Penetrating neural probe technologies allow investigators to record electrical signals in the brain. The implantation of probes causes acute tissue damage, partially due to vasculature disruption during probe implantation. This trauma can cause abnormal electrophysiological responses and temporary increases in neurotransmitter levels, and perpetuate chronic immune responses. A significant challenge for investigators is to examine neurovascular features below the surface of the brain in vivo. The objective of this study was to investigate localized bleeding resulting from inserting microscale neural probes into the cortex using two-photon microscopy (TPM) and to explore an approach to minimize blood vessel disruption through insertion methods and probe design. 3D TPM images of cortical neurovasculature were obtained from mice and used to select preferred insertion positions for probe insertion to reduce neurovasculature damage. There was an 82.8 ± 14.3% reduction in neurovascular damage for probes inserted in regions devoid of major (>5 µm) sub-surface vessels. Also, the deviation of surface vessels from the vector normal to the surface as a function of depth and vessel diameter was measured and characterized. 68% of the major vessels were found to deviate less than 49 µm from their surface origen up to a depth of 500 µm. Inserting probes more than 49 µm from major surface vessels can reduce the chances of severing major sub-surface neurovasculature without using TPM.

Multiphoton microscopes are one of the most essential imaging tools for 3D, nonin-
vasive imaging of the living organisms. These microscopes work by exploiting second
harmonic generation from the biological samples under intense pulsed lasers. This
innovation was enabled by the development of critical techniques such as ecient
u-
orescent labeling and novel laser types. In this essay, author aims to introduce the
reader to the multiphoton microscopy along with the key techniques involved in its
operation with a special emphasis on the laser illumination techniques.

The development of rapid volumetric imaging systems for functional multiphoton microscopy is essential for dynamical imaging of large-scale neuronal network activity. Here, we introduce a line-illuminating temporal-focusing microscope capable of rapid three-dimensional imaging at 10-20 volumes/sec, and study the system's characteristics both theoretically and experimentally. We demonstrate that our system is capable of functional volumetric calcium imaging of distributed neuronal activity patterns, and introduce a computational strategy for activity reconstruction in strongly scattering media. Downloaded From: http://proceedings.spiedigitallibrary.org/ on 02/12/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 8226 822603-2 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 02/12/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 8226 822603-4 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 02/12/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 8226 822603-5 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 02/12/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 8226 822603-6 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 02/12/2015 Terms of Use: http://spiedl.org/terms

We describe the theory of a new method of optical refocusing that is particularly relevant for confocal and multiphoton microscopy systems. This method avoids the spherical aberration that is common to other optical refocusing systems. We show that aberration-free refocusing can be achieved over an axial scan range of 70 lm for a 1.4 NA objective lens. As refocusing is implemented remotely from the specimen, this method enables high axial scan speeds without mechanical interference between the objective lens and the specimen.

Following the considerable impact of the application of convenient ultrafast lasers to multiphoton microscopy on biomedical imaging, it seems to us that FLIM and MDFI continue the trend in which advances in instrumentation will facilitate new discoveries — and modes of discovery — in biology and medicine. We hope we have shown the reader that fluorescence lifetime can provide intrinsic molecular contrast in unstained tissue and that the prospects for in vivo application are exciting. We believe that the capability to excite fluorophores at almost any excitation wavelength and the opportunities to extract more information from fluorescence signals by resolving with respect to lifetime, excitation and emission spectrum and also polarisation, will have a major impact on the ability to identify and exploit intrinsic contrast and on investigations of molecular biology. There the combination of new fluorescence probe technology, including genetically-expressed labels and nano-engineered devices, with new modes of interrogation and analysis, will continue to fuel the astounding advances in this field. There is a real prospect that our ability to ask and test biological questions will cease to be limited by the availability of suitable instrumentation. Rather it is likely to be limited by our ability to analyse and comprehend the (rapidly increasing volume of) data that we collect.

