Zeiss 710 MP microscope
A question was raised whether different wavelengths of light frm 720 to 1040 nm are focused at different depths in the sample. This is important for colocalization and for photo manipulation. For instance, if a tissue is zapped at 720 nm to photo-activate and then zapped at 930 nm for imaging, are the same volumes being illuminated,
These tests suggest, surprisingly, that the wavelengths are focused at the same Z depth. These tests are not perfect because the imaging is superficial as in only right up against the coverslip. Perhaps mixing the beads with collagen and pouring into a MatTek dish to set would be a better test.
It is surprising that there is not a Z offset between colors because there certainly is when doing standard confocal. For the IR laser, I do not know whether there is a lens in the lightpath which Zeiss has programmed to compensate.
Images below showing no shift were collected using the 25X N.A. 0.8 multi immersion DIC lens with Zeiss 1.518 oil.
Click here for stack of images used to make montage above.
9 Jan 2013
comments, questions, suggestions: Michael.Cammer@med.nyu.edu