Our research team develops technologies and protocols for cryo-electron microscopy and cryo-electron tomography. We have developed protocols for micropatterning TEM grids to direct cell growth and positioning for cryo-ET experiments. Whole-cell cryo-electron tomography is used to produce nanometer-level resolution structures of macromolecular complexes within cells preserved in a native frozen hydrated state.
Micropatterning can increase the throughput of whole-cell cryo-ET, cryo-correlative light electron microscopy, and cryo-focused-ion-beam milling experiments. The number and distribution of cells on EM grids are crucial for whole-cell cryo-ET studies. This protocol provides a rapid, reproducible, and broadly adaptable method to direct cell growth on EM grids through micropatterning.
The investigator should be comfortable using pipettes, tweezers, basic cell culture techniques, and fluorescence and transmission electron microscopes. When patterning for the first time, use a well-known cell type and ECM combination and test them on coverslips or MatTek dishes. Research team members involved in this protocol development are:Dr.
Brian Sibert, Mr.Joseph Kim, and Dr.Jae Yang. To begin, transfer the grids to a grid prep holder and glow discharge the grids carbon side up. Next, transfer the grids with the carbon side up to a clean glass slide or coverslip with at least a one centimeter separation between the grids.
Pipette 10 microliters of 0.05 poly-L-lysine onto each grid and incubate the grids in a humid chamber for at least 30 minutes. After incubation, wash each grid three times with 15 microliters of 0.1 molar HEPES at pH 8.5. Incubate the grid in each wash for 30 seconds, then remove most of the liquid from the grid with a pipette without letting the grid dry.
After the final wash, leave each grid in 15 microliters of 0.1 molar HEPES. Next, prepare the PEG-SVA solution and immediately replace the HEPES solution on the grids with 10 microliters of the PEG-SVA solution, then incubate the grids in a humid chamber for at least one hour. After incubation, wash each grid three times with 15 microliters of sterile water, incubating the grid in each wash for 30 seconds.
After the final wash, leave each grid in 15 microliters of water. Place a one microliter drop of water on the center of a new clean coverslip, then carefully transfer the grid from the 15 microliter water drop to the drop on the new coverslip with the carbon side up. Next, carefully place at PDMS stencil over the grid keeping the grid centered and minimizing stencil contact with the carbon foil of the grid.
Then add one microliter of PLPP gel onto the grid and pipette gently to mix. Move the coverslip with the grid to a dark location to dry. The gel will dry in 15 to 30 minutes.
For micropatterning, open the software with a live Brightfield view from the microscope. To design a new template for an initial run, select Add ROI and choose a 3, 000 micrometer circle. Position the circle ROI over the grid using the Brightfield image on the screen as a guide, then press Lock to secure the ROI.
After locking the ROI in place, select Add Pattern and choose the appropriate pattern. Using the replication options, generate copies of the initial pattern to create an eight by eight grid square region. Adjust the spacing and angle as necessary to align the patterns with the grid squares.
Position the pattern in one corner of the grid. Next, duplicate the pattern and place a copy in each of the four corners of the grid moving the microscope stage as necessary for each region. Use the replication options to modify an eight by eight grid square region into a two by eight grid square region to be placed on both sides of the center.
Leave the center four grid squares unpatterned. For each pattern, adjust the total dose if necessary. 30 millijoules per square millimeter is a good starting point.
Under Expert Options, iterate adjusting the angle, position, space between, and ratio of the pattern to refine alignment of the pattern with the grid squares. Move the microscope stage to change the grid region in the Brightfield display. Once the template and patterns are positioned, uncheck all but one of the regions in the action panel of the software.
Use the microscope stage to navigate to that region and focus on the carbon foil. Click on the eyeball icon in the action panel to toggle the pattern overlay display off. Once the grid is in focus, close the Brightfield shutter and press the Play icon at the bottom right corner of the software to begin the patterning process, which can be monitored live.
Repeat this step until all of the regions are patterned. Finally, remove the coverslip with the grid from the microscope and immediately add 10 microliters of sterile PBS onto the grid. After 10 minutes, remove the stencil with tweezers, then wash the grid three times with 15 microliters of PBS and leave each grid in PBS in a dark location.
Prepare 15 microliters of the extracellular matrix for each grid, then replace the PBS from each grid with 15 microliters of the extracellular matrix and incubate the grid in a humid chamber for at least one hour. After incubation, wash each grid five times with 15 microliters of sterile PBS as demonstrated previously. After the final wash, leave each grid in PBS.
Using a fluorescence microscope, detect the fluorophore in the extracellular matrix to confirm patterning and that the carbon foil remains intact. HeLa cells seeded onto micropatterned and unpatterned TEM grids are visible by fluorescent staining using a calcein AM and ethidium homodimer-1 based cell viability assay. Using a mixed collagen and fibrinogen extracellular matrix, HeLa cells readily adhere to patterns across the grid.
The overall morphology of cells that expanded along the pattern is similar to that of cells grown on unpatterned grids. A Brightfield image of RSV infected BEAS-2B cells 18 hours post seeding shows cell adhesion and growth along the central region of the pattern. Most of the infected cells are positioned along the higher density central region of the gradient pattern.
The RSV variants are located close to the periphery of the infected BEAS-2B cells grown on micropatterned grids. Many RSV structural proteins are identified within the tomograms, including the nucleocapsid and viral fusion protein. On micropatterned grids, Drosophila neurons with pan-neuronal GFP expression can be easily tracked by light microscopy due to fluorescent labeling and their location being within the micropatterns.
Neurons on unpatterned grids can also be tracked through GFP signaling by light microscopy. However, locating them in cryo-electron microscopy becomes difficult due to cellular debris and contamination from the media. Due to the dimensions of the neuron cell body and extended neurites, cryo-electron tomography tilt series are collected along thinner regions of the cells with higher magnification.
The neuronal cell membrane, mitochondria, microtubules, actin filaments, vesicular structures, and macromolecules such as ribosomes are well-resolved in higher magnification image montages and slices through the 3D tomogram. Similar subcellular features are observed from 3D tomograms of unpatterned neurons. Applying the stencil to the grid and adding on the PLPP gel is the most sensitive step that can affect patterning results and should be done with caution.
Cryo-CLEM can be used to localize targets of interest within cells for cryo-ET data collection. Cryo-FIB-SEM can be used to thin cells on areas otherwise too thick for data collection.
