Increased nicotinamide adenine dinucleotide pool promotes colon cancer progression by suppressing reactive oxygen species level

Nicotinamide adenine dinucleotide (NAD) exists in an oxidized form (NAD(+)) and a reduced form (NADH). NAD(+) plays crucial roles in cancer metabolism, including in cellular signaling, energy production and redox regulation. However, it remains unclear whether NAD(H) pool size (NAD(+) and NADH) could be used as biomarker for colon cancer progression. Here, we showed that the NAD(H) pool size and NAD(+)/NADH ratio both increased during colorectal cancer (CRC) progression due to activation of the NAD(+) salvage pathway mediated by nicotinamide phosphoribosyltransferase (NAMPT). The NAMPT expression was upregulated in adenoma and adenocarcinoma tissues from CRC patients. The NADH fluorescence intensity measured by two-photon excitation fluorescence (TPEF) microscopy was consistently increased in CRC cell lines, azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC tissues and tumor tissues from CRC patients. The increases in the NAD(H) pool inhibited the accumulation of excessive reactive oxygen species (ROS) levels and FK866, a specific inhibitor of NAMPT, treatment decreased the CRC nodule size by increasing ROS levels in AOM/DSS mice. Collectively, our results suggest that NAMPT-mediated upregulation of the NAD(H) pool protects cancer cells against detrimental oxidative stress and that detecting NADH fluorescence by TPEF microscopy could be a potential method for monitoring CRC progression.11Ysciescopu

Development of high-speed two-photon microscopy for biological and medical applications

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2005.Includes bibliographical references (p. 135-144).Two-photon microscopy (TPM) is one of the most powerful microscopic technologies for in-vivo 3D tissue imaging up to a few hundred micrometers. It has been finding important applications in neuronal imaging, tumor physiology study, and optical biopsy. A practical limitation of TPM is its slow imaging speed (0.3 1 frames/s). We designed high-speed two-photon microscopes (HSTPMs) whose imaging speed is more than 10 times faster than traditional TPM, while their imaging depths, image contrast are comparable to those of TPM. The first high speed system is HSTPM based on polygonal mirror scanner. The scanning speed reaches 13 frames/s for typical tissues using a polygonal mirror scanner. This system is based on single-focus scanning and single-pixel signal collection. The usage of higher input power is required to compensate for the signal reduction due to higher scanning speed. However, since fluorescence signal is ultimately limited by the saturation of fluorophores due to their finite lifetimes, is the signal to noise ratio (SNR) of single focus scanning systems are also ultimately limited at high speed. This problem is circumvented in a second system based on parallelization by scanning specimens with multiple foci of excitation light and collecting signals with spatially resolved detectors. The imaging speed is increased proportional to the number of foci and similar excitation laser power per focus circumventing the problem of fluorophore saturation. However, it has been recognized that this method is severely limited for deep tissue imaging due to photon scattering.(cont.) We quantitatively measured the photon scattering effect and demonstrated that its image resolution is the same as conventional TPM but its image contrast is degraded to the faster signal decay with the increase of imaging depth. We designed a new MMM based on multi-anode photomultiplier tube (MAPMT) which utilizes the advantage of MMM in terms of parallelization but overcomes the emission photon scattering problem by optimizing the design detector geometry. This method achieved equivalent SNR as conventional TPM with imaging speed more than 10 times higher than TPM. We applied these HSTPMs to a number of novel biomedical applications focusing on studying biological problems that needs to resolve the high speed kinetics processes or or the imaging of large tissue sections with subcellular resolution to achieve the requisite statistical accuracy. In the study of transdermal drug delivery mechanisms with chemical enhancers,, large section imaging enables microscopic transport properties to be measured even in skin which is highly topographical heterogeneous. This methodology allowed us to identify the novel transport pathways through the stratum corneum of skin. In the study of tumor physiology, microvasculature in tumor tissue deep below the surface was characterized to be densely distributed and tortuous compared to that of normal tissue. The interaction of leukocyte and endothelium in tumor tissue was measured by imaging the kinetics of leukocyte interaction with blood vessel wall in tumor tissues using HSTPM. The capability of large section imaging was further applied to develop a 3D tissue cytometer with the advantages that cell-cell and cell- extracellular matrix interaction can be quantified in tissues.(cont.) The statistical accuracy of this instrument was verified by quantitatively measuring cell population ratios in engineered tissue constructs composed of a mixture of two cell subpopulations. Further, this 3D tissue cytometer was applied to screen and to identify rare recombination events in transgenic mice that carry novel fluorescent genetic reporters.by Ki Hean Kim.Ph.D

Multiphoton tissue imaging by using moxifloxacin

Multiphoton microscopy has been widely used for in-vivo tissue imaging of various biological studies. However, its application to clinical studies has been limited due to either lack of clinically compatible exogenous contrast agents or weak autofluorescence of tissues. We investigated moxifloxacin as a contrast agent of cells for multiphoton tissue imaging. Moxifloxacin is an FDA approved antibiotic with relatively good pharmacokinetic properties for tissue penetration and intrinsic fluorescence. Two-photon microscopy (TPM) of moxifloxacin treated mouse corneas showed good tissue penetration and high concentration inside the corneal cells [1]. Cell labeling of moxifloxacin was tested in both cultured cells and isolated immune cells. Moxifloxacin tissue applications were tested in various mouse organs such as the skin, small intestine, and brain. Most of tissues were labeled well via topical administration, and only the skin required additional gentle removal of the outermost stratum corneum by tape stripping. TPM of these tissues showed non-specific cell labeling of moxifloxacin and fluorescence enhancement [2]. Although most of experimental results were from mouse tissues, its clinical application would be possible. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence. Moxifloxacin labeling of cultured cells was demonstrated by comparing TPM images with and without moxifloxacin treatment. Bright fluorescence inside cells were observed only with moxifloxacin at the same imaging condition. TPM of the skin dermis visualized many dermal cells with increased fluorescence, and TPM of the villus in the small intestine showed the covering epithelial cells and cells inside the villus clearly. Please click Additional Files below to see the full abstract

