Understanding Filter Turret Registration Problems

Microscope manufacturers all use removable filter cubes in turrets or sliders on their epi illuminated microscopes. When a filter cube is inserted into an individual turret location, the tolerances associated with that fit can often result in a shift in the orientation of the field diaphragm. As seen below, where a filter is inserted into different turret positions, the tolerances associated with a filter cube positioning will result in a shift in the orientation of the field diaphragm. The same effect can also happen when reinserting the same cube in the same turret position.  This makes the process of multi label imaging with different filter cubes frustrating while trying to minimize sample exposure by using a field diaphragm.  

  Image 1 shows two images taken with the same FITC filter cube in different filter turret positions on the same microscope at 100x. The colorized merge of these images show on the right highlights the difference in how a change in position of the cube in the different turret positions effect the resulting image field. 

Image 1 shows two images taken with the same FITC filter cube in different filter turret positions on the same microscope at 100x. The colorized merge of these images show on the right highlights the difference in how a change in position of the cube in the different turret positions effect the resulting image field. 

The images below shows how a filter turret tolerances show up not only as field diaphragm shifts when they are combined, but also create colocalization problems between fluorophores.  

  Image 2 shows two images taken with the same FITC filter cube in different filter turret positions on the same microscope at 40x. The colorized merge of these images show on the right highlights the difference in how a change in position of the cube in the different turret positions effect not only the resulting image field but the colocalization of the cells as well. 

Image 2 shows two images taken with the same FITC filter cube in different filter turret positions on the same microscope at 40x. The colorized merge of these images show on the right highlights the difference in how a change in position of the cube in the different turret positions effect not only the resulting image field but the colocalization of the cells as well. 

  Image 3 shows a zoomed in section of the images shown in Image 2, to better highlight the colocalization problem.

Image 3 shows a zoomed in section of the images shown in Image 2, to better highlight the colocalization problem.