A method for direct imaging of x–z cross‐sections of fluorescent samples

Abstract

The x-z cross-sectional profiles of fluorescent objects can be distorted in confocal microscopy, in large part due to mismatch between the refractive index of the immersion medium of typical high numerical aperture objectives and the refractive index of the medium in which the sample is present. Here, we introduce a method to mount fluorescent samples parallel to the optical axis. This mounting allows direct imaging of what would normally be an x-z cross-section of the object, in the x-y plane of the microscope. With this approach, the x-y cross-sections of fluorescent beads were seen to have significantly lower shape-distortions as compared to x-z cross-sections reconstructed from confocal z-stacks. We further tested the method for imaging of nuclear and cellular heights in cultured cells, and found that they are significantly flatter than previously reported. This approach allows improved imaging of the x-z cross-section of fluorescent samples. LAY DESCRIPTION: Optical distortions are common in confocal microscopy. In particular, the mismatch between the refractive index of the immersion medium of the microscope objective and the refractive index of the sample medium distorts the shapes of fluorescent objects in the x-z plane of the microscope. Here, we introduced a method to eliminate the shape-distortion in the x-z cross-sections. This was achieved by mounting fluorescent samples on vertical glass slides such that the cross-sections orthogonal to the glass surface could be imaged in the x-y plane of the microscope. Our method successfully improved the imaging of nuclear and cellular heights in cultured cells and revealed that the heights were significantly flatter than previously reported with conventional approaches.