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.
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.
The endogenous nonlinear optical (NLO) signals of two-photon excitation fluorescence (TPEF), second harmonic
generation (SHG), and coherent anti-stokes Raman scattering (CARS) have been widely used to image a variety of
biological samples. Different nonlinear optical signals could convey different structural and biomolecular information.
Therefore, it is desirable to combine multiple nonlinear optical signals together for biomedical imaging. However, the
simplification of the sophistical, high cost laser source and the simultaneous excitation and detection of multiple NLO
signals are the challenges for the multimodal NLO microscopy. In this work, we instrument a multimodal nonlinear
optical microscopy system which integrates the multiplex CARS module with the TPEF, SHG microscopy. The
excitation source is the combination of femtosecond Ti:sapphire laser and the broadband near infrared supercontinuum
light from a photonic crystal fiber. The multiplex CARS measurements, covering the vibrational frequency from 2400 to
3300 cm-1, allowed us to detect the pure non-resonant background (NRB) signals and the CARS signals of aliphatic C-H
and O-H bonds simultaneously. The relatively large NRB in the femtosecond laser excited CARS images could be
efficiently suppressed by simple subtraction operation. The TCSPC detection system records the spectral and temporal
characteristics of the TPEF signals and spectrally resolves the CARS signals from different molecular vibrational bonds.
We demonstrate the multimodal imaging capability of the system using C.elegans as the living biological samples.
Reduced nicotinamide adenine dinucleotide (NADH) is a well-known metabolic coenzyme and endogenous fluorophore. In this study, we develop a system that simultaneously measures time- and wavelength-resolved fluorescence to extract free and protein-bound NADH signals from total cellular fluorescence. We analyze temporal characteristics of NADH fluorescence in a mixture of NADH and lactate dehydrogenase (LDH) as well as in living cell samples. The results show that in both the NADH/LDH mixture and cell samples, a fraction of free NADH and protein-bound components can be identified. The extracted free and bound NADH signals are confirmed by time-resolved measurement of anisotropy decay of NADH fluorescence, based on the fact that free NADH is a small fluorescent molecule with much shorter rotational diffusion time than bound NADH. The ratio of free NADH signal to bound NADH signal is very different between normal and cancer cervical epithelial cells. In addition, the ratio changes significantly when the cell samples are treated with a mitochondrial inhibitor or uncoupler, demonstrating that the method is sensitive to monitor cellular metabolic activity. Finally, we demonstrate that the microviscosity for relatively small molecules such as NADH in cells could be extracted from wavelength- and time-resolved NADH fluorescence of living cell samples.
Two-photon microscopy based on endogenous fluorescence provides non-invasive imaging of living biological system.
Reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), keratin, collagen and elastin
are the endogenous fluorophores widely used as the contrast agents for imaging metabolism and morphology of living
cells and tissue. The fluorescence of tryptophan, a kind of essential amino acid, conveys the information on cellular
protein content, structure and microenvironment. However, it can't be effectively excited by the commonly used
Ti:sapphire femtosecond laser. Because each endogenous fluorophore provides limited information, it is desirable to
simultaneously excite fluorescence from as many fluorophores as possible to obtain accurate biochemical and
morphological information on biomedical samples. In this study, we demonstrate that the supercontinuum generation
from a photonic crystal fiber (PCF) excited by an ultrafast source can be used to excite multiple endogenous nonlinear
optical signals simultaneously. By employing the spectral lifetime detection capability, this technology provides a unique
approach to sense the fine structure, protein distribution and cellular metabolism of cells and tissues in vivo. In particular,
with application of acetic acid, a safe contrast agent used for detection cervical cancer for many years, the tryptophan
signals reveal cellular morphology and even cell-cell junctions clearly. Moreover, it was found that the pH value
dependent lifetime of tryptophan fluorescence could provide the qualitative information on the gradient of pH value in
epithelial tissue. Finally, we will demonstrate the potential of our multi-color TPEF microscopy to investigate the early
development of cancer in epithelial tissue.