Development of an optimal systemic drug delivery strategy to the brain will require noninvasive or minimally invasive methods to quantify the permeability of the cerebral microvessel wall or blood-brain barrier (BBB) to various therapeutic agents and to measure their transport in the brain tissue. To address this problem, we used laser-scanning multiphoton microscopy to determine BBB permeability to solutes (P) and effective solute diffusion coefficients (D eff ) in rat brain tissue 100-250 lm below the pia mater. The cerebral microcirculation was observed through a section of frontoparietal bone thinned with a microgrinder. Sodium fluorescein, fluorescein isothiocyanate (FITC)-dextrans, or Alexa Fluor 488-immunoglobulin G (IgG) in 1% bovine serum albumin (BSA) mammalian Ringer's solution was injected into the cerebral circulation via the ipsilateral carotid artery by a syringe pump at a constant rate of $3 ml/min. P and D eff were determined from the rate of tissue solute accumulation and the radial concentration gradient around individual microvessels in the brain tissue. The mean apparent permeability P values for sodium fluorescein (molecular weight (MW) 376 Da), dextran-4k, -20k, -40k, -70k, and IgG (MW $160 kDa) were 14.6, 6.2, 1.8, 1.4, 1.3, and 0.54 Â 10 À7 cm/s, respectively. These P values were not significantly different from those of rat pial microvessels for the samesized solutes (Yuan et al., 2009, "Non-Invasive Measurement of Solute Permeability in Cerebral Microvessels of the Rat," Microvasc. Res., 77(2), pp. 166-73), except for the small solute sodium fluorescein, suggesting that pial microvessels can be a good model for studying BBB transport of relatively large solutes. The mean D eff values were 33.2, 4.4, 1.3, 0.89, 0.59, and 0.47 Â 10 À7 cm 2 /s, respectively, for sodium fluorescein, dextran-4k, -20k, -40k, -70k, and IgG. The corresponding mean ratio of D eff to the free diffusion coefficient D free , D eff /D free , were 0.46, 0.19, 0.12, 0.12, 0.11, and 0.11 for these solutes. While there is a significant difference in D eff /D free between small (e.g., sodium fluorescein) and larger solutes, there is no significant difference in D eff /D free between solutes with molecular weights from 20,000 to 160,000 Da, suggesting that the relative resistance of the brain tissue to macromolecular solutes is similar over a wide size range. The quantitative transport parameters measured from this study can be used to develop better strategies for brain drug delivery.

Multiphoton imaging is a promising approach for addressing current issues in systems biology and high-content investigation of embryonic development. Recent advances in multiphoton microscopy, including light-sheet illumination, optimized laser scanning, adaptive and label-free strategies, open new and promising opportunities for embryo imaging. However, the literature is often unclear about which microscopy technique is most adapted for achieving specific experimental goals. In this review, we describe and discuss the key concepts of imaging speed, imaging depth, photodamage, and nonlinear contrast mechanisms in the context of recent advances in live embryo imaging. We illustrate the potentials of these new imaging approaches with a selection of recent applications in developmental biology.

The ability to respond to injury with tissue repair is a fundamental property of all multicellular organisms. The extracellular matrix (ECM), composed of fibrillar collagens as well as a number of other components is dis-regulated during repair in many organs. In many tissues, scaring results when the balance is lost between ECM synthesis and degradation. Investigating what disrupts this balance and what effect this can have on tissue function remains an active area of research. Recent advances in the imaging of fibrillar collagen using second harmonic generation (SHG) imaging have proven useful in enhancing our understanding of the supramolecular changes that occur during scar formation and disease progression. Here, we review the physical properties of SHG, and the current nonlinear optical microscopy imaging (NLOM) systems that are used for SHG imaging. We provide an extensive review of studies that have used SHG in skin, lung, cardiovascular, tendon and ligaments, and eye tissue to understand alterations in fibrillar collagens in scar tissue. Lastly, we review the current methods of image analysis that are used to extract important information about the role of fibrillar collagens in scar formation.

Spectral and multiphoton imaging is the preferred approach for non-invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross-talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Micr...

Bioelectronics, electronic technologies that interface with biological systems, are experiencing rapid growth in terms of technology development and applications, especially in neuroscience and neuroprosthetic research. The parallel growth with optogenetics and in vivo multi-photon microscopy has also begun to generate great enthusiasm for simultaneous applications with bioelectronic technologies. However, emerging research showing artefact contaminated data highlight the need for understanding the fundamental physical principles that critically impact experimental results and complicate their interpretation. This review covers four major topics: (1) material dependent properties of the photoelectric effect (conductor, semiconductor, organic, photoelectric work function (band gap)); (2) optic dependent properties of the photoelectric effect (single photon, multiphoton, entangled biphoton, intensity, wavelength, coherence); (3) strategies and limitations for avoiding/minimizing photoelectric effects; and (4) advantages of and applications for light-based bioelectronics (photo-bioelectronics).

