Jianan Y. Qu

Laser-induced fluorescence spectroscopy at endoscopy: tissue optics, Monte Carlo modeling, and in vivo measurements

The optical properties (absorption coefficient, scattering coefficient and the anisotropic factor of scattering) and fluorescence characteristics of normal and abnormal bronchial tissue were measured in vitro. After adding additional blood optical properties to in vitro optical properties of tissue, the in vivo bronchial fluorescence was simulated and analyzed by Monte Carlo modeling. The Monte Carlo simulation results showed that with an appropriate illumination and fluorescence collection geometry, the distortion of in vivo fluorescence spectra of tissue caused by variations of optical properties at different wavelengths could be much reduced. Based on these results, a spectrofluorometry system was developed for the collection of in vivo laser-induced fluorescence spectra of tissue during endoscopy. In comparing the in vivo fluorescence spectral shape of bronchial tissue collected by this system with the intrinsic one obtained in vitro, we found no obvious distortion in the in vivo spectra. This was completely consistent with the analysis of Monte Carlo modeling. The in vivo measurement results demonstrated that significant differences in fluorescence intensity between normal and diseased bronchial tissue (dysplasia, carcinoma in situ) can be used to differentiate them from each other. Also, changes in fluorescence intensity are more robust for detecting abnormal tissues than the differences in spectral characteristics.

Optical properties of normal and carcinomatous bronchial tissue

To understand better the optical characteristics and autofluorescence properties of normal and carcinomatous bronchial tissue, we measured the absorption coefficient, scattering coefficient, and anisotropy factor from 400 to 700 nm. We made the measurements by using an integrating sphere with a collimated white-light beam to measure total reflectance and transmittance of samples. The unscattered transmittance of the samples was measured through polarized on-axis light detection. The inverse adding-doubling solution was utilized to solve the equation of radiative transfer and to determine the absorption coefficient and reduced scattering coefficient. The scattering coefficient and anisotropy factor were derived from the unscattered transmittance of the sample and the reduced scattering coefficient. The measured parameters allow us to simulate photon propagation in normal bronchial and tumoral tissue by using Monte Carlo modeling.

Diagnostic imaging of the larynx: autofluorescence of laryngeal tumours using the helium-cadmium laser.

The use of tissue autofluorescence for the detection and localization of cancer of the larynx is described. In this pilot study, eight patients with probable carcinoma of the vocal folds underwent laryngoscopy in which the tissue autofluorescence spectra of normal and pathologically confirmed tumour tissue were acquired in vivo. Fluorescence images of the suspect areas were also acquired using the LIFE system (Xillix Technologies Corp.). The results suggest that the autofluorescence properties of laryngeal tissue, under 442 nm illumination, are similar to those of bronchial tissue and that the LIFE system has the potential to increase the accuracy of staging of cancer of the larynx and also to allow earlier diagnosis of tumours and their recurrence.

Conserved Motif of CDK5RAP2 Mediates Its Localization to Centrosomes and the Golgi Complex

As the primary microtubule-organizing centers, centrosomes require γ-tubulin for microtubule nucleation and organization. Located in close vicinity to centrosomes, the Golgi complex is another microtubule-organizing organelle in interphase cells. CDK5RAP2 is a γ-tubulin complex-binding protein and functions in γ-tubulin attachment to centrosomes. In this study, we find that CDK5RAP2 localizes to the Golgi complex in an ATP- and centrosome-dependent manner and associates with Golgi membranes independently of microtubules. CDK5RAP2 contains a centrosome-targeting domain with its core region highly homologous to the Motif 2 (CM2) of centrosomin, a functionally related protein in Drosophila. This sequence, referred to as the CM2-like motif, is also conserved in related proteins in chicken and zebrafish. Therefore, CDK5RAP2 may undertake a conserved mechanism for centrosomal localization. Using a mutational approach, we demonstrate that the CM2-like motif plays a crucial role in the centrosomal and Golgi localization of CDK5RAP2. Furthermore, the CM2-like motif is essential for the association of the centrosome-targeting domain to pericentrin and AKAP450. The binding with pericentrin is required for the centrosomal and Golgi localization of CDK5RAP2, whereas the binding with AKAP450 is required for the Golgi localization. Although the CM2-like motif possesses the activity of Ca2+-independent calmodulin binding, binding of calmodulin to this sequence is dispensable for centrosomal and Golgi association. Altogether, CDK5RAP2 may represent a novel mechanism for centrosomal and Golgi localization.

