Autofluorescence (AF) spectra of colonic normal and adenocarcinoma tissues are measured under excitation of 337 nm and analyzed by multivariate curve resolution alternating least squares (MCR-ALS) method using non-negativity constraint. Collagen, nicotinamide adenine dinucleotide hydrate (NADH) and elastin are identified as the main contributing biomedical components. Fisher’s discrminant anlysis (FDA) on the concentration profiles of the principle components (PCs) shows acceptable sensitivity, specificty and accuracy for discrminanting the adenocarcinoma tissues from the normal tissues. MCR-ALS is a powerful tool for characterzing the spectra profiles of the main biochemical components in neoplasm transformation.

In the past two decades, two-photon microscopy (TPM) transforms biomedical research, allowing nondestructive high-resolution fluorescent molecular imaging and label-free imaging in vivo and in real time. Here we review the recent advances of TPM technology including novel laser sources, new image acquisition paradigms, and microendoscopic imaging systems. Then, we survey the capabilities of TPM imaging of biological relevant molecules such as nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and reactive oxygen species (ROS). Biomedical applications of TPM in neuroscience and cancer detection are demonstrated.

Employing nonlinear spectral imaging technique based on two-photon-excited fluorescence and second-harmonic generation (SHG) of biological tissue, we combine the image-guided spectral analysis method and multi-channel subsequent detection imaging to map and visualize the intrinsic species in a native rabbit aortic wall. A series of recorded nonlinear spectral images excited by a broad range of laser wavelengths (730-910 nm) are used to identify five components in the native rabbit aortic wall, including nicotinamide adenine dinucleotide (NADH), elastic fiber, flavin, porphyrin derivatives, and collagen. Integrating multi-channel subsequent detection imaging technique, the high-resolution, high contrast images of collagen and elastic fiber in the aortic wall are obtained. Our results demonstrate that this method can yield complementary biochemical and morphological information about aortic tissues, which have the potential to determine the tissue pathology associated with mechanical properties of aortic wall and to evaluate the pharmacodynamical studies of vessels.

Two-photon excited spectroscopies from ex vivo human skin are investigated by using a femtosecond laser and a confocal microscope (Zeiss LSM 510 META). In the dermis, collagen is responsible for second harmonic generation (SHG); elastin, nicotinamide adenine dinucleotide (NADH), melanin and porphyrin are the primary endogenous sources of two-photon excited autofluorescence. In the epidermis, keratin, NADH, melanin and porphyrins contribute to autofluorescence signals. The results also show that the SHG spectra have the ability to shift with the excitation wavelength and the autofluorescence spectra display a red shift of the spectral peaks when increasing the excitation wavelength. These results may have practical implications for diagnosis of skin diseases.

Steady state and time-resolved autofluorescence spectroscopies are employed to study the autofluorescence characteristics of human colonic tissues in vitro. The excitation wavelength varies from 260 to 540 nm, and the corresponding fluorescence emission spectra are acquired from 280 to 800 nm. Significant difference in fluorescence intensity of excitation-emission matrices (EEMs) is observed between normal and tumor colonic tissues. Compared with normal colonic tissue, low nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), and high amino acids and protoporphyrin IX (PpIX) fluorescences characterize high-grade malignant tissue. Moreover, the autofluorescence lifetimes of normal and carcinomatous colonic tissues at 635 nm under 397-nm excitation are about 4.32+-0.12 and 18.45+-0.05 ns, respectively. The high accumulation of endogenous PpIX in colonic cancers is demonstrated in both steady state and time-resolved autofluorescence spectroscopies.

Based on the experimental researches on the fluorescence spectra of 1% erythrocyte solution with various concentration of alcohol excited by violet light-emitting diode LED light at 407 nm, the mechanism of alcohol on the fluorescence spectra of erythrocyte solution is investigated theoretically. The experiment results indicate that induced by the LED light at 407 nm, erythrocyte solution with the concentration of 1% can emitting striking spectra with two fluorescence regions. One is the short-wave fluorescence region from 430 to 525 nm, and the other is the long-wave fluorescence region from 580 to 750 nm. When the concentration of alcohol in erythrocyte solution increasing, the fluorescence intensity of short-wave area decrease while the fluorescence intensity of long-wave area increases. Combining the blood absorb spectra to the experiment results, it is shown that the formation of the short-wave fluorescence area is because the solution transmits the fluorescence spectra and the self-absorption of erythrocyte. The long-wave fluorescence region comes from porphyrin such as protoporphyrin, zinc porphyrin etc.. And there is resonance energy transmission between the short-wave fluorophores and the long-wave ones. According to the experiment results and the physical theory in erythrocyte fluorescence, it is found that alcohol make higher self-absorption ratio of the erythrocyte which improves the resonance energy transmission between fluorophores in the two wave bands. The result will offer experimental and theoretical reference for examining the alcohol content in blood.

High signal-to-noise surface enhanced Raman scattering (SERS) signal of rat serum was obtained in this article. Two methods were presented to enhance the intensity of rat serum SERS signal. The novel colloid silver was synthesized using heating method by microwave and it was introduced to be the active site of SERS, "hot sites" could be detected in the active site. At same time the high numerical aperture (NA) oil immersed object lens was applied in micro-Raman system so that some new peaks come up in SERS spectrum of rat serum because of the evanescent wave was employed to excite the sample.