Most attempts at optical microscopy of bulk living objects have failed because of reflections from optical components and the object surface, and overlapping of unsharp out-of-focus images with the one (weak) sharp in-focus image. The optical signal in the image plane thus consists of a strong d.c. component, a weak noise and a still weaker signal. Most of the first two components can be suppressed by scanning in concordance with both the illuminating and the image-forming rays. In the first scan the focussed-on-object plane is illuminated only in several very fine discrete spots. In the second scan only that light is permitted to reach the image plane which has been reflected in the illuminated "points" of the object plane. Both scans are performed by a single rotating aperture disc and the whole field is covered within 0.05 sec. Because the total number of scanning lines is about 10,000 in the field of the microscope objective - giving a line period for the strongest objectives of 10nm - the resolution is not impaired by this scanning. On the contrary, it follows from theory that the resolution is better because the contrast is also improved.
The following living biological objects have been successfully observed: Eye tissues - cornea (epithelium, stroma, endothelium), lens, all layers of the retina: nerves and nerve fibres: brain cells in whole brain: muscle fibres and nerve endings in striated muscle, heart muscle eel ls (in juvenile mice, through epicardium): stratum corneum of human skin, frog skin eel ls: spermatozoa: blood cells: cartilage, bone and dental tissues: plant eel ls. Fossil teeth and bones, protozoa and bacteria in flint, insects in amber, and fossil plant tissues have also been successfully studied.
Petran, Mojmir; Hadravsky, Milan; Boyde, Alan; and Mueller, Martin
"Tandem Scanning Reflected Light Microscopy,"
Scanning Electron Microscopy: Vol. 4
, Article 9.
Available at: https://digitalcommons.usu.edu/electron/vol4/iss1/9