This PDF file contains the front matter associated with SPIE Proceedings Volume 6442, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

The non-resonant background signal has been the major obstacle in coherent anti-Stokes Raman scattering (CARS) spectroscopy and microscopy. This unwanted background is generated by the electronic response of the sample. It not only obscures the desired signal but also results in spectral interference with the desired vibrationally resonant CARS signal, making it difficult to assign vibrational peaks using characteristic spontaneous Raman spectra. We show that the non-resonant background can be used as a local oscillator for spectral interferometric CARS spectroscopy. Two different techniques are discussed to extract the vibrationally resonant multiplex CARS spectrum and discriminate it against the much larger non-resonant background. The pump, Stokes and probe pulses are all selected inside a single broadband ultrafast pulse (bandwidth ~1800 cm^-1) by a phase- and/or polarization-controlled pulse-shaping technique. The first technique generates two spectral interference CARS signals simultaneously, and the normalized difference of these two signals provides an amplified background-free broadband resonant CARS spectrum over 400-1500 cm^-1. The second method generates a single spectral interference CARS signal by a phase-only pulse shaping. A Fourier transform spectral interferometry (FTSI) method is used to retrieve the Raman-equivalent CARS spectrum from the measured spectral signal. Both methods enhance the resonant CARS signal by utilizing the non-resonant background as a local oscillator for homodyne mixing.

A novel procedure is developed to describe and reproduce experimental coherent anti-Stokes Raman scattering (CARS) data, with particular emphasis on highly congested spectral regions. The approach - exemplified here with high-quality multiplex CARS data - makes use the maximum entropy method for phase retrieval. The retrieved imaginary part of the nonlinear susceptibility is shown to be equal to the spontaneous Raman spectrum. The phase retrieval procedure does not influence the noise contained in the spectra. The conversion of CARS to Raman data permits a quantitative interpretation of CARS spectra. This novel approach is demonstrated for highly congested multiplex CARS spectra of sucrose, fructose and glucose. This novel procedures enables extraction of vibrational information from multiplex CARS data without the use of a priori information of the sample.

Coherent anti-Stokes Raman scattering (CARS) microscopy, which can produce images of specific molecules without staining, has attracted the attention of researchers, as it matches the need for molecular imaging and pathway analysis of live cells. In particular, there have been an increasing number of CARS experimental results regarding lipids in live cells, which cannot be fluorescently tagged while keeping the cells alive. One of the important applications of lipid research is for the metabolic syndrome. Since the metabolic syndrome is said to be related to the lipids in lipocytes, blood, arterial vessels, and so on, the CARS technique is expected to find application in this field. However, CARS microscopy requires a pair of picosecond laser pulses, which overlap both temporally and spatially. This makes the optical adjustments of a CARS microscope challenging. The authors developed a CARS unit that includes optics for easy and stable adjustment of the overlap of these laser pulses. Adding the CARS unit to a laser scanning microscope provides CARS images of a high signal-to-noise ratio, with an acquisition rate as high as 2 microseconds per pixel. Thus, images of fast-moving lipid droplets in Hela cells were obtained.

Since the first introduction of Raman microscope in 1973, optical and laser technology has made a tremendous step forward. However, despite of the obvious advantages of being a very informative and nondestructive method of studying biological samples, spontaneous Raman scattering suffers from a series of limitations such as a fluorescent background and a low signal level. Nonlinear Raman spectroscopy and, in particular, spectroscopy of coherent anti-Stokes Raman scattering (CARS) can resolve most of the problems associated with conventional Raman spectroscopy. In this report, the most critical issues of the CARS microspectroscopy setup design are reviewed and several exciting potential applications of the broadband CARS microspectroscopy, where the CARS microscopy has an advantage with respect to Raman microscopy, are outlined.

A new, synchronously pumped picosecond OPO for CARS microscopy is presented. It is based on non-critically phasematched interaction in LBO pumped by a frequency-doubled modelocked Nd:Vanadat laser at 532 nm. Within the parametric process a tuneable pair of two different wavelengths in the NIR range is generated (Signal <680 ...990 nm, Idler 1150...>2450 nm). In this system they are extracted from the cavity at the same mirror and therefore propagating collinear at the same beam path. Due to the mechanism of their generation there is no jitter between Signal and Idler. Though the wavelengths are different the GVD is negligible for this picosecond pulse duration. As a result the two pulse trains are spatially and temporally perfectly matched. The pulses generated are close to transform limit with about 5-6 ps pulse duration, excellent beam quality (M² < 1,1) and high pointing stability. The output power for Signal and Idler is about 1 W each @ 4 W pump power. The tuning mechanism is split into two parts - temperature tuning for rough variations and fast angular BRF tuning for the fine adjustment of the output wavelength. The perfect spatial and temporal overlap make the described OPO an ideal and nearly hands-free laser source for CARS microscopy with a tuneable energy difference 1,400 ... >10,000 cm^-1. The absolute wavelength range is resulting in high penetration depth and low photo damage of the analyzed samples. Finally some CARS-images are presented and the latest results and methods for further sensitivity enhancements are shown.

