The overall goal of this technique is to study the supramolecular organization of photosynthetic membranes using cryo-scanning electron microscopy of freeze-fractured leaf tissue samples. This method can help answer key questions in cell biology, such as the morphological characterization of cells and organelles, the distribution of membrane-integral protein complexes, and localization of biominerals. The main advantage of this technique is that freeze-fracture is performed on leaf tissues, and so thylakoid membrane supramolecular organazation is investigated within the native organelle and cellular context.

While this method is demonstrated here for a biological sample, it can also be applied to such as gels, polymers, and drug delivery systems. Generally, individuals new to these procedures should be aware that not all samples will be well-frozen or properly fractured. To carry out cryofixation of leaf tissues by high-pressure freezing, begin by using the corner of a razor blade to scratch the bottom of the 0.1-millimeter cavity of a 0.1-to-0.2-millimeter aluminum platelet.

Prepare at least a dozen platelets for six samples. Then use absolute ethanol to wash the platelets, and keep them in a clean dish. After preparing the high-pressure freezing machine, fill the samples box with liquid nitrogen.

With a razor blade, cut a small piece of leaf from the plant and use tweezers to place the tissue inside a microcentrifuge tube half filled with one hexadecane. To infiltrate the intercellular spaces of the leaf tissue, insert the tip of the infiltration device, prepared as described in the text protocol into the hole in the lid of the microcentrifuge tube, and pull the plunger to create vacuum. After about 30 seconds, release the plunger.

Next, using tweezers, remove the leaf piece from the tube. Use the razor blade to cut a piece small enough to fit into a platelet. And if necessary, trim the leaf to a thickness of less than 0.2 millimeters.

To prevent tissue collapse, leaves are infiltrated with liquid prior to high-pressure freezing. Sample thickness is kept below 200 microns in order to maximize the chance for obtaining a vitrified sample. Place a clean platelet with the scratch side up into the freezing machine's holder.

And then place the cut piece of leaf into the platelet. Use one hexadecane to fill the remaining space of the platelet before using another platelet with the scratches facing the leaf to close the sandwich. Close and tighten the holder.

Freeze the sample by pressing the jet button. Then, quickly transfer the holder into liquid nitrogen. Use precool tweezers to remove the sandwich from the holder, taking care not to fracture it.

There are two critical steps in the procedure:freeze-fracture, which is a random process and its success can only be assessed in the cryo-SEM'and double-layer coating, which is required for obtaining high-magnification images with minimal beam damage. While preparing and programming the freeze-fracture machine according to the manufacturer's instructions, place the platinum carbon, or Pt/C gun, at 45 degrees, and the carbon gun at 90 degrees. Cool the freeze-fracture system stage to minus 140 or minus 160 degrees Celsius.

Then attach the vacuum cryo transfer shuttle to the machine and use liquid nitrogen to fill the shuttle chamber. Insert a freeze-fracturing specimen holder into the loading station, and with liquid nitrogen flood the loading station chamber. Next, using high-precision-grade tweezers, load a frozen sandwich onto the holder and tighten it in place.

Then flip the holder over to check that the sandwich is secured in place, and repeat for a second sandwich. Detach the cooled shuttle from the freeze-fracture machine and connect it to the loading station. Then open the shuttle valve, introduce the retracting arm into the holder, lock it in place, and reintroduce the arm with the holder into the shuttle.

After reattaching the shuttle to the freeze-fracture machine introduce the holder into the chamber, using the retracting arm. Now align the freeze-fracture microtome knife to height such that it will knock off the top platelets of the sandwiches. With the microtome knife, quickly break the sandwiches.

And then immediately raise up the knife to prevent contamination of the samples by debris clinging to the knife. Position the cooled shutter above the newly-exposed plane to shield the samples from possible contamination by water molecules in the chamber. Next, to coat the samples with platinum, press Setup to show Layer 1 on the screen.

Then turn on the Pt/C gun by pressing the GUN 1 button followed by the ON button. Examine the evaporation rate, and once it reaches 0.02 to 0.04 nanometers per second, press the Measure button to monitor the thickness of the deposited layer while simultaneously exposing the samples. To coat with carbon, select GUN 2 followed by the ON button.

