The overall goal of this procedure is to image the distribution of metals in a population of cells. This is accomplished by first preparing sterile silicone nitride windows and next growing cells on those windows. Then the cells are fixed, rinsed into a compatible buffer and dried.
The final step is to image the cells at the beam line. Ultimately, x-ray fluorescence microscopy is used to show the distribution of metals in the cell. Generally, individuals new to this method will struggle as the windows are very fragile.
Visual demonstration of this method is critical as growing cells on the windows with minimal cell aggregation, as well as the rinsing steps are tricky because cells do not adhere well to the x-ray fluorescent substrates. The first task is to isolate the window. First, using a stereoscope, make sure that a pair of reverse fine tip tweezers are straight aligned and clean.
Next, slightly open the capsule containing the window. Squeeze one end containing the window while rotating the other end. Don't squeeze the window itself.
Then carefully grip the frame with the tweezers while gently squeezing the capsule near the opened end. Apply pressure to make the capsule wider where the window edges should come out and remove the window. Now prepare some adhesive tape.
Place a piece of tape on a clean slide and cut a quarter inch strip of tape from the long side of the slide. Then place a slide of tape on the short side and remove the section of tape with the tweezers. Apply the tape to the edge of the window with enough force to adhere it and being careful not to cover the window opening with tape.
Using the tweezers, set the window into the bottom of a cultured dish and press the tape firmly to the dish. Then repeat the process to tape down the other side of the window. The dish should now be sterilized by UV for an hour.
This protocol requires that cells be grown to 50 to 70%confluence on dishes containing the attached window. Any vital stains applied and the cells should be imaged before proceeding with fixation. Remove the media by gentle aspiration tilting the dish at a 45 degree angle.
Then rinse the dish gently with DPBS. Replace the removed liquid immediately by gently adding fresh 4%P-F-A-P-B-S at a 45 degree angle and towards the side of the dish. Slowly relax the angle to allow the fixative solution to cover the windows.
Keep the fixative on the cells for 20 minutes after the fixation. Remove the liquid by gentle aspiration. Using the same gentle technique as before, gently replace the liquid with pipes.
Sucrose solution. The technique for removing and replacing the liquid is critical because the cells do not adhere well to the substrates. This is where many first timers have had their experiments fail.
Then gently replace the pipe sucrose solution to wash away any residual fixative. Now, remove the window by tweezing the tape. If the windows get detached from the tape, float the window to the surface with added solution and remove it with the reverse tweezers.
Now, without touching the window, thoroughly dob any solution from the edge. Also, dob the indented area using a rounded fold of tissue. The goal is to have as little crystallization of salt or buffer on the window as possible.
Lastly, carefully place the window onto the grid mat propped up on an edge against the ridge in the mat. The window should touch as little of the mat as possible. Once the sample is dried, the verify the presence of cells on the windows using a light microscope.
After confirming that cells are on the windows, pack the windows for transportation to the synchrotron beam line. Gently tape the windows on two edges to a glass slide. Then tape the glass slide to a plastic Petri dish and tape the Petri dish shut.
Up to this point, the steps may be carried out at any typical lab, the following steps would best be done at a synchrotron beamline. Unpacking the window begins with removing the tape gently with tweezers. Then the window is transferred to an aluminum holder provided by the beamline collaborators at the synchrotron.
Secure the window with a thin even coat of nail polish along its edge. Now, insert the aluminum holder into a kinematic mount. Attach the mount onto a bracket connected to the motorized stage that is inside the sample chamber and at the beamline of the x-ray microscope.
After exiting the lead line to x-ray microscope instrument area, set up the scan using beamline specific software. Choose the appropriate area of the sample to scan while viewing the position of the x-ray beam on the sample. Using a camera equipped with a video crossed hair and pre aligned with a downstream scintillator camera, additional details are provided in the text protocol.
Using these techniques in the x-ray images of a sample, it is possible to see that there is variation in the elements present indicating that fixation preserve these aspects of the cells, such as with this human HSY five Y cell. However, if buffer remained present during drying, extensive crystal formation creates structural damage to the cells and interferes with the collection of the x-ray fluorescent spectra. This can be seen in a poorly processed RET B 1 0 3 cell.
After watching this video, you should have a good understanding of how to prepare a adherent cells by chemical fixation for x-ray fluorescence imaging. Don't forget that working with formaldehyde and biological materials can be extremely hazardous and precautions such as proper PPE and following institutional guidelines should always be taken while performing this procedure.