We report a wide-field Coherent Anti-Stokes Raman Scattering (CARS) microscopy technique based on non-phasematching illumination and imaging systems. This technique is based on a non-collinear sample illumination by broad laser beams and recording image of sample at anti-Stokes wavelength using full-frame image detector. An amplified Ti:Sapphire laser and an Optical Parametric Amplifier (OPA) provided picosecond pump and Stokes beams with energies sufficient for CARS generation in an area of 100 μm in diameter. The whole field of view of the microscope was illuminated simultaneously by the pump and Stokes beams, and CARS signal was recorded onto a cooled CCD, with resolution determined by the microscope objective. Several illumination schemes and several types of thin sample preparations have been explored. We demonstrated that CARS image of a 100x100 μm sample can be recorded with submicrometer spatial resolution using just a few laser pulses of microJoule energies.

We report in vivo molecular imaging of mouse sciatic nerve by epi-detected coherent anti-Stokes Raman Scattering (E-CARS) microscopy with vibrational selectivity, high signal-to-background ratio, 3D spatial resolution, and real-time imaging capability. The large CARS signal from the CH₂ stretch vibration allows highly sensitive and selective imaging of the myelin membrane which possesses a high lipid to protein ratio. The underlying contrast mechanism of in vivo CARS is explored by 3D imaging of fat cells that surround the nerve as well as dermal adipocytes in the mouse ear. Simultaneous E-CARS imaging of myelinated axons and second harmonic generation imaging of the surrounding collagen fibers were performed in vivo without any labeling. Finally, we show that CARS microscopy is able to distinguish between healthy myelin and disintegrated myelin induced by lysolecithin based on decrease in E-CARS intensity as well as loss of dependence on excitation polarization. Our system provides a multimodality in vivo imaging tool for studying neurodegenerative disorders.

We present a new Coherent Anti-Stokes Raman Scattering (CARS) microscopy technique for label-free imaging of biomolecules in living cells; dual-CARS microscopy. The use of three synchronized laser pulses in a dual-pump/dualdetection configuration enables imaging of two species with different molecular vibrations simultaneously, as well as acquisition of images free of non-resonant background. We show the power of the method by imaging deuterated nonadecane slowly diffusing into a suspension of living yeast cells in medium, clearly distinguishing the medium and the lipid droplets in the cells by probing the CH₂ vibration from the D-nonadecane by probing the CD vibration. In addition, images of lipid stores in living C. elegans nematodes free of non-resonant background are shown. This results in a significant enhancement of the image contrast, allowing the visualization of emerging, low-density lipid stores in a dauer larva, difficult to distinguish in conventional CARS microscopy. The separation of the non-resonant background is shown to be beneficial also when monitoring molecules with weak vibrational modes. The improved sensitivity obtained is illustrated by probing the C=C vibration in polyunsaturated lipids extracted from fish. This enables the monitoring of the degree of unsaturation of lipids, a high value of which is reported in foods known to have positive effects on human health.

We present a time domain Fourier transform coherent Raman microscopy. We show that with an added local electric field, the signal can be amplified by an order of magnitude through homodyne detection. Our approach requires a single broadband pulse to modulate, probe and amplify Raman coherence with passive phase stability to obtain high-resolution coherent anti-Stokes Raman (CARS) spectra for microscopy and microspectroscopy applications.

We have developed a fast, sensitive multiphoton microscope employing a tunable femtosecond laser and an electron-multiplying CCD camera (EMCCD) in a non-descanned detection scheme. In one configuration, the microscope provides "standard" multiphoton fluorescence images with a relatively high acquisition rate (limited only by the speed of the scanners) over a broad band of emission wavelengths. In a second configuration, the use of a diffractive optic element allows acquisition of one full set of spectrally-resolved images after only one complete scan. Increased speed is achieved in this configuration by setting the integration time of the camera such that an entire line is scanned at once, with the spectrum at each point being perpendicular to the scanned line. In both configurations, a suitable optical arrangement permits acquisition of high resolution images for both multiphoton excitation of fluorescence and widefield transmission microscopy. To illustrate the performances of our microscope we present spectrally-resolved images of individual fluorescent molecules spread over a surface as well as fluorescence images of yeast cells expressing a membrane receptor tagged with a variant of the green fluorescent protein.

Here we present the first results of a new multi photon excitation microscopy setup which extends the excitation wavelengths far beyond one micron. A synchronously pumped femtosecond-OPO (OPO PP-Automatic, APE) pumped by a femtosecond-Ti:Sapphire oscillator is used as the light source. Up to 500nm tuning can be achieved in the NIR (for instance 1100-1600nm) with fixed pump frequency and a single optics set. Automated tuning makes it an ideal tool for MPE-, SHG- and THG microscopy, which is demonstrated in combination with an optimized scanner / microscope / detection system. Together with the Ti:Sapphire pump laser (Coherent Chameleon) excitation wavelengths from 700 to 1600nm are achieved. A single-beam scanner (LaVision BioTec) was optimized for Ti:Sapphire and OPO wavelength ranges including dispersion compensation for maintaining the short pulses at the sample site as well as an overall transmission of 30-38% for the OPO range (measured up to 1400nm). Measurements on human dermis with excitation above 1 micron, compared to lower wavelengths, showed doubling of the penetration depths, strongly reduced photo damage, and by 30fold increased excitation efficiency and 10fold reduced photobleaching of red fluorescent dyes, including RFP and Cy5.5. 1100nm-excited SHG efficiency of collagen was 10 to 30fold stronger, compared to 880 nm, yet autofluorescence was decreased by up to 4 times resulting in a significantly improved signal-to-noise ratio for the detection of fluorescent dyes. The resolution is slightly reduced in comparison to Ti:Sapphire excitation, which corresponds well to the longer excitation wavelength used.