Once the evaporation rate is approximately 0.1 nanometers per second, press the Measure button and at the same time move the shutter to expose the samples. When the coating is complete, warm the stage to minus 120 degrees Celsius. To prepare the scanning electron microscope for cryo work, from the main menu select Stage, Stage Initialize, and confirm.

Under the Vacuum tab, click the Vent button to vent the microscope chamber. Then select Tools, Goto Panel, and open the Stage Points List panel. Select the CRYO EXCHANGE position to move the stage to the proper coordinates for accepting the sample holder.

While wearing a clean pair of gloves, open the specimen chamber and insert the cryostage onto the main stage of the microscope. Close the chamber, and under the Vacuum tab, click the Pump button. Next, cool the microscope by filling the microscope duor with liquid nitrogen.

Once the temperature of the stage drops below minus 120 degrees Celsius, activate the heat function on the cryo transfer unit to warm the stage to minus 120 degrees Celsius. Now attach the cryo transfer shuttle to the microscope and transfer the sample holder using the retracting arm into the microscope cryo stage. Using the joystick, carefully move the specimen holder close to the objective lens.

In the Apertures tab, select the 10-micron aperture. Then, in the Gun tab, double-click the EHT Target field, enter 10 kilovolts for the EHT target, and click OK.Then, to turn on accelerating voltage, click the bottom EHT tab and select EHT On.In the Detectors tab, select InLens from the drop-down list. Then click on the Scanning tab and choose a scan speed equal to one, which is a fast scan to minimize beam damage, and a store resolution of 1024 by 768 pixels.

In the Noise Reduction dropdown list, select Line Average, and adjust the value of n to 20 to 30 lines for searching. Then, by clicking the Control and Tab keys on the keyboard, use the centerpoint function to move the sample to areas of interest while simultaneously magnifying to identify regions with fractured cells. After identifying regions with fractured cells, scan the sample while magnifying to find a fractured chloroplast.

Then change the line average n value to greater than 100, for noise reduction. In the Scanning tab, press Freeze to stop the scanning. By pressing Control Shift Tab on the keyboard, the center-feature function is used to zoom into interesting chloroplast-fracture faces;and high-magnification images are acquired using the same parameters as before.

Finally, press the Save TIFF button to save the image. Carry out image analysis according to the text protocol. This figure shows a cryo-SEM image of a platelet containing a high-pressure frozen freeze-fractured Craterostigma Pumilum leaf piece with a large region of fractured cells.

In this example, the leaf piece is missing;but some leaf tissue remained, attached to the knife grooves on the platelet. After a successful fracture is found, low-magnification images of cells are acquired to identify regions of interest, such as fractured chloroplasts. Images of single-fractured chloroplasts are acquired to identify thylakoid membrane fracture faces.

Shown in this example is a high-magnification image of thylakoid membranes in C.Pumilum leaves. The four different fracture faces can be distinguished. The exoplasmic fracture faces of stacked and unstacked membranes, EFs and EFu respectively, and the protoplasmic faces of stacked and unstacked membranes, PFs and PFu respectively.

Photosystem II, or PS2, is located in both the EFs stacked grana membrane regions and EFu stromalamellar membrane regions. When hydrated, PS2 density is approximately three times higher in the EFs than in the EFu, which differentiates the two faces. During dehydration of C.Pumilum, the density of PS2 in the EFs gradually decreases, reaching roughly half of its density at hydrated conditions.

Notably, upon further dehydration to five to 10%relative water content, PS2 complexes organize into rows and arrays. Development of the freeze-fracture replica technique paved the way for studying the size of membrane protein complexes and their arrangement within biological membranes. The combination of freeze-fracture with high-pressure freezing in cryo-SEM allows investigating various kinds of samples at several size scales, from the tissue and cellular levels down to the macromolecular level at a near-native state.

In addition to the technique shown here, methods such as Energy-Dispersive x-ray Spectroscopy, EDS, can also be applied in the cryo-SEM;for instance, for alkalizing biominerals.