In vivo fluorescence imaging of conjunctival goblet cells

Conjunctival goblet cells (GCs) are specialized epithelial cells that secrete mucins onto the ocular surface to maintain the wet environment. Assessment of GCs is important because various ocular surface diseases are associated with their loss. Although there are GC assessment methods available, the current methods are either invasive or difficult to use. In this report, we developed a simple and non-invasive GC assessment method based on fluorescence imaging. Moxifloxacin ophthalmic solution was used to label GCs via topical administration, and then various fluorescence microscopies could image GCs in high contrasts. Fluorescence imaging of GCs in the mouse conjunctiva was confirmed by both confocal reflection microscopy and histology with Periodic acid-Schiff (PAS) labeling. Real-time in-vivo conjunctival GC imaging was demonstrated in a rat model by using both confocal fluorescence microscopy and simple wide-field fluorescence microscopy. Different GC densities were observed in the forniceal and bulbar conjunctivas of the rat eye. Moxifloxacin based fluorescence imaging provides high-contrast images of conjunctival GCs non-invasively and could be useful for the study or diagnosis of GC related ocular surface diseases.11Ysciescopu

In vivo 3D measurement of moxifloxacin and gatifloxacin distributions in the mouse cornea using multiphoton microscopy

Moxifloxacin and gatifloxacin are fourth-generation fluoroquinolone antibiotics used in the clinic to prevent or treat ocular infections. Their pharmacokinetics in the cornea is usually measured from extracted ocular fluids or tissues, and in vivo direct measurement is difficult. In this study multiphoton microscopy (MPM), which is a 3D optical microscopic technique based on multiphoton fluorescence, was applied to the measurement of moxifloxacin and gatifloxacin distribution in the cornea. Intrinsic multiphoton fluorescence properties of moxifloxacin and gatifloxacin were characterized, and their distributions in mouse cornea in vivo were measured by 3D MPM imaging. Both moxifloxacin and gatifloxacin had similar multiphoton spectra, while moxifloxacin had stronger fluorescence than gatifloxacin. MPM imaging of mouse cornea in vivo showed (1) moxifloxacin had good penetration through the superficial corneal epithelium, while gatifloxacin had relatively poor penetration, (2) both ophthalmic solutions had high intracellular distribution. In vivo MPM results were consistent with previous studies. This study demonstrates the feasibility of MPM as a method for in vivo direct measurement of moxifloxacin and gatifloxacin in the cornea.1175Ysciescopu

Open-top axially swept light-sheet microscopy

Open-top light-sheet microscopy (OT-LSM) is a specialized microscopic technique for high throughput cellular imaging of large tissue specimens including optically cleared tissues by having the entire optical setup below the sample stage. Current OT-LSM systems had relatively low axial resolutions by using weakly focused light sheets to cover the imaging field of view (FOV). In this report, open-top axially swept LSM (OTAS-LSM) was developed for high-throughput cellular imaging with improved axial resolution. OTAS-LSM swept a tightly focused excitation light sheet across the imaging FOV using an electro tunable lens (ETL) and collected emission light at the focus of the light sheet with a camera in the rolling shutter mode. OTAS-LSM was developed by using air objective lenses and a liquid prism and it had on-axis optical aberration associated with the mismatch of refractive indices between air and immersion medium. The effects of optical aberration were analyzed by both simulation and experiment, and the image resolutions were under 1.6 & micro;m in all directions. The newly developed OTAS-LSM was applied to the imaging of optically cleared mouse brain and small intestine, and it demonstrated the single-cell resolution imaging of neuronal networks. OTAS-LSM might be useful for the high-throughput cellular examination of optically cleared large tissues

Reassignment of Scattered Emission Photons in Multifocal Multiphoton Microscopy

Multifocal multiphoton microscopy (MMM) achieves fast imaging by simultaneously scanning multiple foci across different regions of specimen. The use of imaging detectors in MMM, such as CCD or CMOS, results in degradation of image signal-to-noise-ratio (SNR) due to the scattering of emitted photons. SNR can be partly recovered using multianode photomultiplier tubes (MAPMT). In this design, however, emission photons scattered to neighbor anodes are encoded by the foci scan location resulting in ghost images. The crosstalk between different anodes is currently measured a priori, which is cumbersome as it depends specimen properties. Here, we present the photon reassignment method for MMM, established based on the maximum likelihood (ML) estimation, for quantification of crosstalk between the anodes of MAPMT without a priori measurement. The method provides the reassignment of the photons generated by the ghost images to the original spatial location thus increases the SNR of the final reconstructed image.RO1 EY017656Singapore-MIT Alliance for Research and TechnologyNIH P41EB0158715 R01 NS0513204R44EB012415NSF CBET-0939511MIT Skoltech InitiativeDavid H. Koch Institute for Integrative Cancer Research at MI

Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.1152sciescopu

Spectral-domain optical coherence phase and multiphoton microscopy

We describe simultaneous quantitative phase contrast and multiphoton fluorescence imaging by combined spectral-domain optical coherence phase and multiphoton microscopy. The instrument employs two light sources for efficient optical coherence microscopic and multiphoton imaging and can generate structural and functional images of transparent specimens in the epidirection. Phase contrast imaging exhibits spatial and temporal phase stability in the subnanometer range. We also demonstrate the visualization of actin filaments in a fixed cell specimen, which is confirmed by simultaneous multiphoton fluorescence imaging. © 2007 Optical Society of America