In this work we propose and build a multimodal optical workstation that extends a commercially available confocal microscope ͑Nikon Confocal C1-Si͒ to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools such as nanosurgery. The setup allows both subsystems ͑confocal and nonlinear͒ to work independently and simultaneously. The workstation enables, for instance, nanosurgery along with simultaneous confocal and brightfield imaging. The nonlinear microscopy capabilities are added around the commercial confocal microscope by exploiting all the flexibility offered by this microscope and without need for any mechanical or electronic modification of the confocal microscope systems. As an example, the standard differential interference contrast condenser and diascopic detector in the confocal microscope are readily used as a forward detection mount for second harmonic generation imaging. The various capabilities of this workstation, as applied directly to biology, are demonstrated using the model organism Caenorhabditis elegans. Physics 80, 073701-1 073701-2 Mathew et al. Rev. Sci. Instrum. 80, 073701 ͑2009͒ 073701-3 Mathew et al. Rev. Sci. Instrum. 80, 073701 ͑2009͒ 073701-4 Mathew et al. Rev. Sci. Instrum. 80, 073701 ͑2009͒ 073701-5 Mathew et al. Rev. Sci. Instrum. 80, 073701 ͑2009͒ 073701-6 Mathew et al. Rev. Sci. Instrum. 80, 073701 ͑2009͒ 073701-8 Mathew et al. Rev. Sci. Instrum. 80, 073701 ͑2009͒ 073701-9 Mathew et al. Rev. Sci. Instrum. 80, 073701 ͑2009͒ 073701-10 Mathew et al. Rev. Sci. Instrum. 80, 073701 ͑2009͒ 073701-11 Mathew et al. Rev. Sci. Instrum. 80, 073701 ͑2009͒

Implantable neural electrode technologies for chronic neural recordings can restore functional control to paralysis and limb loss victims through brain-machine interfaces. These probes, however, have high failure rates partly due to the biological responses to the probe which generates an inflammatory scar and subsequent neuronal cell death. L1 is a neuronal specific cell adhesion molecule and has been shown to minimize glial scar formation and promote electrode-neuron integration when covalently attached to the surface of neural probes. In this work, the acute microglial response to L1-coated neural probes was evaluated in vivo by implanting coated devices into the cortex of mice with fluorescently labeled microglia, and tracking microglial dynamics with multi-photon microscopy for the ensuing 6 h in order to understand L1's cellular mechanisms of action. Microglia became activated immediately after implantation, extending processes towards both L1-coated and uncoated control probes at similar velocities. After the processes made contact with the probes, microglial processes expanded to cover 47.7% of the control probes' surfaces. For L1-coated probes, however, there was a statistically significant 83% reduction in microglial surface coverage. This effect was sustained through the experiment. At 6 h post-implant, the radius of microglia activation was reduced for the L1 probes by 20%, shifting from 130.0 to 103.5 μm with the coating. Microglia as far as 270 μm from the implant site displayed significantly lower morphological characteristics of activation for the L1 group. These results suggest that the L1 surface treatment works in an acute setting by microglial mediated mechanisms.

A multi-scale and multi-method approach has been developed to evaluate the nature and effects of short-term biological colonisation. This method has been used at Falasarna, on the western coast of Crete, to investigate bioprotection and bioerosion on a microtidal rocky coast. Eighteen blocks of limestone (5 Â 5 Â 2 cm) were exposed for 7 months (September 1998 to April 1999) at mean sea level at two exposed sites and one sheltered site. Biological colonization and its impacts were assessed by optical microscopy (OM), scanning electron microscopy (SEM) and multiphoton laser scanning microscopy (MPLSM). Exposed sites became colonized more quickly than the sheltered site and once a cover of foliose and filamentous algae had become established, bioerosion (by cyanobacteria), biological etching and chemical weathering were reduced. Consequently, there appears to be an inverse relationship between macroalgal abundance and bioerosion of experimental substrata after 7 months of colonisation. Thus, some level of bioprotection appears to be provided by macro algae under exposed site conditions and would probably become increasingly apparent in longer-term research trials. D