We demonstrated that the dispersion of scanning microscope optics (including a Zeiss 40x/1.2 Apochromat objective) can be compensated by means of chirped mirrors over a bandwidth of 170 nm at 800 nm. The interferometric autocorrelation trace recorded at the focus of the microscope objective with a two-photon diode indicated a pulse duration of < 12 fs. The propagation time difference of the system can be minimized by proper choice of the components, enabling sub-12-fs pulse delivery with a completely filled 40x/0.8 Zeiss Achroplan water immersion objective.

We report on a new fiber delivery system for coupling a compact, turn-key infrared femtosecond laser into a multiphoton microscope. Using a hollow core photonics crystal fiber with minimized dispersion, we achieve beam propagation on distances up to 4 meters. We measured a pulse duration close to 300 fs at the output of the microscope, for a femtosecond laser pulse duration of 200 fs. We present experimental parameters and system optimisation, and compare images obtained with fiber coupling versus direct coupling of the femtosecond laser.

In this study we investigate visible fluorescence of cytotoxic bio-markers (molecular probes) based on the derivatives of piperidone at femtosecond infrared pulsed laser excitation. The subject of this investigation is the origin of the fluorescence. Does it originate from the excited state absorption, which occurs only at slow, nanosecond excitation, or is it due to intrinsic multi-photon absorption? In the past, it has been shown indirectly, through the laser photolysis study, that the contribution of the excited state absorption is minimal for several compounds of such type. The results of direct experiments with an infrared femtosecond fiber laser as an excitation source described here support this hypothesis. The observed dependence of the fluorescence on the pump power indicated the contribution of not only two-photon, but multi-photon routes of excitation. Additionally, it was shown that the spectral features of the fluorescence correlate with the presence of glycine, an amino acid that is one of the building blocks of proteins in a cell. The implication of this result is, in addition to their anticancer action, the compounds can possibly be used for fluorescent diagnostics of cancer and multi-photon fluorescent microscopy of malignant cell cultures using portable infrared fiber lasers as excitation sources.

Sample induced optical aberrations in slices of rat brain tissue have been corrected with a deformable membrane mirror. The aberration correction required by the DMM was determined using a genetic algorithm with the intensity at a point in the sample as a fitness value. We show that by optimising on the intensity of a single point in the sample we are able to improve the axial resolution across the whole field of view of the image at a fixed sample depth. The ratio between the corrected axial resolution and the diffraction limited resolution is on average 2.7 for a 50 μm thick rat brain tissue sample and 12 for a 380 μm thick sample across the whole field of view. The uncorrected ratio being 4.1 and 15.5 respectively. Using a single aberration correction per depth, compared to a point-by-point aberration correction, will significantly decrease scan times and therefore reduce photobleaching and phototoxic effects enabling more rapid microscopy with active aberration correction.

In this study, we present two different approaches that allow multi-wavelength fluorescence lifetime measurements in the time domain. One technique is based on a streak camera system, the other technique is based on a time-correlated singlephoton- counting (TCSPC) approach. The setup consists of a confocal laser-scanning microscope (LSM 510, Zeiss) and a Titanium:Sapphire-laser (Mira 900D, Coherent) that is used for pulsed one- and two-photon excitation. Fluorescence light emitted by the sample is dispersed by a polychromator (250is, Chromex) and recorded by a streak camera (C5680 with M5677 sweep unit, Hamamatsu Photonics) or a 16 channel TCSPC detector head (PML-16, Becker & Hickl) connected to a TCSPC imaging module (SPC-730/SPC-830, Becker & Hickl). With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. We applied these methods to Förster resonance energy transfer (FRET) measurements and discuss the advantages over fluorescence techniques that are already well established in the field of confocal microscopy, such as spectrally resolved intensity measurements or single-wavelength fluorescence lifetime measurements.

The average fluorescence lifetime of GFP in solution is a function of the refractive index of its environment. Here, we demonstrate that this also appears to be the case for GFP-tagged proteins in cells. Using TCSPC-based FLIM with a scanning confocal microscope, we image GFP-tagged proteins in fixed cells in different media. We find that the average fluorescence lifetime of GFP in cells is shortened, as glycerol or sucrose are added to the medium. This is the case for GFP-tagged MHC proteins with the GFP located inside the cytoplasm, and also for GPI-anchored GFP which is located outside the cell membrane. We observe a linear relationship between the inverse average lifetime of GFP in fixed cells and the square of the refractive index of the medium. Implications of this phenomenon when using Total Internal Reflection Fluorescence (TIRF) microscopy will also be discussed as a shortening of the lifetime is seen close to the glass prism used to produce the evanescent wave in TRIF.

SLIM (spectral fluorescence lifetime imaging) is a highly sophisticated new technique, which combines spectral resolved and time resolved detection. Real spectral information is achieved by using a grating in front of a PML-array, which allows time-correlated single photon counting (TCSPC). Whereas spectrally resolved fluorescence imaging alone has a reasonable sensitivity, the specificity of fluorescence detection can be improved by considering the fluorescence lifetime. SLIM was realized on the basis of a laser scanning microscope. The fluorescence light from the second descanned detection channel was coupled into a 600 μm multimode fibre. The end of the fibre was put into the input focal plane of an MS125 spectrograph (grating of 600 lines/mm). A PML-16 multichannel PMT module, containing a 16 channel multi-anode PMT and the TCSPC routing electronics was attached to the output of the spectrograph. The grating yields a 200 nm spectral range spread over the 16 channels of the detector. The spectral bandwith of the PMT channels was about 12 nm. For fluorescence excitation, a Ti:Sa laser or alternatively a ps diode laser was used. The various possibilities which SLIM offers to improve cell diagnosis will be discussed as well as successfully realized applications. These include cancer diagnosis with endogenous and exogenous fluorophores, and FRET measurements for multiple protein interactions.

