I was most interested to read the article “Fluorescent microspheres enable adaptive-optics microscopy” (February, p. 53) Although an adaptive optical system may well be an effective way to improve deep tissue imaging, it is usually best to limit the work the adaptive system has to perform, which can result in tissue depths much greater than the 40 µm shown in the article.
For example, using an objective lens that is telecentric in object space results in an optical system that is invariant in astigmatism and coma as one focuses at different depths into tissue. This leaves only spherical aberration. It is also possible to eliminate changes in spherical aberration by using an immersion liquid index matched to the tissue index. As one images deeper into the tissue, the depth of the immersion fluid is reduced to maintain a constant depth to the object plane. Hence all optical aberrations can be held constant as one focuses into tissue. The only induced wavefront errors left are due to inhomogeneities in the tissue and spherical aberration due to errors in index matching.
Using this method, I have built and demonstrated a confocal microscope prototype with resolution of 2 µm to depths of 800 µm working at a frame rate of 30 Hz. This was achieved in vivo with unstained tissue. At these depths scattering within the tissue can become the major factor in limiting image quality.
Richard Fisher, Optical Consultant
www.richardfisher.com
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