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On the basis of an infrared femtosecond Cr:forsterite laser, we developed a semiquantitative method to analyze the microscopic distribution of bilirubins. Using 1230 nm femtosecond pulses, we selectively excited the two-photon red fluorescence of bilirubin dimers around 660 nm. Autofluorescences from other endogenous fluorophores were greatly suppressed. Using this distinct fluorescence measure, we found that poorly differentiated hepatocellular carcinoma (HCC) tissues on average showed 3.7 times lower concentration of bilirubins than the corresponding nontumor parts. The corresponding fluorescence lifetime measurements indicated that HCC tissues exhibited a longer lifetime (500 ps) than that of nontumor parts (300 ps). Similarly, oral cancer cell lines had longer lifetimes (>330 ps) than those of nontumor ones (250 ps). We anticipate the developed methods of bilirubin molecular imaging to be useful in diagnosing cancers or studying the dynamics of bilirubin metabolisms in live cells.

This paper aims to evaluate the clinical characteristics of ovarian cancer patients with cerebral metastases. Ten ovarian cancer patients with brain metastases were retrospectively identified from a total of 539 ovarian cancer patients. Their characteristics before and at the time of diagnosis of cerebral metastases were analysed. The survival of them was also measured. Ten (1.9%) of the 539 ovarian cancer patients had brain metastases in the study period. Nine had stage III or IV tumours with either moderate or poor histological differentiation. The mean time from diagnosis of ovarian cancer to documentation of central nervous system metastasis was 24.3 months, which was 11.1 months if other sites of metastasis were involved before cerebral relapse. All of the patients with intra-cranial tumours suffered from associated neurological defects and relived by treatments. The median survival time after diagnosis of central nervous system involvement was 3 months. In this study, all ovarian cancer patients with cerebral metastases had clinical neurological symptoms. Physicians should pay more attention to ovarian cancer patients with neurological defects and arrange brain imaging studies for the early diagnosis of brain metastases and prompt management to improve quality of life.

Water samples from both hot and artesian springs in Kuantzeling in west-central Taiwan have been collected on a regular basis from July 15, 1999 to the end of August 2001 to measure cation and anion concentrations as a tool to detect major earthquake precursors. The data identify chloride and sulfate ion anomalies few days prior to major quakes and lasting a few days afterward. These anomalies are characterized by increases in Cl- concentrations from 34.9% to 41.2% and 71.5% to 138.1% as well as increases in SO42- concentrations from 232.7% to 276.8% and 100.0% to 155.1% above the means in both hot and artesian springs. The occurrence of these anomalies is probably explained first as stress/strain-induced pressure changes in the subsurface water systems which then generate precursory limited geochemical discharges at the levels of subsurface reservoirs. Therefore, finally leading to the mixing of previously separated subsurface water bodies occurs. This suggests that the hot and artesian springs in the Kuantzeling area are possible ideal sites for recording strain changes serving well as earthquake precursors.

Using 1230nm femtosecond pulses, we selectively excited the 660nm two-photon red fluorescence of bilirubin oxidants. This distinct fluorescence measure can be used to evaluate metabolic state of bilirubins in cancer cells.

Without a labeling, we demonstrated that lipid granules in leukocytes have distinctive third harmonic generation (THG) contrast. Excited by a 1230nm femtosecond laser, THG signals were generated at a significantly higher level in neutrophils than other mononuclear cells, whereas signals in agranular lymphocytes were one order smaller. These characteristic THG features can also be observed to trace the newly recruited leukocytes following in vivolipopolysaccharide (LPS) challenge. Furtherm ore, using video-rate TH G microscopy, we also captured images of blood cells in human capillaries. Quite different from red-blood-ce lls, every now and then, round and granule rich blood cells with strong THG contrast appeared in circulation. The corresponding volume densities in blood, evaluated from their frequencies of appearance and the velocity of circulation, fall within the physiological range of human white blood cell counts. These results suggested that labeling-free THG imag ing may provide timely tracing of leukocyte movement and hematology inspection without disturbing the normal cellular or physiological status. Keywords: Cell tracking, harmonic generation microscopy, third harmonic generation, leukocyte, lymphocyte, neutrophil, monocyte, red blood cells

The understanding of the interaction between tumors and surrounding microenvironment in vivo is an important first step and basis for pathway-targeting cancer therapy. To in vivo observe the dynamic development of tumor cells and validate the efficacy of therapy in microscopic scales, people commonly performed multi-photon fluorescence microscopy through an invasive window chamber setup. However, under such system, the cancer cells can't be identified and long-term tracked without a fluorescence labeling. Exploiting the intrinsic third harmonic generation (THG) and two-photon fluorescence (2PF) contrasts of melanin, we demonstrated in vivo identification of melanoma and tracked its development without labeling. It was achieved with a least invasive femtosecond Cr:forsterite laser and a laser scanning nonlinear microscopy system with 3D sub-micron spatial resolution. Combined with molecular probes or reporters, we anticipate thus developed platform a powerful tool to reveal molecular insights of tumor microenvironments, enhance our understanding of tumor biology, and trigger new therapeutic approaches.

We demonstrated that the intensity levels and lifetimes of two-photon autofluorescences (2PAF) in human liver tissues can be exploited to diagnose hepatocellular carcinoma (HCC). Excited by an infrared femtosecond laser, we suppressed the two-photon autofluorescences of most endogenous fluorophores and made red autofluorescences more specific to particular molecules in the cryo-sectioned human livers. Using such fluorescence contrast, we found HCC tissues have reduced levels of 2PAF and longer fluorescence lifetime.

Using third harmonic generation (THG) microscopy, we demonstrate that granularity differences of leukocytes can be revealed without a label. Excited by a 1230 nm femtosecond laser, THG signals were generated at a significantly higher level in neutrophils than other mononuclear cells, whereas signals in agranular lymphocytes were one order of magnitude smaller. Interestingly, the characteristic THG features can also be observed in vivo to track the newly recruited leukocytes following lipopolysaccharide (LPS) challenge. These results suggest that label-free THG imaging may provide timely tracking of leukocyte movement without disturbing the normal cellular or physiological status.