MULTIPHOTON MICROSCOPIC IMAGING OF MOUSE INTESTINAL MUCOSA BASED ON TWO-PHOTON EXCITED FLUORESCENCE AND SECOND HARMONIC GENERATION

Multiphoton microscopy(MPM),based on two-photon excited fuorescence and second harmonic generation,enables direct noninvasive visualization of tissue architecture and cell morphology in live tissues without the administration of exogenous contrast agents.In this paper,we used MPM to image the microstructures of the mucosa in fresh,unfixed,and unstained intestinal tissue of mouse.The morphology and distribution of the main components in mucosa layer such as columnar cells,goblet cells,intestinal glands,and a little collagen fibers were clearly observed in MPM images,and then compared with standard H&:E images from paired specimens.Our results indicate that MPM combined with endoscopy and miniaturization probes has the potential application in the clinical diagnosis and in vivo monitoring of early intestinal cancer.

RAMAN SPECTROSCOPY OF HUMAN HEMOGLOBIN FOR DIABETES DETECTION

Glycat ed hemoglobin(HbAlc)has been increasingly accepted as the gold standard for diabetesmonit oring.In this study,Raman spect roso was tentatively emplo yed for human hemoglobin(b)bioc hemical analysis aimed at de veloping a a simple blood test for diabetes monitoring.Ramanspectroscopy measurementsperformed oWereglobimples of patients(n=39)withconfirmed diabetes and healthyvolulhetentatiassignments of the measuredRaman bands were perfortwogegroups.Meanwhile,principal componentaminant analysis(LDA)wereemployed to develop effective weell1ormal controls andpatients with diabetes.Asat wo groups demonstrated twodistinct clust ers with a sensitivity and specificity d 73%,respectively.Then the effectiveness of the diagnostic algorithm based onechnique was confirmed by receiveroperating characteristic(ROC)curve.The area under the ROC curve was 0.92,indicating a good diagnostic result.In summary,our preliminary results demonstrate that proposing Raman spectroscopy can provide a significant potential for the noninvasive detection of diabetes.

CHARACTERIZATION OF SIGNAL CONDUCTIONALONG DEMYELINATED AXONS BY ACTION-POTENTIAL-ENCODED SECOND HARMONICGENERATION

Action-potential encoded optical second harmonic generation(SHG)has been recently proposedfor use in det ecting the axonal damage in patients with demnyelinat ing diseases.In this study,thecharact erization of signal conduction along axons of two different levels of denyelination wasstudied via a modified Hodgkin Huxley model,because some types of demyelinating disease,i.e.primary progressive and secondary progesive multiple scleross,are dificult to be distinguishedby magnetic resonance imaging(MRI),we focused on the diferences in signal conduction between two diferent demyelinated axons,such as the first-level demyelination and the second.level demyelination.The spatio-temporal distribution of action potentials along denyelinatedaxons and conduction properties including the refractory period and frequency encoding in theset wo patterns were investigated.The results showed that denyelination could induce the decreaseboth in the amplitude of action potentials and the ability of frequency coding,Furthermore,t hesignal conduction velocity in the second-level dernyelination was about 21%slower than that inthe first-level demyelination.The refractory period in the second-level demyelination was about32%longer t han the first-level.Thus,detecting the signal conduction in demnyelinat ed axons byaction-potential encoded optical SHG could greatly improve the assessment of demyelinatingdisorders to classify the patients.This technique also offers a potential fast and noninvasiveoptical approach for monitoring membrane potential.

NONLINEAR SPECTRAL IMAGING OF ELASTIC CARTILAGE IN RABBIT EARS

Elastic cartilage in the rabbit external ear is an important animal model with attractive potential value for researching the physiological and pathological states of cartilages especially during wound healing.In this work,nonlinear optical microscopy based on two-photon excited fluor-escence and second harmonic generation were employed for imaging and quantifying the intact elastic cartilage.The morphology and distribution of main components in elastic carti lage incuding cartilage cells,collagen and elastic fibers were clearly observed from the high-resolution two-di mensional nonlinear optical images.The areas of cell nuclei,a parameter related to the pathological changes of nornal or abnormal elastic cartilage,can be easily quantifed.Moreover,the three-dimensional structure of chondrocytes and matrix were displayed by constructing threedimensional image of cartilage tissue.At last,the enmission spectra from cartilage were obtained and analyzed.We found that the different ratio of collagen over elastic fibens can be used to locate the observed position in the elastic cartilage.The redox ratio based on the ratio of nicotinamide adenine dimucleotide(NADH)over flavin adenine dinucleotide(FAD)fuorescence can also be calculated to analyze the metabolic state of chondrocytes in different regions.Our results demonstrated that this technique has the potential to provide more accurate and comprehensive information for the physiological states of elastic cartilage.

DIAGNOSTIC APPLICATION OF MULTIPHOTON MICROSCOPY IN EPITHELIAL TISSUES

Epithelial cancer comprises more than 85%of human cancers.The detection and treatment at the early stage has been demonstrated to apparently improve patient survival.In this review,we summarize our recent research works on the diagnostic application of epithelial tissue based on multiphoton microscopy(MPM),including identification of the layered structures of esophagus,oral cavity,skin and bronchus tissues,establishment of the diagnostic features for distinguishing gastric normal tissue from cancerous tissue,linking collagen alteration and ectocervical epithelial tumor progression for evaluating epithelial tumor progression,and differentiating normal,inflammatory,and dysplastic ectocervical epithelial tissues.These results provide the groundwork for developing MPM into clinical multiphoton endoscopy.

NON-LINEAR SPECTRAL IMAGING MICROSCOPY STUDIES OF HUMAN HYPERTROPHIC SCAR

Skin scar is unique to humans,the major significant negative outcome sustained after thermal injuries,traumatic injuries,and surgical procedures.Hypertrophic scar in human skin is investigated using non-linear spectral imaging microscopy.The high contrast images and spectroscopic intensities of collagen and elastic fibers extracted from the spectral imaging of normal skin tissue,and the normal skin near and far away from the hypertrophic scar tissues in a 10-year-old patient case are obtained.The results show that there are apparent differences in the morphological structure and spectral characteristics of collagen and elastic fibers when comparing the normal skin with the hypertrophic scar tissue.These differences can be good indicators to differentiate the normal skin and hypertrophic scar tissue and demonstrate that non-linear spectral imaging microscopy has potential to noninvasively investigate the pathophysiology of human hypertrophic scar.