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Journal of the Optical Society of America B

Accumulated photon-echo spectroscopy for stained tissue samples

Kiyoshi Uchikawa, M. Sakamoto, Akitoshi Koreeda, S. Saikan

Published Jun 1, 2000• 0 citations

Abstract

We performed phase relaxation-time measurements of the accumulated photon echo for normal and cancerous tissue samples of human liver, that were stained with several kinds of dye. Each dye interacted selectively with a specific kind of biomolecule in the tissues, for example, DNA, proteins, or lipids. The cancerous tissues showed a tendency to have shorter relaxation times than the normal tissues had. The difference of relaxation time observed between the normal and the cancerous tissues depended on the dyes that were used for staining, and the difference became most clear when the tissues were stained with a cyanine dye derivative, i.e., YO-PRO3 Iodide, which has a high selectivity for DNA. We performed the relaxation-time mapping of the accumulated photon echo (photon-echo imaging) for a liver tissue sample stained by YO-PRO3 Iodide. The photon-echo imaging was successful in showing a region of a highly differentiated hepatocellular carcinoma in the tissue sample, while fluorescence-intensity mapping could not identify the cancerous region in the same tissue sample. These results indicated a possibility that the reduction of accumulated photon-echo relaxation time observed in the cancerous tissues was caused by a change of microscopic dynamics of DNA.

DOI: 10.1364/josab.17.001058

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We performed phase relaxation-time measurements of the accumulated photon echo for normal and cancerous tissue samples of human liver, that were stained with several kinds of dye. Each dye interacted selectively with a specific kind of biomolecule in the tissues, for example, DNA, proteins, or lipids. The cancerous tissues showed a tendency to have shorter relaxation times than the normal tissues had. The difference of relaxation time observed between the normal and the cancerous tissues depended on the dyes that were used for staining, and the difference became most clear when the tissues were stained with a cyanine dye derivative, i.e., YO-PRO3 Iodide, which has a high selectivity for DNA. We performed the relaxation-time mapping of the accumulated photon echo (photon-echo imaging) for a liver tissue sample stained by YO-PRO3 Iodide. The photon-echo imaging was successful in showing a region of a highly differentiated hepatocellular carcinoma in the tissue sample, while fluorescence-intensity mapping could not identify the cancerous region in the same tissue sample. These results indicated a possibility that the reduction of accumulated photon-echo relaxation time observed in the cancerous tissues was caused by a change of microscopic dynamics of DNA.

The fluorescence detection technique of incoherent accumulated photon echo is applied to the investigation of animal and human tissues. The correlation between the kind of animal tissue and the dephasing time is determined. The dephasing times of normal and cancerous human tissues are measured by this technique. The cancer tissues exhibit shorter dephasing times than the normal tissues, and this is interpreted as being caused by the large number of conformational substates of cancer tissues. The dephasing time of a benign-tumor tissue is also measured and shows a value similar to that of the normal tissue. The dephasing time of a metastasizing cancer region is also measured. The applicability of photon-echo spectroscopy to diagnosis is noted.

An analytic theory and experimental studies of the accumulated photon echo is presented. We define a temporal correlation function

\tilde{G}

of the excitation field E ( t ) as

\tilde{G}(\tau,t)=(1/T_{1})\int_{0}^{\infty}\mathrm{d}t'E(t-t')E(t-t'-\tau)\cdot\exp(-t'/T_{1})

, where T 1 is the population relaxation time of the sample, and call the width of

\tilde{G}

with respect to τ a temporal correlation time

\tilde{\tau}_{\text{c}}(t)

. It is proved that the echo intensity is written by the temporal correlation functions when (1) T 1 and the duration of the excitation light τ p are much longer than

\tilde{\tau}_{\text{c}}(t)

in order to make the function

\tilde{G}((\tau,t)

well-defined, (2) T 1 and τ p are much longer than the homogeneous dephasing time T 2 , and (3) one can find a time interval Δ t such that

\tilde{G}(\tau,t)

does not change much between t and t + Δ t , where

\varDelta t{\gg}\tilde{\tau}_{\text{c}}

. Then we can show that the time resolution of the accumulated photon echo is determined by the averaged

\tilde{\tau}_{\text{c}}(t)

over an interval of the integration of the echo signal. This discussion is valid for any excitation source if the above conditions are satisfied, and applies to almost all cases of the practical accumulated photon echo experiment. A discussion is also made on the spurious spike on the echo decay curve near zero delay time that appeared in an experiment with a high time resolution in Nd 3+ -doped silicate glass. It is shown that the most probable cause for this spike is uncorrelated inhomogeneous broadenings of the resonant lines of neodymium ions.

The time resolution of a heterodyne-detected accumulated photon echo has been improved significantly by using a dye laser whose lasing frequency is repetitively scanned. A time resolution of less than 30 fsec was obtained, even with a non-mode-locked cw dye laser. This technique is applicable provided that the absorption recovery time of the sample is longer than the repetition period of the frequency-scanning dye laser. The absorption recovery times of several dyes that were doped with polyvinyl alcohol have also been measured.

Fourier-transform spectroscopy using the technique of femtosecond accumulated photon echo with incoherent light has been performed in dye-doped polymers. Several types of interference effect or quantum beat between the zero-phonon line and other spectral components including the phonon sideband, the vibronic lines, and the photoproduct state have been observed. Based on the density matrix incorporating the transition-frequency modulation, it is pointed out that the factorization approximation for the relaxation function holds rigorously in the accumulated photon echo. We also clarified that the Fourier cosine transform of the heterodyne-detected echo signal provides the hole-burning spectrum.