Concentration measurements of multiple analytes in human sera by near-infrared laser Raman spectroscopy

Our primary goal in this study is to demonstrate that near-infrared Raman spectroscopy is feasible as a rapid and reagentless analytic method for clinical diagnostics. Raman spectra were collected on human sera by use of a 785-nm excitation laser and a single-stage holographic spectrometer. A partial-least-squares method was used to predict the analyte concentrations of interest. The prediction errors of total protein, albumin, triglyceride, and glucose in human sera ranged from 1.0% to 10%, which are highly acceptable for clinical diagnosis, of their mean physiological levels. For investigating the potential application of near-infrared Raman spectroscopy in screening of therapeutical drugs and substances of abuse the concentrations of acetaminophen, ethanol, and codeine in water solution were measured in the same fashion. The errors of the Raman tests for acetaminophen and ethanol are lower than their toxic levels in human serum, and the sensitivity for detection of codeine fails to reach its toxic level.

Autofluorescence spectroscopy of epithelial tissues

Autofluorescence of rabbit and human epithelial tissues were studied by using a depth-resolved fluorescence spectroscopy system with multiple excitations. Keratinization was found to be common in the squamous epithelium. Strong keratin fluorescence with excitation and emission characteristics similar to collagen were observed in the topmost layer of the keratinized squamous epithelium. The keratin signal created interference in the assessment of the endogenous fluorescence signals (NADH/FAD fluorescence in epithelium and collagen fluorescence in stroma) associated with the development of epithelial precancer. Furthermore, the keratinized epithelial layer attenuated the excitation light and reduced the fluorescence signals from underlying tissue layers. The autofluorescence of columnar epithelium was found to be dominated by NADH and FAD signals, identical to the autofluorescence measured from nonkeratinized squamous epithelium. The study also demonstrated that a fluorescence signal excited at 355 nm produced sufficient contrast to resolve the layered structure of epithelial tissue, while the signal excited at 405 nm provided the information for a good estimation of epithelial redox ratios that are directly related to tissue metabolism. Overall, the depth-resolved measurements are crucial to isolate the fluorescence signals from different sublayers of the epithelial tissue and provide more accurate information for the tissue diagnosis.

Two-photon autofluorescence microscopy of multicolor excitation

We developed a two-photon autofluorescence lifetime imaging system with excitations selected from the supercontinuum generated from a photonic crystal fiber. The system excites multiple endogenous fluorophores, such as nicotinamide adenine dinucleotide (NADH) and tryptophan, simultaneously and produces coregistered two-photon autofluorescence images of a biological sample. The technology provides a unique approach to investigate the cellular metabolic activity and protein expression in cells that are potentially important for noninvasive precancer diagnostics. We demonstrated that by taking the tryptophan fluorescence as a reference the ratio of NADH to the tryptophan signal serves as a sensitive indicator of cellular metabolism. The ratio can also clearly differentiate normal cells from cancer cells. The tryptophan fluorescence lifetime images of cells shows that the lifetime of tryptophan fluorescence, varying over a wide range, may be highly dependent on the expression and structure of the protein that tryptophan is packed in.

Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence

A time-resolved spectroscopic imaging system is built to study the fluorescence characteristics of nicotinamide adenine dinucleotide (NADH), an important metabolic coenzyme and endogenous fluorophore in cells. The system provides a unique approach to measure fluorescence signals in different cellular organelles and cytoplasm. The ratios of free over protein-bound NADH signals in cytosol and nucleus are slightly higher than those in mitochondria. The mitochondrial fluorescence contributes about 70% of overall cellular fluorescence and is not a completely dominant signal. Furthermore, NADH signals in mitochondria, cytosol, and the nucleus respond to the changes of cellular activity differently, suggesting that cytosolic and nuclear fluorescence may complicate the well-known relationship between mitochondrial fluorescence and cellular metabolism.

Sensing cell metabolism by time-resolved autofluorescence

We built a time-resolved confocal fluorescence spectroscopy system equipped with the multichannel time-correlated single-photon-counting technique. The instrument provides a unique approach to study the fluorescence sensing of cell metabolism via analysis of the wavelength- and time-resolved intracellular autofluorescence. The experiments on monolayered cell cultures show that with UV excitation at 365 nm the time-resolved autofluorescence decays, dominated by free-bound reduced nicotinamide adenine dinucleotide signals, are sensitive indicators for cell metabolism. However, the sensitivity decreases with the increase of excitation wavelength possibly due to the interference from free-bound flavin adenine dinucleotide fluorescence. The results demonstrate that time-resolved autofluorescence can be potentially used as an important contrast mechanism to detect epithelial precancer.

Depth-resolved fluorescence spectroscopy of normal and dysplastic cervical tissue

A portable confocal system with the excitations at 355nm and 457nm was instrumented to investigate the depth-resolved fluorescence of cervical tissue. The study focused on extracting biochemical and morphological information carried in the depth-resolved signals measured from the normal squamous epithelial tissue and squamous intraepithelial lesions. Strong keratin fluorescence with the spectral characteristics similar to collagen were observed from the topmost keratinizing layer of all tissue samples. It was found that NADH and FAD fluorescence measured from the underlying non-keratinizing epithelial layer were strongly correlated to the tissue pathology. This study demonstrates that the depth-resolved fluorescence spectroscopy can potentially provide more accurate diagnostic information for determining tissue pathology.