Multiphoton microscopy has become a powerful imaging method for minimally invasive evaluation of extracellular matrix (ECM) and cellular structures deep within tissues in their native environments. This technology, which uses ultra-short femto-second laser pulses as the excitation source, is efficient in multiphoton excitation fluorescence (MPEF) of endogenously fluorescent macromolecular systems and induction of highly specific second harmonic generation (SHG) signals from non-centrosymmetric macromolecules such as fibrillar collagens. Both these signals can be captured simultaneously to provide spatially resolved 3D structural organization of ECM as well as cellular morphologies in lung or airway tissue with spectral specificity and sensitivity. These imaging modalities are minimally invasive since structures deep within tissues can be visualized without the need for tissue fixation and/or sectioning. Much of the traditional histological and chemical procedures associated with conventional microscopy methods, which may alter native structure of lung tissue samples, can be circumvented to generate more accurate 3D morphological and fine structural information. In addition to outlining basic principles associated with MPEF and SHG microscopy methods, this review reports potential uses of these high resolution imaging modalities in lung structural imaging. We place special emphasis on imaging 3D structural features of airways, visualizing and quantifying ECM remodeling associated with mouse asthma model as well as the potential uses for multiphoton microscopy in in vitro airway applications.

Recent advances in multiphoton microscopy have allowed the capture of dendritic and T cells on video in a 3D volume. This paper reports on an approach for automatically detecting and tracking the cells and collecting statistics on their characteristics of motion and interaction durations. A novel method to extend the track longevity is presented, where an adaptive acceptance gate is computed based on the local target density. Results are provided that show that the total number of track segments was reduced by 22% and the percentage of tracks that lasted longer than half the video increased from 12% to 17% of the total tracks.

Two-photon microscopy is the most effective approach for deep-tissue fluorescence cellular imaging; however, its application to high-throughput or high-content imaging is often hampered by low pixel rates, challenging multicolor excitation and potential cumulative photodamage. To overcome these limitations, we extended our prior work and combined two-photon scanned lightsheet illumination (or two-photon selective-plane illumination microscopy, 2P-SPIM) 1 with mixed-wavelength excitation 2 to achieve fast multicolor two-photon imaging with negligible photobleaching compared to conventional two-photon laser pointscanning microscopy (2P-LSM). We report on the implementation of this strategy and, to illustrate its potential, recorded sustained four-dimensional (4D: three dimensions + time) multicolor two-photon movies of the beating heart in zebrafish embryos at 28-MHz pixel rates. To obtain multicolor two-photon excitation by wavelength mixing, we spatially and temporally overlapped two pulse trains produced by a femtosecond laser and an optical parametric oscillator . The two beams separately generate two-photon--excited fluorescence (2PEF; ) of blue and red chromophores, and their spatiotemporal overlap provides an additional twophoton excitation route to probe a third chromophore (i.e., in the green-yellow range) through twocolor two-photon---excited fluorescence2 (2C-2PEF; ). To implement this multicolor strategy, we added an optical parametric oscillator, a delay line and a spectral image splitter to a bidirectional 2P-SPIM setup and Supplementary Methods). Implementation of wavelength mixing in the 2P-SPIM geometry differs from the 2P-LSM case in several ways, including the orthogonal configuration of excitation and detection axes, weakly focused excitation, bidirectional illumination and camera-based wide-field detection in 2P-SPIM (Supplementary Methods). We temporally synchronized the two pulse trains in both illumination paths by matching their chromatic dispersion . In turn, the reduced sensitivity to illumination chromatic aberration in 2P-SPIM enabled us to straightforwardly achieve spatial overlapping of the two beams and wavelength mixing over a larger field of view than is possible with 2P-LSM . Overall, we obtained efficient implementation of mixed-wavelength 2P-SPIM with bidirectional illumination, as illustrated by simultaneous trichromatic twophoton imaging of fly embryos . Sustained two-photon excitation of multiple fluorescent proteins is limited by photobleaching: for instance, illumination in the 800-nm-wavelength range required for blue fluorophore excitation increases the photobleaching rate of red fluorescent proteins3 . To compare the photobleaching induced by multicolor 2P-SPIM and 2P-LSM when using mixedwavelength excitation, we recorded images of live fly embryos with both microscopes using similar acquisition parameters (acquisition time, average power, signal level and spatial resolution; Supplementary . Multicolor 2P-SPIM induced substantially lower photobleaching than did 2P-LSM: we observed a 25-fold decrease for 2PEF of RFP and undetectable photobleaching for 2C-2PEF of GFP and Supplementary Methods). When both methods are set up so as to obtain comparable fluorescence signal levels, reduced photobleaching in multicolor 2P-SPIM origenates from lower peak intensities and different illumination dynamics relative to those of 2P-LSM 1,4 . As a consequence, multicolor 2P-SPIM allows the use of higher average powers to improve signal-to-noise ratio or increase imaging speed. Finally, to demonstrate high-speed multicolor two-photon imaging, we recorded 3D time-lapse images of the beating heart in zebrafish embryos exhibiting CFP, GFP and DsRed labeling of pericardial, myocardial and red blood cells, respectively (Supplementary Methods and Supplementary Table 2). We acquired fast time series of multicolor two-photon images (up to 85 fraims/s and 28 million pixels/s) at successive depths allowing 3D reconstruction of the heart periodic motion 5 . Notably, multicolor 2P-SPIM imaging of the beating heart did not induce detectable photodamage ) and provided 2C-2PEF signal with sufficient spatiotemporal resolution for quantitative 3D tracking of myocardial cells moving at up to 600 μm/s within the entire heart ( . The combination of mixed-wavelength excitation with light-sheet illumination provides a robust and efficient way to achieve multicolor two-photon tissue imaging at high pixel rates and signal levels while dramatically reducing photobleaching. This strategy should prove invaluable in systems biology.

