The overall goal of the following experiment is to introduce fluorescently labeled small molecules or proteins into the cytoplasm of a cultured mammalian cell, and to observe the induced morphological changes from micrometer to multi nanometer resolution. This is achieved by first culturing mammalian cells on grided glass cover slips and micro injecting them with a fluorescently labeled small molecule or protein of interest. Next, the cells are fixed in formaldehyde and observed and imaged using fluorescence microscopy.
Then the cells are fixed with glutaraldehyde, sectioned, stained, and imaged using electron microscopy. Finally, images taken during brightfield fluorescent and electron microscopy are combined and aligned. Ultimately, the correlation of these images allows the researcher to image at high resolution perturbations induced by the small molecule or protein of interest.
Hi, I'm Dr.Evan Reddick in the laboratory of Dr.Neil Alto in the microbiology department at the University of Texas Southwestern Medical Center in Dallas. Today I'm gonna talk to you about the M Clem procedure or the microinjection correlative light and electron microscopy. The main advantage of this procedure over standalone techniques such as microinjection or microscopy, is that here they're performed in concert and aided to the use of a grided glass cover slip.
It's this gridded cover slip that allows researchers to return to the same cell during different parts of the protocol. In addition, this technique affords the researcher the ability to observe induce subcellular architectural changes from a wide variety of injectable small molecules or proteins of interest, and to do so from micrometer to multi nanometer resolution. To prepare for microinjection culture cells such as normal rat kidney or immortalized cervical cancer cells on photo etched glass grided cover slips affixed two live cell dishes incubate the cells at 37 degrees Celsius and 5%CO2.
Depending on the experimental timeline to be used, adjust the co fluency of the cells. A fully confluent plate will make identification of micro injected cells in the electron microscope very difficult. Therefore, seed cells such as they reach about 50%co fluency on the day of a short experiment and around 25%co fluency.
On the day of a longer experiment, Prepare fluorescently labeled protein DNA or small molecules in the appropriate buffer to a final volume of 50 microliters. Depending on the Fluor ofor of choice, add a tracking dye such as Texas red or cascade blue. Add a concentration of 0.5 to one milligrams per milliliter.
Filter the solution through a 0.22 micron centrifugal filter. After pulling microinjection needles using manufacturer's guidelines, carefully pipette for microliters of solution into the backend of a needle. Load the needle into the micro manipulator arm of a commercial microinjection device attached to an inverted microscope.
Place a dish of cultured cells into the dish holder of the inverted microscope. Find cells growing within a photo etched grid and note the grids alphanumeric identifier. This will be used in all subsequent steps.
It's important to choose cells for microinjection that are adhered close to the center of the cover slip, and to avoid those near the periphery. This is important as it will ensure an efficient transfer of your cells of interest to the epon resin centerpiece used later for electron microscopy Manual, manually lower the injection arm over the cover slip and use the micro manipulator to lower the needle until the tip gently touches the surface of the cells. Micro inject every other cell within the chosen grid for cells grown at about 50%Co fluency expect around 80 cells per 600 square micrometer grid, which will give an N of about 30 to 40 experimental and controlled cells.
After injecting, place the dish back into the incubator for a desired length of time to carry out fluorescent microscopy. Discard the growth medium and fix the cells with formaldehyde in PBS at room temperature for 10 minutes. Replace the fixative with one XPBS.
Then document the arrangement of the cells by taking five to 10 bright field images of the chosen grid at low and high magnification. To identify the micro injected cells, take fluorescent images at the excitation and emission wavelengths of the inert injection dye eye. Using a confocal microscope, collect a Zack of images of micro injected cells at high magnification within the same previously imaged grid.
High magnification is needed in order to correlate fluorescent images with subcellular structures that will be identified using electron microscopy. After confocal imaging, use a cover slip removal fluid to separate the cover slip from the dish and place it cell side up in a fresh culture dish containing one XPBS. Proceed to processing for electron microscopy.
Be after following standard EM protocols. To fix stain and dehydrate the samples, prepare epon resin and place the cover slip cell side down on top of an eon resin filled tube. Allow the resin to polymerize at 60 degrees Celsius for 24 hours.
To remove the cover slip from the epon resin. Quickly immerse it into liquid nitrogen. The temperature shock from 60 degrees Celsius to negative 196 degrees Celsius.
Dislodges the cover slip from the resin tube. Wait five to 10 minutes, then retrieve the tube, which is now free of the cover slip. Under a dissecting scope, inspect the surface of the resin to locate the grid of interest.
Using a sterile razor blade or scalpel, trim the epon block down such that only the grid of interest is at the apex of the block with an ultra microtone cut serial sections of the trimmed block use urinal acetate and lead citrate to contrast the sections using standard protocols. Relocate the desired cells in the electron microscope using the relative positioning of cells as a guide. Using the high resolution fluorescent images captured earlier.
Identify regions of interest in the electron micrograph that correspond to the fluorescent signal. Use landmarks such as the position of hetero chromatin and the nuclear envelope to determine which fluorescent Zack image corresponds to the EM slice. Currently being imaged with an imaging program such as Image J or Adobe Photoshop, reduce the opacity of the fluorescent image to 50%and overlay it onto the corresponding TEM image.
Align the images by adjusting the scale shown. Here are the results from microinjection and correlative light and electron microscopy. This image shows the brightfield micrograph of NRK cells growing on a laser etched glass gridded cover slip.
Note the b and m background letters used for identifying the grid containing injected cells. Arrows indicate the two cells identified and imaged with electron microscopy. Shown here is a fluorescent image of the inert tracking dye cascade blue used to identify micro injected cells.
Here is an overlay of the brightfield and fluorescent signals of amine labeled small molecule. This panel represents the correlation of brightfield fluorescent and electron microscopy of an injected cell. The arrowhead indicates fluorescent puncta imaged at higher magnification in this panel.
After watching this video, you should have a good understanding of how to micro inject a protein of interest and track its subcellular morphological changes that it induces using the procedure of microinjection Clem or correlative light and electron microscopy.