While the growth and structure of amyloid fibers with ß-sheet secondary structure has been widely investigated in recent years, the mechanism of self-assembly remains poorly understood. Multiple intermediate species have been proposed to play important roles in the self assembly process, yet many of these remain poorly defined or have not been clearly observed. Fluorescence microscopy and spectroscopy should provide powerful tools to amyloid formation mechanisms, although given the tight packing of molecules within amyloid structures one must be concerned about the extent to which the coupling of fluorescent probes will interfere with the amyloid formation process. We have performed systematic characterization of the self assembly and interactions between a model amyloid forming peptide, residues 16-22 from the amyloid beta peptide, together with two different rhodamine conjugated forms of this same peptide sequence. We observe that in some cases, the fluorescent dye does appear to alter the morphology of assembled amyloid structures. We also report on amyloid formation using mixtures of labeled and unlabeled peptides which does not perturb the morphology of the amyloid fibers and tubes, and appears to provide an excellent system for further investigation of amyloid formation.

Multiphoton tomography with the clinical femtosecond laser system DermaInspect has become an important non-invasive high resolution imaging tool for skin research, melanoma detection, and in situ drug monitoring of pharmaceutical and cosmetical components. The detection of endogenous fluorophores and SHG active biostructures such as mitochondrial NAD(P)H, melanin in melancytes and basal cells, as well as the extracellular matrix components elastin and collagen is achieved with submicron resolution when using high NA focusing optics. So far, the working distance was limited to 200 µm. In addition, the focusing optics was large in diameter (2-3 cm). Here we report for the first time on clinical deep-tissue high-resolution imaging with a novel high NA rigid GRIN microendoscope which extends the potential of clinical multiphoton tomography significantly. We performed the very first clinical in vivo measurements with two-photon endoscopes and studied wounds of patients with ulcus cruris.

The high degree of structural order in skeletal muscle allows imaging of this tissue by Second Harmonic Generation (SHG). Biochemical and colocalization studies have gathered an increasing wealth of clues for the attribution of the molecular origin of the muscle SHG signal to the motor protein myosin. Thus, SHG represents a potentially very powerful tool in the investigation of structural dynamics occurring in muscle during active production of force and/or shortening. A full characterization of the polarization-dependence of the SHG signal represents a very selective information on the orientation of the emitting proteins and their dynamics during contraction, provided that different physiological states of muscle (relaxed, rigor and active) exhibit distinct patterns of SHG polarization dependence. Here polarization data are obtained from single frog muscle fibers at rest and during isometric contraction and interpreted, by means of a model, in terms of an average orientation of the SHG emitters which are structured with a cylindrical symmetry about the fiber axis. The setup is optimized for accurate polarization measurements with SHG, combined with a line scan imaging method allowing acquisition of SHG polarization curves in different physiological states. We demonstrate that muscle fiber displays a measurable variation of the orientation of SHG emitters with the transition from rest to isometric contraction.

Third Harmonic Generation (THG) from the vicinity of interfaces, using focused laser beams can be obtained virtually from any inhomogeneous medium. Its sensitivity to the presence and extent of inhomogeneity in the focal volume has already found a variety of applications ranging from material characterization to label free three-dimensional microscopy of biological samples. In this presentation, we demonstrate a number of new applications of THG in the microscopy of food samples and living cells. Also, we report on an anomalous behavior in the THG z-response. So far the observations and theoretical predictions supported a single peak of THG signal, with the peak position corresponding to the interface. We have observed an anomalous behavior where a single interface can give rise to two peaks located across the interface. The simulations, which we carried out using a paraxial theory of THG and measurements done on typical normally dispersive materials, suggest that this anomalous behavior is due to a particular combination of χ⁽³⁾ and the magnitude of dispersion.

The deep-tissue penetration and submicron spatial resolution of multi-photon microscopy and the high-detection efficiency and nanometer spectral resolution capability of a spectrograph were combined to study the intrinsic emission of mouse skin post mortem biopsy and section, and in vivo tissue samples. The different layers of skin could be clearly distinguished based on both their spectral signature and morphology. Auto fluorescence could be detected from both cellular and extra cellular structures. In addition SHG from collagen and a narrowband spectral emission band related to collagen were observed. Visualization of the spectral images in RGB color allowed us to identify tissue structures such as epidermal cells, lipid-rich keratinocytes and intercellular structures, hair follicles, collagen, elastin, and dermal fibroblasts. The results also showed morphological and spectral differences between the mouse skin post mortem biopsy and in vivo samples which explained by biochemical differences, specifically of NAD(P)H. Overall, spectral imaging provided a wealth of information not easily obtainable with present conventional multi-photon imaging methods.

