Hi, I'm David Carlson, a Graduate student in the lab of James Evans in Biochemistry and molecular Biology at the University of California Davis. In this video, I'll show you how to build a simple and get effective cryo light stage for correlative cryo light electron microscopy. A cryo stage consists of a transparent viewing stream, a cutout for the objective lenses, a liquid nitrogen doer, a heat sink for viewing your sample and a sample transfer mount.
The majority of materials used were purchased at a local hardware and grocery store. They consist of a nine inch pipe pan, nine inch cake pan, four pairs of chopsticks, great stuffed spray foam, a four centimeter by eight centimeter by one centimeter block of aluminum drilled with mounting holes for number 10, bolts five. Number ten three quarter inch flathead slotted bolts and nuts, three number 4 3 8 inch round slotted bolts and nuts, 15 number 10 washers, two transparent plastic clipboards water, and a cryo EM grid box holder and handling rod, which can be purchased from Ted Pella.
Place the pie dish on the microscope stage under the objective lenses. Place the aluminum block near the edge of the dish and focus on the surface of the aluminum. Check that the objective lenses can freely swing in and out over the brim of the pie dish.
If not, reposition the aluminum block and check again. Once the location of the aluminum block is determined, mark the position of the mounting bolts with a marker. Position the cryo grid box next to the aluminum block and with a marker, record the position of the notch and each side of the cryo grid box with the layout of the bolts recorded on the surface of the pie dish.
Use a sharp screw and hammer to tap starter holes on each one of the marked dots using a number 17 drill bits. Drill out the holes for mounting the aluminum block. Rounding each hole to smooth the edges.
Next, using number 35 drill bit to drill and round the holes for mounting the cryo grid box, the pie dish. Prepped and drilled. Insert number ten three quarter inch flat heads slotted bolts into the holes on the aluminum block and place two number 10 SAE washers over each of the aluminum block.
Mounting holes on the pie dish. Next, mount the block using your fingers to start each bolt and tighten them down with a drill. Turn the pie dish over and place a number 10 washer on each bolt and secure them with a nut.
Use a wrench to ensure all nuts are tightly secured. Next, from the back of the pie dish, place number four, round head slotted. Bolts through the cryo grid box mounting holes and secure them with a nut from the top of the pie dish.
With the top of the cryo stage complete, we demonstrate how to transfer the cryo grid box in the absence of both the plastic clipboard and liquid Nitrogen. Next, we start the preparation for the insulating foam mold. Start by taping the chopsticks and bundles of two.
Then tape each chopstick bundle to the rim of the cake pan opposite one another. The purpose of the chopsticks is to give uniform thickness of foam insulation. Next wet the cake pan and bottom surface of the pie dish with water.
Start by adding a single layer of great stuff. Spray foam over the entire bottom of the cake pan and spray with water. Add a second layer of great stuffed spray foam And spray with water.
Press the pie dish evenly and firmly into the cake pan. Flip the combined dishes over and plus a heavy weight on top of the dishes. Let the foam cure overnight.
Once the foam is cured, use a serrated knife to cut the dish free and remove excess foam from the sides of the dish. Remove the chopstick spacers and separate the pie dish from the cake pan. The foam should stay with the pie dish.
Next, put the cryo stage under the objective lens and place a plastic clipboard over it. With a marker, trace the path of the objective lenses as they swing in and out. Mark an X away from the semicircle close to the edge of the pie dish.
With the second clipboard, mark the location of the crowd grid box mount. Cut out the hole for the crowd grid box using a three-quarter inch hole saw and drill, press on the other clipboard. Cut out the X with a three-quarter inch hole saw.
Remove the semi-circular area by cutting several Times with a three inch hole saw and drill press Now the fabrication is complete. The materials needed For sample preparation include a cryo grid box and handling rod tweezers, cryo grid box transfer block, torn chem wipe cooling cup liquid et ethan cup 400 mesh R two one holy carbon EM grids, styrofoam box, and a pneumatic plunge freezer. Vertical down the styrofoam box, cryo grid box, transfer block, cooling cup and liquid et ethan cup with liquid nitrogen.
Next, place the cryo grid box into the cryo grid box transfer block. Using the tweezers to hold the EM grid carbon side up. Apply two microliters of cell suspension at two times 10 to the six cells per milliliter to the top of the grid.
Load the tweezers onto the pneumatic plunger and pull the liquid from the grid surface by holding a piece of torn chem wipe to the backside of the EM grid and plunge into liquid Ethan slurry. Quickly move the grid from the liquid ethane into the liquid nitrogen and place it in a cryo grid box. To use the cryo stage, Start by filling the dish with liquid nitrogen and cover it with a clipboard.
After transferring the cryo grid box through the whole cut in the plastic clipboard, remove one grid from the box and place it on the viewing surface of the aluminum block. After moving the cryo stage into position on the microscope, place the second clipboard on top of the first and carefully. Slide the first clipboard out.
Insert the objective lenses and use the microscope as normal. Start by scanning and low magnification for the grid center indicated by three metal tabs. Here, focus at 50 x.
You can see a thin layer of vitreous ice covering the grid changing to fluorescence. You can see the CFP histone marker localized to the nucleus of the cell. Next I switch objectives to 100 x and Focus on individual yeast cells.
To Remove the cryo stage from the microscope, place the first clipboard on top of the second and carefully slide the bottom clipboard out. Remove the cryo stage from the microscope and replace the grid into the cryo grid box using the whole cut in the clipboard. Lastly, screw the cryo grid box handling rod into the cryo grid box and transfer it to liquid nitrogen for viewing at a later date.
By electron microscopy stage stability is very important. In microscopy, we found that the majority of image instability in our system comes from the added weight of the cryo stage on the microscope stage. In the absence of liquid nitrogen, we can see a large amount of instability when focused on the aluminum block.
By adding support to the front of the microscope stage, the instability is greatly reduced. Here I show a support structure consisting of an adjustable screw mechanism that I place between the microscope stage and base. The Benefit of extra support is also seen in the cryo light microscopy showing that the instability comes from the added weight and not the liquid nitrogen bubbles.
As You can see, this cryo light stage is an inexpensive and useful way to gather fluorescent information of frozen hydrated cells. Scanning in brightfield and darkfield allows you to map areas of uniform ice. Once we locate these areas of interest, we can use the fluorescence channel to see our fluoro for marking proteins of interest in this case, A CFP hisone marker.
After capturing the images in cryo fluorescence, we can use the low magnification images to map and find the same grid, square, and image the same cells in cryo-electron microscopy. As you can See, this cryo light stay is useful and inexpensive way to gather fluorescent images of frozen hydrogen samples. The same region can also be observed in cryo EM by following standard cryo EM transfer and imaging techniques.
In this case, the cryo light microscopy data acts as a reference map to identify exact region for correlative study. This stage can be applied to correlative imaging of many types of samples, including whole cells in vitro reactions of macro molecular complexes or isolated organelles and thin cryo sections of tissue. Thank you for watching and I hope you find this cryo light microscopy stage as useful as we do.
