at 0°a nd single-axis tilt series are collected with 1.5°i ncrement between -65°and 65°for frozen samples. Autofocus is performed before image acquisition periodically. The general periodicity switch angle is 30°as focus would be checked every five images when the tilt angle is lower than 30°and every two images when the tilt angle is higher than 30°. In order to track the targets during the tilting, ''tracking before'' is usually performed when the tilt angle is lower than 10°and ''tracking after'' is performed for other tilt angles.

Data alignment and reconstruction
For the tomographic data with fiducial gold, use IMOD software package to do alignment and reconstruction. Execute the Protomo software package (Winkler and Taylor 2006) for the alignment and reconstruction for tomographic data without gold particles. Low-pass and high-pass filtering might be used for the noisy images, which is particularly helpful for the cross-correlation calculation during alignment. With Protomo, four or five iterations is enough for the 2 k 9 2 k images and the weighted back-projection algorithm is used to generate the final 3D reconstruction from raw images. Apply median filtering or nonliner anisotropic diffusion filtering distributed in IMOD to enhance the contrast and signal-to-noise ratio of the tomograms. Further analysis of subvolume averaging and segmentation can be performed in Dynamo and Amira, respectively.

DISCUSSION
The CorrSight system can sustain cryo-LM imaging for a long duration of up to 5 h. The Maps software can save all the acquired data automatically in the project and avoid data loss due to unexpected interruption such as software crashing. More importantly, the packed Autogrids matches well with cryo-SEM, cryo-TEM with autoloader, and Cryo Transfer Workstation. The application of AutoEMation software makes the correlation much easier. For the manual verification during the correlation, fluorescent beads can be used if there are no distinguishable particles. Verification with highmagnification imaging is necessary before cryo-ET data collection although the target is preexposed with a small amount of dose, as acquisition of each tilt series would take nearly 1 h. The protocol in this study applies mainly to relatively thin samples, which can be prepared by plunge freezing by Vitrobot or similar apparatus and imaged with cryo-TEM directly. If the samples are too thick for electron beam to penetrate, ultrathin sectioning under LN2 environment needs to be executed before the sample can be loaded into the cryo-TEM. Sometimes high-pressure freezing should be performed instead of fast frozen for large cells or tissues, to get vitreous ice for the whole volume (Mahamid et al. 2015).
Besides the cryo-sectioning by diamond knife (Cortese et al. 2013), milling by cryo-focused ion beam (cryo-FIB) recently becomes the priority to get artifactfree, thin, frozen-hydrated lamella via fabrication (Arnold et al. 2016;Beck and Baumeister 2016;Lucic et al. 2013). After cryo-LM imaging, the locations with fluorescence signal can be specifically milled by FIB to generate the thin lamella, followed by cryo-EM highresolution data collection (Fukuda et al. 2014). In addition, the resolution of LM imaging needs to be improved by integrating a super-resolution LM platform and a well-designed cryo module, which would be very powerful for the correlation of light and electron microscopy. In the future, more integrated and automated system to bring all the above-mentioned steps in a seamless pipeline would make the correlative cryo-EM more powerful and robust.