When imaging anisotropic samples with a laser scanning optical microscope, the results are often affected by the polarization of the excitation light source. Quantifying the polarization dependence of biological fibrous material such as muscle and collagen allows us to gain molecular information at length scale below the resolution of optical microscopes. One problem associated with rotating the direction of linearly polarized excitation light for an epi-illuminated laser scanning microscope is due to the reflective properties of the main dichroic mirror. Depending on the direction of the incident polarization, the dichroic mirror can induce different amount of phase retardation, thus altering the desired output polarization. In this work, we theoretically determined the needed combination of wave plates and their angular positions to compensate for the effect of the dichroic mirror, thus achieving any arbitrary linear polarization angle for the excitation incident on sample.

Using the technique of second-harmonic generation (SHG) microscopy we obtained large area image of type I collagen from rat tail tendon as it is heated from 40°C to 70°C for 0 to 180 minutes. The high resolution images allowed us to investigate the collagen structural change. We observed that heating the tendon below the temperature of 54°C does not produce any change in the averaged SHG intensity. At the heating temperature of 54°C and above, we find that increasing the heating temperature and time leads to decreasing SHG intensity. As the tendon is heated above 54°C, a decrease in the SHG signal occurs uniformly throughout the tendon, but the regions where the SHG signal vanishes form a tiger-tail like pattern. By comparing the relative SHG intensities in small and large areas, we found that the denaturation process responsible for forming the tiger-tail like pattern occurs at a higher rate than the global denaturation process occurring throughout the tendon. Our results show that second-harmonic generation microscopy is effective in monitoring the thermal damage to collagen and has potential applications in biomedicine.

Multiphoton optoporation of vital cells was performed using a femtosecond pulsed laser in the near infrared (NIR). Exogenous materials such as macromolecules and exogenes were transported into the targets via laser assisted transient opening of the cell membrane. This method is also appropriate for nanoprocessing and optoporation inside 3D stem cell structures without photodestructive collateral effects which was confirmed with TUNEL-assay (DNA strand breaks) and tests for reactive oxygen species (ROS).

We present a simple and robust way to reject out-of-focus background when performing deep two-photon excited fluorescence (TPEF) imaging in thick tissue. The technique is based on the use of a deformable mirror (DM) to introduce illumination aberrations that preferentially degrade TPEF signal while leaving TPEF background relatively unchanged. A subtraction of aberrated from unaberrated images leads to background rejection. We present a heuristic description of our technique, which we corroborate with experiment. Images of a labeled mouse olfactory bulb are compared with standard TPEF microscopy images, demonstrating significant out of focus TPEF background rejection with our technique. Finally we improve our technique by developing a faster aberration modulation mechanism that performs background subtraction line by line rather than frame by frame. In this manner, the overall image acquisition rate of our technique is the same as that of a standard TPEF microscope.

We investigated human cutaneous basal cell carcinoma ex-vivo samples by combined time resolved two photon intrinsic fluorescence and second harmonic generation microscopy. Morphological and spectroscopic differences were found between malignant skin and corresponding healthy skin tissues. In comparison with normal healthy skin, cancer tissue showed a different morphology and a mean fluorescence lifetime distribution slightly shifted towards higher values. Topical application of delta-aminolevulinic acid to the lesion four hours before excision resulted in an enhancement of the fluorescence signal arising from malignant tissue, due to the accumulation of protoporphyrines inside tumor cells. Contrast enhancement was prevalent at tumor borders by both two photon fluorescence microscopy and fluorescence lifetime imaging. Fluorescence-based images showed a good correlation with conventional histopathological analysis, thereby supporting the diagnostic accuracy of this novel method. Combined morphological and lifetime analysis in the study of ex-vivo skin samples discriminated benign from malignant tissues, thus offering a reliable, non-invasive tool for the in-vivo analysis of inflammatory and neoplastic skin lesions.

Even though multi-photon fluorescence microscopy offers higher resolution and better penetration depth than traditional fluorescence microscopy, its use is restricted to the detection of molecules that fluoresce. Two-photon absorption (TPA) imaging can provide contrast in non-fluorescent molecules while retaining the high resolution and sectioning capabilities of nonlinear imaging modalities. In the long-wavelength water window, tissue TPA is dominated by the endogenous molecules melanin and hemoglobin with an almost complete absence of endogenous two-photon fluorescence. A complementary nonlinear contrast mechanism is self-phase modulation (SPM), which can provide intrinsic signatures that can depend on local tissue anisotropy, chemical environment, or other structural properties. We have developed a spectral hole refilling measurement technique for TPA and SPM measurements using shaped ultrafast laser pulses. Here we report on a microscopy setup to simultaneously acquire 3D, high-resolution TPA and SPM images. We have acquired data in mounted B16 melanoma cells with very modest laser power levels. We will also discuss the possible application of this measurement technique to neuronal imaging. Since SPM is sensitive to material structure we can expect SPM properties of neurons to change during neuronal firing. Using our hole-refilling technique we have now demonstrated strong novel intrinsic nonlinear signatures of neuronal activation in a hippocampal brain slice. The observed changes in nonlinear signal upon collective activation were up to factors of two, unlike other intrinsic optical signal changes on the percent level. These results show that TPA and SPM imaging can provide important novel functional contrast in tissue using very modest power levels suitable for in vivo applications.

