Doesn’t matter if it’s fixed or alive

Many people have been asking if the fixation procedure affects the Refractive Index (RI) of cells. This week we tried to verify it. We compared the RI values of seven living and eight fixed Fibroblastic Reticular cells for different structures: plasma membrane, nucleus and organelles.

The graph 1 shows the results obtained.

Graph 1. Comparison of the RI between living cells (n=7) and fixed cells (n=8) for three different structures: plasma membrane, nucleus and organelles.

 

We could identify for each structure a specific RI range which is slightly different for the two conditions (<0.001). We can conclude that it is possible to identify a RI range proper of each cellular sub-structure allowing the definition of special stains common to both living and fixed cells.

 

The following pictures (Figure 1, 2) show the digital staining obtained with our software STEVE for both cases.

final image_fixed cells

Figure 1. Fixed Fibroblastic Reticular Cell. On the left panel, the refractive index distribution within the cells is displayed while on the right panel the 3D reconstruction is shown. The nucleus is digitally stained with blue, the organelles with red and the plasma membrane is green.

final image_living cells

Figure 2. Live Fibroblastic Reticular Cell. On the left panel, the refractive index distribution within the cells is displayed while on the right panel the 3D reconstruction is shown. The nucleus is digitally stained with blue, the organelles with red and the plasma membrane is green.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The results obtained in this study confirm that different cell structures correspond to a specific RI range independently from fixation. This will allow us to create a Cell Cloud in the future, where every user of the 3D Cell Explorer will be able to upload his/her own stain panel and compare them with the other users.

 

Many thanks to Chris for this blog post!