Two-photon fluorescence and confocal reflected light imaging of thick tissue structures

Ki Hean Kim; Peter T. C. So; Irene E. Kochevar; Barry R. Masters; Enrico Gratton

doi:10.1117/12.307081

29 April 1998 Two-photon fluorescence and confocal reflected light imaging of thick tissue structures

Ki Hean Kim, Peter T. C. So, Irene E. Kochevar, Barry R. Masters, Enrico Gratton

Proceedings Volume 3260, Optical Investigations of Cells In Vitro and In Vivo; (1998) https://doi.org/10.1117/12.307081
Event: BiOS '98 International Biomedical Optics Symposium, 1998, San Jose, CA, United States

Abstract

The technology of two-photon excitation has opened a window of opportunity for developing non-invasive medical diagnostic tools capable of monitoring thick tissue biochemical states. Using cellular endogenous chromophores, (beta) -nicotinamide- adenine dinucleotide phosphate [NAD(P)H], the cellular metabolic rates in living human skin were determined. Although important functional information can be obtained from the fluorescence spectroscopy of endogenous chromophores, these chromophores are rather poor contrast enhancing agent for mapping cellular morphology. First, most endogenous chromophores are confined to the cellular cytoplasm which prevents the visualization of other cellular organelles. Second, there is significant variability in the distribution and the quantum yield of endogenous chromophores which depends on tissue biochemistry but prevents consistent comparison of cellular morphology. On the other hand, the deep tissue cellular morphology has been imaged with excellent resolution using reflected light confocal microscopy. In reflected light microscopy, the image contrast originates from the index of refraction differences of the cellular structures. The organelle boundaries with significant index differences such as the plasma membrane and the nucleus envelope can be consistently visualized. A combination of morphological and functional information is required for a thorough tissue study. This presentation describes the development of a new microscope which is capable of simultaneously collecting both two-photon fluorescence and confocal reflected light signals. Promising biomedical applications include the non-invasive diagnosis of skin cancer and the study of wound healing.

Citation Download Citation

Ki Hean Kim, Peter T. C. So, Irene E. Kochevar, Barry R. Masters, and Enrico Gratton "Two-photon fluorescence and confocal reflected light imaging of thick tissue structures", Proc. SPIE 3260, Optical Investigations of Cells In Vitro and In Vivo, (29 April 1998); https://doi.org/10.1117/12.307081

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