Microscopy!

Today (November 8th), we worked with cells from the blood-brain barrier using microscopy! By definition, microscopy is "investigation using a microscope." To refresh my knowledge of the microscopy processes we use, JP gave me this link to read. In our work, we used Alexa 488 and Hoechst 33342 (also known as DAPI). Before I arrived, JP fixed the cells and added the primary antibody. We had to decide whether or not to permeablize the cells before adding the DAPI. We decided to try imaging the cells without permeablization first. We added a DAPI solution to the sample, incubated the cells, and then removed the excess solution. We then washed the sample a couple times to remove excess dye.

Once the sample was ready to be imaged, we had to decide what wavelengths of lasers to use to image them. To do this, we used a program called SpectraViewer. The resulting graphs are shown below.

 The vertical line on the graphs represents the wavelength of the selected laser. The green curve represents the Hoechst 33342 and the red curve represents the Alexa 488. The shaded regions represent the region that will be recorded. We decided to use the 405 nm laser for the Hoechst 33342 and the 488 nm laser for the Alexa 488.

To image the cells, we used a Zeiss Spectral Confocal Microscope (seen in the image below). It was so cool to be able to see the cells magnified to such a high degree!

The nuclei were very clearly visible, but the staining from the Hoechst 33342 was not visible. After viewing the images, we decided that it would be necessary to permeabilize the cells and re-add the Hoechst.

I won't be able to go to RPI next week because I will be in Kansas City for my national championship horse show, but I can't wait to return on the 22nd to see the images from the cells and continue our work!