The overall goal of this method is to obtain correlative light and electron microscopy, or CLEM data, by combining fluorescence immunocytochemical information with ultra-structural morphology from transmission electron microscopy in a single image. This method can help answer key questions in the field of immunocytochemistry and pathology, such as what subcellular structure is a particular protein of interest associated with. The main advantage of this technique is that it uses the same quantum dot nanoparticle probe to obtain both the live microscopy signal from the target protein and the electron microscopy structural localization.
After fixing, embedding and sectioning human somatostatinoma tumor tissue according to the text protocol, prepare a section adhesive solution by placing 20 centimeters of clear adhesive tape into a five milliliter bottle containing 2.0 milliliters of acetone and let it stand for 10 minutes. Remove the tape and dip a nickel grid into the solution, then remove the grid and allow it to air dry. Once dry, use the dull side of the grid to pick up an ultra thin section.
It's critical to establish the correct etching time for the chosen resin formulation. 30 minutes works well for this formulation, but it may need to be recalculated for harder or softer formulations. Prepare one milliliter of fresh saturated sodium metaperiodate etching solution in distilled water and place droplets of the solution on a clean lab film surface.
Place completely dry grids with sections onto the droplets and incubate at room temperature for 30 minutes. Then, transfer the grids to distilled water droplets for 60 seconds to wash them off. To carry out antibody and quantum dot, or QD labeling, on a clean lab film surface, pipette drops of 0.05 molar glycene and PBS.
Place the grids on the droplets and incubate for 10 minutes to block residual aldehyde on the sections. To remove the aldehyde block, briefly blot the edge of the grid. Then place the grid on a droplet of one percent normal goat serum, or NGS, and one percent BSAC in PBS for 10 minutes.
Prepare one milliliter of blocking solution by adding 10 microliters of NGS and 100 microliters of BSAC to 890 microliters of PBS. Then, condition the sections by placing them on a droplet of antibody diluent for 10 minutes. Next, use antibody diluent to dilute anti-somatostatin polyclonal antibody one to 10, and place the grids on droplets of the solution.
Incubate in a moist chamber at room temperature for one hour. Following the incubation, remove the antibody reagent by blotting the edge of the grid. Then use antibody diluent to wash the sections two times for five minutes each.
After diluting secondary antibody one to 10 and preparing drops, incubate the grids on the droplets at room temperature for one hour. Then, remove the antibody solution and wash the sections two times. Dilute streptavidin conjugated QDs one to 10 and incubate the samples on drops of the solution in the dark, at room temperature for one hour.
Then use antibody diluent to wash the grids twice, each time for five minutes, and blot the edge of the grids. Next, wash the grids in distilled water for two minutes before blotting dry the edges. Place the immunostained grid section in a droplet of water on a glass slide and use a glass cover slip to cover it.
To carry out fluorescence light microscopy, insert a filter cube with the following characteristics and use 365 nanometer LED illumination. Place the immunostained section on the immunofluorescence microscope stage. Use light microscopy to evaluate the labeling pattern, the level of any non-specific labeling and to establish that negative controls are clear.
Next, identify ROIs in relation to grid bars. Then set a full colored digital camera to FL auto color and set white balance to 3, 200 K.Set the camera to automatic exposure mode with a gamma setting between 0.45 and 1.00 to optimize the appearance of the images, and analog gain set to one X.Click the camera icon in the ZEN Two Light software graphical interface to live. Then focus on the image and click Snap in the ZEN Two Light software graphical interface to capture the image to the frame store.
Save the images as tf files to avoid compression and pixelation. Prepare a print showing the position of the ROI in relation to the grid bars or other significant tissue landmarks. Then remove the grid from the slide, wash in distilled water, and gently blot the edges dry.
To carry out transmission electron microscopy, transfer the grid to the microscope for examination using an accelerating voltage of 100 kilovolts. Begin with low magnification of approximately 1, 400X to navigate around the grid and find the ROIs with those found with light microscopy. Observe individual QDs at 70, 000X magnification or above.
They will appear as irregular crystalline structures with moderate electron density. Then, using a digital camera with a monochrome 1, 392 by 1, 040 pixel sensor in the imaging software's graphical interface, click the camera icon to on and acquire images. Move the camera icon to the off position to store the image in the frame store.
To carry out CLEM imaging, use a print from the light microscopy imaging to locate corresponding ROIs by TEM. Tissue architectural features are useful for navigation around the section. Capture an image of the corresponding ROI by TEM.
Next, to prepare a CLEM image, ensure that the TEM image is correctly oriented and enlarged to the same size and resolution as the light microscope image, in this case, 300 pixels per inch. Extend the size of the light microscopy image canvas to double its size. Then copy and paste the TEM image beside the fluorescence image.
To create a fluorescence overlay in Photoshop CS2, click on the rectangular marquee tool, select the fluorescent image, and create a duplicate layer from it. Adjust the layer style blending options to 30 to 40%transparency. Then click on the move tool and drag the fluorescence overlay layer over the corresponding TEM image, and align the images.
After aligning the images, flatten the image to produce the final overlay. Then use the rectangular marquee tool to select the new overlay image and copy it to a new canvas. Finally, save the image as a tf file.
In this fluorescence microscopy image, individual somatostatinoma tumor cells contain abundant secretory granules and were positively labeled for the somatostatin hormone. The nuclei appeared as dark holes and at low magnification, variably intense granular orange QD fluorescence was seen in the cytoplasm. The ROI chosen by light microscopy was recognized and imaged using TEM by referring to a 20X light microscopy image as indicated here.
At higher magnification, the cell demonstrates bright fluorescence and intense QD labeling in cytoplasmic granules. The overlaying of fluorescence data on TEM images, as in this example, suggests strong positive labeling corresponds to granules containing moderately electron dense material within the vesicle limiting membrane. Finally, as seen here at higher magnification, the moderately electron dense granules showed intense QD nanocrystal immunolabeling.
Once mastered, this technique can be done in eight hours if performed properly. While attempting the procedure, it's important to remember to handle the grids carefully, by the edges only. Touching the section while picking up the grid may detach the section from the grid.
Following this procedure, other methods like multiplex immunolabeling can be performed in order to answer additional questions, such as does your protein of interest co-localize with another protein. After its development, this technique paved the way for researchers in the field of pathology to explore protein localization in archival human tissue process for electron microscopy. After watching this video, you should have a good understanding of how to combine immunocytochemical information from fluorescence light microscopy with ultra structure from transmission electron microscopy.
Don't forget that working with osmium tetroxide can be extremely hazardous, and precautions such as wearing protective equipment, working in a fume hood and proper waste disposal should be taken while performing this procedure.
