The overall goal of the following experiment is to produce cross sections of yeast communities to examine the spatial distribution of different fluorescent cell populations within a community. This is achieved by first freezing the community and then fixing it using methanol to preserve the spatial distribution of cells in the community. As the second step, the methanol is evaporated to allow penetration of the embedding OCT compound.
The embedded community is then frozen in preparation for cryosectioning. Next, a cryo ome sections. The community and cross-sections can be imaged under a fluorescence microscope Results are obtained that show the spatial distribution of fluorescent cell populations inside a community.
The main advantage of this technique over existing methods like optical sectioning, is that it reveals the spatial distribution of populations deep inside these communities. To begin this procedure, grow yeast communities on a membrane filter. This protocol corresponds to a typical community of less than two times 10 to the eight cells on a six millimeter diameter membrane filter next in a minus 20 degrees Celsius freezer.
To preserve a greater degree of fluorescence, use a piece of watman 5 4 1 filter paper to cover the bottom of a one centimeter diameter. Well, this filter paper is used as a carrier to transfer the community In later stages, add one milliliter of minus 20 degrees Celsius, 100%methanol to the well, and leave the well at minus 20 degrees Celsius for the temperature to equilibrate. Now, freeze the yeast community by using tweezers to dip the membrane filter with the community in liquid nitrogen for at least 15 seconds.
Next, transfer the frozen community to the well with minus 20 degrees Celsius. Methanol in the freezer ensure that the community is kept horizontal. When submerging it in the methanol wait for 20 minutes, the membrane filter will start to disintegrate in the methanol.
After 20 minutes, transfer the community using the carrier watman filter paper from the methanol well to a cold Petri dish kept at minus 20 degrees Celsius. When transferring, keep the community on top of the carrier Watman filter. Draw away extra drops of methanol using another piece of thick watman paper before putting it in the cold Petri dish.
This will ensure that the methanol evaporation time is consistent from sample to sample. Wait four to six hours for the methanol to evaporate. Ending the evaporation at the right time is crucial for the success of this procedure.
To avoid over drying the sample, check the state of the yeast community periodically during evaporation, While the methanol is evaporating. Make a rectangular cubic aluminum foil container that is large enough such that the sample has at least about two millimeter margins from each side. Add a drop of OCT to the bottom of this container to ensure that the sample is surrounded by OCT from all sides.
When evaporation is complete, take the sample out of the minus 20 degrees Celsius freezer. Cut away the parts of the filter paper not covered by the yeast community. It is important to perform this step quickly.
To avoid over drying the sample. Place the community on the bottom surface of the aluminum foil container and immediately cover the sample with OCT to three to four millimeters above community height. Wait 10 minutes.
Finally, place the OCT embedded sample on a metallic plate on dry ice to freeze store the frozen communities at minus 20 degrees Celsius or minus 80 degrees Celsius. In preparation for sectioning frozen communities, set the temperature of the cryo section chamber to minus 25 degrees Celsius. Leave the samples in the chamber until its temperature reaches the temperature of the chamber.
Then remove the foil from the sample and cut off the excess OCT. Apply a drop of OCT on a cryo mount and mount the sample onto the cryo mount. Spread the OCT uniformly on the side of the sample with exposed watman filter paper so that the OCT thickness is two to three millimeters.
Ensuring an OCT margin of two to three millimeters on all sides of the sample helps to preserve the integrity of sections. Let the OCT freeze inside the cryostat chamber before sectioning. Use the sides of the OCT sample block to align the sample with respect to the blade.
If the sample is not aligned with respect to the blade, sectioning will start from a corner or side of the sample. Adjust the mount to ensure that the blade cuts a perfectly vertical face of the sample block. Typical sections for yeast communities are about 14 micrometers thick.
After cutting a section, use a microscope slide kept at room temperature to collect the section. By slowly moving the slide to the cut section, the section will melt and transfer to the microscope. Slide up to 18 sections can be transferred onto one slide.
Store the slides at cold temperatures, preferably minus 20 degrees Celsius before imaging. Loss of signal is minimal when kept for one week at four degrees Celsius for maximum fluorescent signal. Imaging should be done immediately after sectioning.
Representative fluorescence images of vertical cross sections of yeast communities grown under identical conditions are shown here. The community consists of two phototropic strains of visier tagged with the fluorescent proteins. M cherry visualized as red and Y-E-G-F-P visualized as green competition for shared resources, including space leads to columnar patterns as displayed in their vertical cross sections.
Resolutions down to a single cell can be resolved in cross sections. This figure compares cross sections obtained with methanol fixing shown in the bottom panel and without methanol fixing shown in the top panel. The fluorescent signal is preserved in community sections without fixing, but the integrity of the community is compromised.
As a result, some cells float away around the edge of the section and the overall yield is lower compared to fixed communities. Methanol fixing keeps community cells together, but causes shrinkage as observed in the decrease in community height. This figure shows representative brightfield and fluorescence vertical cross-sections of a yeast community consisting of two Protopic strains of visier teed with DS RED and YFP fluorescent proteins.
Cells on top of the community form a crown that appears distinct from mother cells, characterized by stronger scattering in brightfield and brighter fluorescence. These patterns are consistent with reported patterns of differentiation in yeast colonies. After watching this video, you should have a good understanding of how to use cryosectioning for examining the fluorescence su spatial patterns within yeast communities.
