Hemoglobin, one of the most important proteins in blood, is responsible for oxygen transportation in almost all vertebrates. Recently, we discovered two-photon excited hemoglobin fluorescence and achieved label-free microvascular imaging based on the hemoglobin fluorescence. However, the mechanism of its fluorescence emission still remains unknown. In this work, we studied the two-photon excited fluorescence properties of the hemoglobin subunits, heme/hemin (iron (II)/(III) protoporphyrin IX) and globin. We first studied the properties of heme and the similar spectral and temporal characteristics of heme and hemoglobin fluorescence provide strong evidence that heme is the fluorophore in hemoglobin. Then we studied the fluorescence properties of hemin, globin and methemoglobin, and found that the hemin may have the main effect on the methemoglobin fluorescence and that globin has tryptophan fluorescence like other proteins. Finally, since heme is a centrosymmetric molecule, that the Soret band fluorescence of heme and hemoglobin was not observed in the single photon process in the previous study may be due to the parity selection rule. The discovery of heme two-photon excited fluorescence may open a new window for heme biology research, since heme as a cofactor of hemoprotein has many functions, including chemical catalysis, electron transfer and diatomic gases transportation.
We demonstrate that two-photon excited endogenous fluorescence enables label-free morphological and functional imaging of various human blood cells. Specifically, we achieved distinctive morphological contrast to visualize morphology of important leukocytes, such as polymorphonuclear structure of granulocyte and mononuclear feature of agranulocyte, through the employment of the reduced nicotinamide adenine dinucleotide (NADH) fluorescence signals. In addition, NADH fluorescence images clearly reveal the morphological transformation process of neutrophils during disease-causing bacterial infection. Our findings also show that time-resolved NADH fluorescence can be potentially used for functional imaging of the phagocytosis of pathogens by leukocytes (neutrophils) in vivo. In particular, we found that free-to-bound NADH ratios measured in infected neutrophils increased significantly, which is consistent with a previous study that the energy consumed in the phagocytosis of neutrophils is mainly generated through the glycolysis pathway that leads to the accumulation of free NADH. Future work will focus on further developing and applying label-free imaging technology to investigate leukocyte-related diseases and disorders.
We explore diagnostic utility of a multicolor excitation multimodal nonlinear optical (NLO) microscopy for noninvasive detection of squamous epithelial precancer in vivo. The 7,12-dimenthylbenz(a)anthracene treated hamster cheek pouch was used as an animal model of carcinogenesis. The NLO microscope system employed was equipped with the ability to collect multiple tissue endogenous NLO signals such as two-photon excited fluorescence of keratin, nicotinamide adenine dinucleotide, collagen, and tryptophan, and second harmonic generation of collagen in spectral and time domains simultaneously. A total of 34 (11 controlled and 23 treated) Golden Syrian hamsters with 62 in vivo spatially distinct measurement sites were assessed in this study. High-resolution label-free NLO images were acquired from stratum corneum, stratum granulosum-stratum basale, and stroma for all tissue measurement sites. A total of nine and eight features from 745 and 600 nm excitation wavelengths, respectively, involving tissue structural and intrinsic biochemical properties were found to contain significant diagnostic information for precancers detection (p<0.05 ). Particularly, 600 nm excited tryptophan fluorescence signals emanating from stratum corneum was revealed to provide remarkable diagnostic utility. Multivariate statistical techniques confirmed the integration of diagnostically significant features from multicolor excitation wavelengths yielded improved diagnostic accuracy as compared to using the individual wavelength alone.
Real time and in vivo monitoring leukocyte behavior provides unique information to understand the physiological and pathological process of infection. In this study, we demonstrate that two-photon excited reduced nicotinamide adenine dinucleotide (NADH) fluorescence provides imaging contrast to distinguish granulocyte and agranulocyte. By using spectral and time-resolved NADH fluorescence, we study the immune response of human neutrophils against bacterial infection (Escherichia coli). The two-photon excited NADH fluorescence images clearly review the morphological changes from resting neutrophils (round shape) to activated neutrophils (ruffle shape) during phagocytosis. The free-tobound NADH ratio of neutrophils decreases after ingesting disease-causing pathogen: Escherichia coli. This finding may provide a new optical tool to investigate inflammatory processes by using NADH fluorescence in vivo.
Most human cancers arise from epithelium, the superficial layer covering the exterior of body or lining the internal body
cavities. Endogenous fluorophores such as aromatic amino acids, reduced nicotinamide adenine dinucleotide (NADH),
flavoprotein (FAD), keratin, collagen, and elastin can provide abundant information to reveal the changes in
biochemistry, metabolism, and morphology of living tissues. Thus, autofluorescence spectroscopy and microscopy have
been recognized as potential tools for discrimination of cancer from normal tissues. However, current fluorescence
diagnostic studies mostly rely on spectral analysis or morphological differentiation. It is challenged since the emission
spectra of endogenous fluorophores are broad and usually overlapping with each other and the fluorescence intensity
could be affected by many factors. In this study, we instrumented a nonlinear optical microscopy system to characterize
the morphologic and biochemical features in the epithelial precancer in vivo. The 7,12-dimethylbenz(a)anthracenetreated
hamster cheek pouch were used as a living animal carcinogenesis model. And the autofluorescence signals of
NADH, collagen and elastin were recorded by a time- and spectral- resolved detection system. The results show that
there are obvious differences in the morphology of three-dimensional autofluorescence images between normal and
precancerous epithelial tissues. The fluorescence lifetime of NADH and the SHG signal from collagen could provide
additional approaches to identify cancer from normal tissue.
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