KEYWORDS: Luminescence, Green fluorescent protein, Fluorescent proteins, Femtosecond phenomena, Signal detection, Tissues, Microscopy, Mirrors, Two photon excitation microscopy, In vivo imaging
Endogenous fluorophores, such as reduced nicotinamide adenine dinucleotide (NADH), keratin, and tryptophan, have
been used as contrast agents for imaging metabolism and morphology of living cells and tissues. Multilabeling which
maps the distribution of different targets is an indispensable technique in many biomedical and biochemical studies.
Therefore, two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with in vivo
fluorescence labeling techniques such as genetically encoded fluorescent protein could be a powerful tool for imaging
living cells and tissues. However, the challenge is that the excitation and emission wavelengths of these endogenous
fluorophores and fluorescence labels are very different. A
multi-color ultrafast source is required for the excitation of
multiple fluorescence molecules. In this study, we developed a two-photon imaging system with excitations from the
pump femtosecond laser and the selected Supercontinuum generated from a photonic crystal fiber (PCF). Multiple
endogenous fluorophores and fluorescent proteins such as NADH, tryptophan, green fluorescent protein (GFP), and
yellow fluorescent protein (YFP) were excited in their optimal wavelengths alternately or simultaneously. A time- and
spectral-resolved detection system was used to record the TPEF signals. This detection technique separated the TPEF
signals from multiple sources in time and spectral domains. Cellular organelles such as nucleus, mitochondria,
microtubule and Endoplasmic Reticulum (ER), were clearly revealed in the TPEF images.
We demonstrate that using time-resolved two-photon excitation endogenous fluorescence microscopy, the cadmium (Cd)-induced cellular toxic level can be assessed by the free-to protein-bound reduced nicotinamide adenine dinucleotide (free/bound NADH) ratio in a living cell. NADH fluorescence excited at 730 nm is captured at different times following exposure to cadmium at a variety of concentrations. The temporal characteristics of NADH fluorescence from mitochondrial and nuclear compartments are analyzed, respectively. The results show that cadmium induces a significant increase of the free/bound NADH ratio in mitochondria and nucleus, caused by the inhibition effect on the electron transport chain (ETC) and the stimulating effect on the glycolysis pathway, respectively. It is found that induction of metallothionein (MT) in cells occurs after 4 h of exposure to a sublethal concentration of Cd and reaches a peak at 6 h. More importantly, the increase in MT level can effectively suppress the elevation of the free/bound NADH ratio caused by a subsequent exposure to a higher concentration of Cd, indicating that MT plays a key role in protecting cells from Cd-induced toxicity. Our findings show that the free/bound NADH ratio can potentially be used as a sensitive indicator of toxic and carcinogenic actions induced by Cd.
We instrumented a combined fluorescence spectroscopy and imaging system to characterize the single- and two-photon excited autofluorescence in epithelial tissue. Single-photon fluorescence (SPF) are compared with two-photon fluorescence (TPF) measured at the same location in epithelial tissue. It was found that the SPF and TPF signals excited at corresponding wavelengths are similar in nonkeratinized epithelium, but the SPF and TPF spectra in the keratinized epithelium and the stromal layer are significant different. Specifically, the comparison of SPF signals with TPF signals in keratinized epithelial and stromal layers shows that TPF spectral peaks always have about 15-nm redshift with respect to SPF signals, and the TPF spectra are broader than SPF spectra. The results were generally consistent with the SPF and TPF measurements of pure nicotinamide adenine dinucleotide, flavin adenine dinucleotide, keratin and collagen, the major fluorophores in epithelium and stroma, respectively. The double peak structure of TPF spectra measured from keratinized layer suggests that there may be an unknown fluorophore responsible for the spectral peak in the long wavelength region. Furthermore, the TPF signals excited in a wide range of wavelengths provide accurate information on epithelial structure, which is an important advantage of TPF over SPF spectroscopy in the application for the diagnosis of tissue pathology.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.