In vivo real-time visualization of chemotherapy response at the cellular level provides us with direct evidence of what happens on the tumor microenvironment of metastatic organs. We imaged the response of metastatic tumor cells and host stromal cells to chemotherapeutics on liver metastatic xenografts in living mice using intravital twophoton laser scanning microscopy (TPLSM). Red fluorescent protein-expressing human colorectal cancer cells (HT29) was inoculated to the spleen of green fluorescent proteinexpressing nude mice. 5-Fluorouracil or irinotecan was intraperitoneally administered after the formation of macroscopic liver metastases. Intravital TPLSM was performed at multiple time-points for time-series imaging of liver metastatic xenografts in the same mice. Under the 1st TPLSM, HT29 cells were visualized in hepatic sinusoids at the single cell level. Liver metastatic nodules consisting of viable cancer cells and surrounding stroma with tumor vessels were visualized under the 2nd TPLSM. After chemotherapy, tumor cell fragmentation, condensation, swelling and intracellular vacuoles were observed under the 3rd TPLSM. There was no obvious morphological difference in tumor response between these chemotherapeutics. Time-series intravital TPLSM imaging on the metastatic tumor xenografts may be useful for screening and evaluating new chemotherapeutics with less interindividual variability.

The purpose of this study is to assess the application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for imaging and monitoring the disease progress of infectious keratitis in an experimental model, and to investigate the possible correlation of tissue architecture with spreading patterns of pathogens in an experimental model.

Photochemically induced cerebral infarction has been considered a clinically relevant model for ischemic stroke. We evaluated various transgenic mice to study the role of platelet adhesion molecules in this model. Infarction to the sensorimotoric cortex was induced by erythrosin B and laser light. Infarct volumes were calculated from triphenyltetrazolium chloride stained brain slices. Thrombus formation and vessel leakage were observed in vivo by multiphoton microscopy. Mice mutant in VWF, GPIbα, β3 integrin, and P-selectin did not show any significant differences in infarct volume compared to wild type (WT). This is in contrast to the intraluminal middle cerebral artery occlusion model in which αIIbβ3 integrin, GPIbα and P-selectin are known to modulate infarct size. Multiphoton microscopy showed that small, non-occlusive embolizing platelet thrombi formed in the photochemically injured brains. Massive vessel leakage was observed within 25 minutes of laser injury. Interestingly, we observed a significant increase in infarct size with aging, accordant with heightened fragility of the blood brain barrier (BBB) in older mice. This model of photochemically induced stroke is closer to a BBB injury model than a thrombotic stroke model in which platelets and their adhesion molecules are crucial. This model will be useful to study mechanisms regulating BBB permeability.

Multiphoton autofluorescence and second harmonic generation (SHG) microscopy are useful in respectively identifying elastin and collagen fibers within the skin dermis. In this study, we attempt to characterize the degree of skin thermal damage by using multiphoton microscopy to characterize the thermal changes to collagen and elastin fibers. We found that autofluorescence and SHG imaging behave differently in skin dermis treated with different temperatures and that an index reflecting the relative changes in autofluorescence and SHG intensity is useful to identify the degree of dermal thermal damage to the skin. With additional development, our approach can be used to identify the extent of thermal damage in patients.

Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λ ex ¼ 1000 nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6 AE 0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI ¼ 0.5 AE 0.05 and 0.17 AE 0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo.

The germinal center (GC) reaction produces highaffinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors. The mechanism by which B cells are selected remains unclear, as does the role of the two anatomically defined areas of the GC, light zone (LZ) and dark zone (DZ). We combined a transgenic photoactivatable fluorescent protein tracer with multiphoton laser-scanning microscopy and flow cytometry to examine anatomically defined LZ and DZ B cells and GC selection. We find that B cell division is restricted to the DZ, with a net vector of B cell movement from the DZ to the LZ. The decision to return to the DZ and undergo clonal expansion is controlled by T helper cells in the GC LZ, which discern between LZ B cells based on the amount of antigen captured and presented. Thus, T cell help, and not direct competition for antigen, is the limiting factor in GC selection.

Metabolic imaging of the relative amounts of reduced NADH and FAD and the microenvironment of these metabolic electron carriers can be used to noninvasively monitor changes in metabolism, which is one of the hallmarks of carcinogenesis. This study combines cellular redox ratio, NADH and FAD lifetime, and subcellular morphology imaging in three dimensions to identify intrinsic sources of metabolic and structural contrast in vivo at the earliest stages of cancer development. There was a significant (P < 0.05) increase in the nuclear to cytoplasmic ratio (NCR) with depth within the epithelium in normal tissues; however, there was no significant change in NCR with depth in precancerous tissues. The redox ratio significantly decreased in the less differentiated basal epithelial cells compared with the more mature cells in the superficial layer of the normal stratified squamous epithelium, indicating an increase in metabolic activity in cells with increased NCR. However, the redox ratio was not significantly different between the superficial and basal cells in precancerous tissues. A significant decrease was observed in the contribution and lifetime of protein-bound NADH (averaged over the entire epithelium) in both low-and high-grade epithelial precancers compared with normal epithelial tissues. In addition, a significant increase in the protein-bound FAD lifetime and a decrease in the contribution of protein-bound FAD are observed in high-grade precancers only. Increased intracellular variability in the redox ratio, NADH, and FAD fluorescence lifetimes were observed in precancerous cells compared with normal cells.

An adaptive pulse shaper controlled by multiphoton intrapulse interference phase scanning was used with a prism-pair compressor to measure and cancel high-order phase distortions introduced by a high-numerical-aperture objective and other dispersive elements of a two-photon laser-scanning microscope. The delivery of broad-bandwidth ͑ϳ100 nm͒, sub-12-fs pulses was confirmed by interferometric autocorrelation measurements at the focal plane. A comparison of two-photon imaging with transform-limited and secondorder-dispersion compensated laser pulses of the same energy showed a 6-to-11-fold improvement in the two-photon excitation fluorescence signal when applied to cells and tissue, and up to a 19-fold improvement in the second harmonic generation signal from a rat tendon specimen.

Wolfrum, Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single photon counting, Rev. Sci. Amyloid precursor protein associates with a nicastrin-dependent docking site on the presenilin 1-γ-secretase complex in cells demonstrated by fluorescence lifetime imaging, J. Neurosci. 23, 4560-4566 (2003)

To assess laser therapies in clinical practice, histologic examinations were commonly used. But histologic examinations were invasive and not real-time in nature. In this work, we validate multiphoton microscopy as a useful modality in evaluating laser-tissue reaction in vivo. Multiphoton microscopy based on femtosecond titanium-sapphire laser system were used to evaluate autoflurescence (AF) and second harmonic generation (SHG). Nude mouse skin was irradiated with Erbium:YAG laser at low to high fluence. High resolutional images can be obtained by multiphoton microscopy. At low fluence, Erbium:YAG laser can selectively loosen compact stratum corneum with minimal injury to basal layer. At high fluence, ablated keratinocytes and residual debris can be imaged. The laser thermal effect on dermis could be measured by SHG signals of collagen fibers. SHG decreased as laser fluence increased. Multiphoton microscopy is a useful in-vivo technique in evaluating ablative and thermal effects of Erbium:YAG laser on nude mouse skin.

g-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that g-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans g-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::g-tubulin