The benefits of two-photon fluorescence microscopy of biological samples are vast arising from the utilisation of low energy light. The two-photon absorption cross sections (σ₂) of the di-cation free-base and metallated forms of hematoporphyrin derivative (HpD), hematoporphyrin IX (Hp9) and a boronated protoporphyrin (BOPP) are obtained to ascertain their effectiveness as fluorophores for use in two-photon microscopy. The open-aperture Z-scan and the two-photon induced fluorescence (TPIF) techniques, each capable of providing information regarding the nonlinear absorption, are employed to determine σ₂ of the various porphyrins at an excitation wavelength of 800 nm. A significant disparity in the determined values of σ₂ using the two methods is observed. This is largely attributed to the common requirement of higher concentrations used in the open aperture Z-scan method compared with TPIF techniques. Values of σ₂ obtained from the Z-scan experiments are in the order of 10 GM, whilst those obtained from the TPIF experiments are in the order of 200 GM. Insertion of either protons or metal ions into the macrocycle does not enhance the σ₂ of the porphyrins. Successful two-photon induced fluorescence imaging of BOPP free-base loaded G6 glioma cells is achieved, confirming the usefulness of this porphyrin in two-photon microscopy.

Zebra Finches are songbirds which constitute a model for neuro-ethologists to study the neuro-mechanisms of vocal recognition. For this purpose, in vivo and non invasive monitoring of brain activity is required during acoustical stimulation. MRI (Magnetic Resonance Imaging) or NIRS (Near InfraRed Spectroscopy) are suitable methods for these measurements, even though MRI is difficult to link quantitatively with neural activity and NIRS suffers from a poor resolution. In the particular case of songbirds (whose skin is thin and quite transparent and whose skull structure is hollow), two-photon microscopy enables a quite deep penetration in tissues and could be an alternative. We present here preliminary studies on the feasability of two-photon microscopy in these conditions. To do so, we chose to image hollow fibers, filled with Rhodamine B, through the skin of Zebra finches in order to evaluate the spatial resolution we may expect in future in vivo experiments. Moreover, we used the reflectance-mode confocal configuration to evaluate the exponential decrease of backreflected light in skin and in skull samples. Following this procedure recently proposed by S.L. Jacques and co-workers, we planned to determine the scattering coefficient μ_s and the anisotropy g of these tissues and make a comparison between fixed and fresh skin and skull samples for future Monte Carlo simulations of the scattering in our particular multi-layered structure.

Recently, two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex in living mice. In this work we study the in vivo spine rearrangements after an acute and selective damage. For this purpose, we have used a near-IR femtosecond pulsed laser to combine two-photon microscopy imaging with microdissection operation on fluorescently-labeled neurons. Three-dimensional reconstructions of dendrites expressing fluorescence protein have been performed in the cortex of YFP-H and GFP-M transgenic living mice. Afterwards, single dendrites have been laser-dissected irradiating the structure with a high femtosecond laser energy dose. By using a chronically implanted glass window we performed long-term imaging in the area of the dissected dendrite. We will show that laser ablation can be performed with micrometric precision and without visible collateral damage to nearby neuronal structures. Also, we will evidence the morphological changes of the dendritic branches and dendritic spines after this specific perturbation inside the intact neuronal network. Laser microdissection of selected structures of the neuronal branching in vivo represents a promising tool for neurobiological research.

A powerful advantage of multiphoton microscopy is its ability to image endogenous fluorophores such as the ubiquitous coenzyme NADH in discrete cellular populations. NADH is integral in both oxidative and non-oxidative cellular metabolism. NADH loses fluorescence upon oxidation to NAD⁺; thus changes in NADH fluorescence can be used to monitor metabolism. Recent studies have suggested that hypo metabolic astrocytes play an important role in cases of temporal lobe epilepsy (TLE). Current theories suggest this may be due to defective and/or a reduced number of mitochondria or dysfunction of the neuronal-astrocytic metabolic coupling. Measuring NADH fluorescence changes following chemical stimulation enables the quantification of the cellular distribution of metabolic anomalies in epileptic brain tissue compared to healthy tissue. We present what we believe to be the first multiphoton microscopy images of NADH from the human brain. We also present images of NADH fluorescence from the hippocampus of the kainate-treated rat TLE model. In some experiments, human and rat astrocytes were selectively labeled with the fluorescent dye sulforhodamine 101 (SR101). Our results demonstrate that multiphoton microscopy is a powerful tool for assaying the metabolic pathologies associated with temporal lobe epilepsy in humans and in rodent models.

Broad two-photon cross sections of fluorescent proteins allow excitation with a single wavelength of a tunable femtosecond pulsed laser but the brightness is sub-optimal and the cross-talk prevents sensitized emission FRET imaging in heterologous systems. We present a novel arrangement of a resonant scanning microscope capable of fast interline dual wavelength femtosecond excitation of pairs of fluorophores. This allows optimal and selective excitation of mCerulean and mCitrine as well as FRET imaging using the principles of sensitized emission 3-cube imaging. Performance of the system in thin and thick specimens is discussed.

In this work, intravital multiphoton microscopy is applied to the imaging of liver metabolism with the least invasiveness. We observed intravital dynamics of the uptake, processing and excretion of the organic anion species, 6-carboxyfluorescein diacetate (6-CFDA) in the hepatobiliary system. This is achieved by the use of multiphoton microscopy and an in vivo hepatic imaging chamber which allows us to image the dynamics of hepatic metabolism. Multiphoton images revealed that the hepatic processing of 6-CFDA is completed within approximately 50 minutes. The images reveal the liver metabolism of the uptake and processing of 6-CFDA from the hepatocytes, and the subsequent excretion into bile canaliculi. Our results suggest that this approach is a promising technique for investigating intravital hepatic physiology, diseases, and metabolism.

