Figure 1.
Principle of illumination of ultramicroscopy.
We only illuminate the focus region from the side, hence other parts of the sample are not bleached. This allows for the detection of fast bleaching fluorescence signals.
Figure 2.
A: Original image data from hippocampus region, blood vessels and nerve cells with dendrites are visible. The stripe-shaped illumination artifact is caused by shadow-producing impurities in the tissue sample. We illuminated from the right side. B: image after nonlinear horizontal smoothing, C: image after dividing image A by B. This stripe-removal algorithm corrects the non-homogeneous illumination. (all images are contrast adjusted, scale bars 50 microns).
Figure 3.
View on the sample data (500×500×300 microns) of the hippocampus CA1 region, displayed with computer graphics. The section is about 0.5 mm under the surface of the excised hippocampus.
Figure 4.
The formalin-induced fluorescence bleaches relatively fast, but with ultramicroscopy it can be preserved long enough for recording.
Figure 5.
An original image from the z-stack.
The image displays a worse contrast on the sides left and right, because the illumination beam is less focused there and illuminates a thicker part of the sample. We analyzed the beam shape and the depth-resolution with this illumination technique in a previous paper [15]. (scale bar: 100 micron).
Figure 6.
A maximum-filtering followed by a minimum-filtering closes all ‘holes’ in the intensity graph and smoothes the graph to the background level. The large holes are somata, small holes are dendrites.