Correlative light and electron microscopy is a completely stable microscopic technique which can combine the localization information provided by fluorescent microscopy and the ultra structure by electron microscopy. However, the main challenge of this technique is to preserve the fluorescence or fluorescent probes after EM simple preparation. Using this fluorescent protein, we can achieve Epon post embedding correlative light under the electron microscope, which can maintain fluorescence and ultra structure simultaneously.
To begin, take a mouse brain tissue block and trim its surface into a right angled trapezoid. Using a diamond knife with an ultra microtome, cut sections at a thickness of 100 nanometers. Separate a single section from the section band and float it in the water bath.
Add a drop of double distilled water to the center of the gold nanoparticle coated cover glass. Use a sample loop to pick up a section and invert it onto the surface of the water drop. Remove the sample loop, leaving the section on the surface of the water drop.
Then using a marker pen, draw an annulus around the section on the opposite side of the cover glass. To begin, take an ultra thin section of mouse brain tissue expressing mScarlet. Add 10 microliters of mounting buffer onto the ultra thin section on the cover glass and place a new clean cover glass onto the mounting buffer.
Put the two cover glasses with the ultra thin section into an imaging chamber. Locate the section using the annulus marker in the bright-field imaging mode of an inverted fluorescence microscope and move to one corner of the section. Turn on the white light and capture the bright-field image.
Then turn off the white light, activate the laser, and capture fluorescent images of the same field of view. Deactivate the laser and move to the adjacent field of view with a 10%overlap. Capture both the bright-field and fluorescent image of the second field of view and record the imaging path for stitching the field of view together.
Now, use the navigation map to target cells of interest. Activate the laser and capture 300 frames of fluorescent images of the cell of interest. Then deactivate the laser.
Afterwards, activate the white light and take approximately 100 sequential bright-field images. To prepare the sections for electron microscopy, fill a glass jar with double distilled water. Remove the cover glasses, holding the ultra thin sections from the imaging chamber, and separate them with tweezers.
Clamp the cover glass with the section. Wash off the mounting buffer with double distilled water and allow it to dry. Using a single-sided blade, score a hash around the ultra thin section.
Drop 10 microliters of 12%hydrofluoric acid at each corner of the score. Slowly place the cover glass into the water. Then float the pioliform film and ultra thin section on the water surface.
Place an uncoated slot grid on the section to capture it in the grid center. Cover a glass slide with parafilm. Pick up the grid with the pioliform film and allow it to air dry at room temperature.
To begin, capture the bright-field and fluorescent images of mouse brain tissue expressing mScarlet. Locate the raw data to create a navigation map of several bright-field images of different fields of view in image J.Then open image J, navigate through plugins, click stitching and select grid or collection stitching. Click type grid, snake by column.
Select order up and left and click set slice size. Navigate to select data path and click set file name, followed by check display fusion. To import sequential bright-field images of a specific cell, click file and select import, followed by image sequence.
For the sum intensity projection of these images, go to image, select stacks, click Z projection, and then click sum slices. Then select edit, and click invert to invert the sum intensity projection image and enhance the visibility of gold nanoparticles. Next, import sequential fluorescence images.
To create a sum intensity projection image, click image and select stacks. Then go to Z projection, click sum slices and save the images in TIFF format. Open the sum intensity projection images of both bright-field and fluorescence images.
Go to image, select color, and click merge channels to merge the sum intensity projection of the bright-field image with the fluorescence image. Afterwards, select image and click type, followed by RGB color to convert the format of the composite image into RGB. The gold nanoparticles will appear green and the fluorescent protein will appear red.
Then save the image as a TIFF file. To begin, open the registration software and search for EasyClemv0. Once found, click EasyClemv0, click image or sequence, and select open to import the electron microscopy image and composite images of bright-field and fluorescent images.
In the EasyClemv0 interface, click on 2D XYT to select the non-rigid 2D or 3D as the alignment mode. In the EasyClemv0 window, click on the dropdown box to the right of the select image that will be transformed and resized, likely FM, to choose composite RGB color TIFF, then click on the dropdown box to the right of the select image that will not be modified, likely EM, to choose six image TIFF. Click start to initiate the registration process.
In the electron microscopy image window six image TIFF click on a gold nanoparticle to place point one on it. In the fluorescence microscopy image window composite RGB color TIFF, click on the corresponding gold nanoparticle to put point one on it. Then click on update transformation to confirm the alignment of fluorescence and electron microscopy signals of gold nanoparticles and complete the registration by clicking stop.
To export the aligned image stack containing four channel images, click on the overlaid image, select image or sequence, and click save as. Now open image J, click file, and select open to import the overlaid image. Go to image, click stacks, and then stack two images to separate it into four channel images.
Afterwards, select image and click color, followed by merge channels to merge the three channels into a composite image. To convert the composite image into a correlative light and electron microscopy image, select image, then select type and click RGB color.