It is well known that the efficiency and selectivity of two-photon excited fluorescence (TPEF) process can depend on various parameters of the ultrashort pulses, such as the pulse intensity and phase, which interact with the specimen. In order to completely understand this dependence and to obtain optimal TPEF images, techniques like Collinear Frequency Resolved Optical Gating (CFROG) arrangement can be implemented in a microscope for complete pulse characterisation at the sample plane. However, this adds complexity that that additional forward collecting optics is required as well as a suitable frequency doubling crystal. Here we report a simple way to characterize the pulses within a multiphoton microscope that do not require forward collecting optics. This is achieved by taking advantage of the fact that backward propagating second harmonic generation (SHG) signal can be easily generated from starch granules. Since both the fluorescence and SHG signals can be collected using the same detection scheme the experimental arrangement is considerably simplified. Starch, being a non- toxic and non-soluble material does not affect living cells allowing the pulse characteristics to be measured in situ, without the need to move the sample. We obtained real-time SHG-autocorrelations traces by using a single starch granule that was placed alongside the living HeLa cells (GFP labeled) being imaged. Furthermore by placing a spectrometer at the output port of the microscope, a spectrally resolved SHG autocorrelation was acquired allowing complete characterisation of the pulse to be carried out. The temporal intensity and phase profile were retrieved using CFROG technique. Marginal analysis was carried out to ensure that the experimental data was successfully acquired.

Articular cartilage possesses an extensive extracellular matrix consisting of a highly organised network of collagen fibres embedded in a much finer mesh of proteoglycans and other glycoproteins. Many fundamental issues of cartilage biomechanics, its ageing and the development of osteoarthritis concern the detailed organisation of this matrix. Here we investigate the application of multi-photon microscopy to characterise the structure of the extracellular matrix. In reflection mode both second harmonic Generation (SHG) and two photon fluorescence (TPF) imaging modalities reveal differences in the pericellular and inter-territorial matrix in normal tissue and additional changes in degenerative lesions. The SHG signal from the surface zone is dependent on the direction of polarization of the laser excitation beam but the TPF signal is not. The former can be quantified to determine fibre orientation although the pattern is less well resolved than in tendon, reflecting the less regular orientation of the finer fibres. Nevertheless, previously unreported subtle variations in fibre orientation over the surface of the cartilage can be observed. In order to characterise variations with depth we carried out polarization sensitivity experiments at depths up to 180 microns into the tissue. At greater depths the polarization sensitivity is affected by the birefringence and dichroism of the overlying tissue and we have quantified these effects to allow correction of the data.

Intrinsic second harmonic generation (SHG) signals can be used to visualize the three-dimensional structure of cardiac and skeletal muscle with high spatial resolution. Fluorescence labeling of complementary sarcomeric proteins, e.g. actin, indicates that the observed SHG signals arise from the myosin filaments. Recently, the myosin rod domain or LMM - light meromyosin - has been reported to be the dominant source of this SHG signal. However, to date, mostly negative and indirect evidence has been presented to support this assumption. Here, we show, to our knowledge, the first direct evidences that strong SHG signals can be obtained from synthetic paracrystals. These rod shaped filaments are formed from purified LMM. SDS-PAGE protein analysis confirmed that the LMM crystals lack myosin head domains. Some regions of the LMM paracrystals produce a strong SHG signal whereas others did not. The SHG signals were recorded with a laser-scanning microscope (Leica SP2). A ps laser tuned to 880 nm was used to excite the sample through an 63x objective of 1.2 NA. In order to visualize the synthetic filaments - in addition to SHG imaging -, the LMM was labeled with the fluorescent marker 5-IAF. We were able to observe filaments of 1 to 50 μm in length and of up to 5 μm in diameter. In conclusion, we can show that the myosin rod domain (LMM) is a dominant source for intrinsic SHG signals. There seems, however, a signal dependence on the paracrystals' morphology. This dependence is being investigated.

Multiphoton tomography offers a painless method to examine patients under natural physiological conditions in vivo. Multiphoton excitation induces a weak autofluorescence of naturally endogenous fluorescent bio-molecules, such as flavines, NAD(P)H, metal-free porphyrines, components of lipofuscin, elastin and keratin. Additionally, collagen can be detected by second harmonic generation (SHG). Due to the nonlinearity, the effects occur only in a very tight focus, where the photon density is high enough. This leads to high axial and lateral resolution of <1μm without any need of a confocal detection and avoids out-of-focus damage. The limited depth range, given by the working distance of the focusing optics, is overcome with a gradient index-lens (GRIN-lens) based endoscope. In this work we present the first results of clinical applications in vivo of gradient-index lens endoscopes. Images of e.g. elastin and collagen (SHG) in the dermal layer of human skin are presented.

Adult human and rat pancreas stem cells as well as tumor spheroids were irradiated with femtosecond laser pulses in the near infrared (NIR) spectral range at high transient GW/cm² and TW/cm² intensities. The cellular response to the laser exposure was probed by the detection of modifications of NAD(P)H autofluorescence, the formation of reactive oxygen species (ROS) and DNA strand breaks (TUNEL-assay), and viability (live/dead test). The results confirm that long-term scanning of stem cells can be performed at appropriate laser exposure parameters without a measurable impact on the cellular metabolism and vitality. In addition, it was proven that a targeted inactivation of a particular single stem cells or a single tumour cell inside a 3D cell cluster using single point illumination at TW/cm² laser intensities can be performed without affecting neighbouring cells. Therefore multiphoton microscopes can be considered as biosafe tools for long-term analysis of stem cells as well as highly precise optical knocking out of single cells within cell clusters and tissues.

Light delivery by fiber can directly excite the desired region. In recent years a number of methods have been proposed for fiber-based multiphoton microscopy. Of particular interest is the type of fiber used for both excitation with femtosecond pulses and method of collection of emitted fluorescence. In our efforts towards the development of a compact multiphoton microendoscope, we have conducted a study comparing the performance of three types of fibers (single mode fiber, hollow core photonic crystal fiber, and double clad fiber) in order to provide an objective comparison between various excitation approaches. In this study, we have demonstrated the ability of multiphoton microscopy and second harmonic generation microscopy for high resolution intensity and life time imaging of the oral mucosa and submucosa in vivo based on the use of double clad photonic crystal fiber.

The combination of multi photon laser scanning microscopy with transgenic techniques has set the stage for in vivo studies of long term dynamics of the central nervous system in mice. Brain structures located within 100?m to 200?m below the brain surface can be observed minimum-invasively during the post-adolescent life of the animal. However, even when selecting the most appropriate microscope optics available for the purpose, trans-cranial observation is compromised by the aberrations induced by the cranium and the tissue interposed between the cranium and the actual focus. It still is an un-resolved task to calculate these aberrational effects or to, at least, estimate quantitatively the distortions they induce onto the recorded images. Here, we report about measurements of the reflection, the absorption, and the effects on the objective point spread function of the mouse cranium as a function of the thickness of the cranium, the locus of trans-cranial observation and the wavelength. There is experimental evidence for pronounced Second Harmonic Generation (SHG) effects.

The current advances in fluorescence microscopy coupled with the development of new fluorescent probes such as visible fluorescent proteins (VFP) allow Förster (fluorescence) resonance energy transfer (FRET) to be used to study protein-protein interactions in living specimens. For FRET to occur, the donor emission spectrum should overlap the acceptor absorption at least about 30%. This spectral overlap generates donor and acceptor spectral bleedthrough (DSBT and ASBT) in the FRET channel (or acceptor channel) while exciting the double labeled cell with donor excitation wavelength. The spectral imaging microscopy helps to avoid the DSBT into FRET channel by linear unmixing. But the ASBT is the same spectral region of the FRET signal which cannot be removed by linear unmixing. To obtain a quantitative estimation of the C/EBPα protein dimerization in GHFT1-5 living cell nucleus, we need to remove the ASBT from the FRET channel. So, we developed an algorithm that removes the ASBT signal from the spectral FRET (sFRET) image. The E% estimated using the processed spectral FRET (psFRET) algorithm provides excellent comparison with other techniques such as confocal and lifetime FRET microscopy. This psFRET algorithm was also characterized with FRET standards, based on constructs with known-length amino acid linkers.

Layer-by-Layer or self-assembly techniques can be used to prepare Fluorescent polymer samples on glass coverslips serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general for sectioning microscopy. Layers can be realized as ultra-thin (<< 100 nm) or thin (approx. 100 nm) characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to directly measure the z-response of different scanning optical microscopes. In two-photon excitation microscopy z-sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized in a Sectioned Imaging property chart (SIPchart), we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the z-response of the system over a full field of view area. These samples are also useful for monitoring photobleaching behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be effciently used for real time alignment of the microscope.

Raman spectrum of Platymonas subcordiformis was studied by FT-Raman spectroscopy. The results show that the optimum experiment conditions is that making sample lose solvent with centrifuge, excitation laser power for 360mW and accumulating 70 times. The main peaks of the spectrum are located at 556cm^-1, 615cm^-1, 880cm^-1, 960 cm^-1, 1112cm^-1, 1457cm^-1, 1523cm^-1, 2986cm^-1, respectively. These peaks can be assigned to protein, instauration acid and ester, etc., which are the main compositions of Platymonas subcordiformis. The precise measurements of algae Raman Spectroscopy could be used for biological samples research, such as developing a new optical taxonomic methodology to distinguish different algae species, and a rapid, non-destructive detection way of stress effects.

Multiphoton tomography based on femtosecond laser NIR (near infrared) pulses was used to perform non-invasive optical sectioning of skin with high spatial and intracellular resolution. Scar formation due to formation of collagen fibers is an important aspect during wound healing processes in skin and tissues and was monitored in vivo using the system DermaInspect. Multiphoton tomography was performed of a dermal wound after nevi extraction. The healing process and the aggregation of collagen fibers could be long term monitored due to the autofluorescence of endogenous fluorophores and SHG of collagen. The system DermaInspect might become a high resolution diagnostic tool for dermatological diagnostics and monitoring therapeutic effects.

Multiphoton stimulated autofluorescence microscopy and Magnetic resonance imaging (MRI) address different molecular properties of the sample and reach to a different length scale. MRI maps density or mobility of nuclei (here: hydrogen), and targets at whole objects from the scale of sub-millimetres to meters. Multiphoton imaging profits from the nonlinear absorption of light in the focus of a femtosecond laser source stimulating the autofluorescence of biomolecules. As this effect relies on a high light intensity the accessible field of view is limited, but the resolution is very high. Studying a plant embryo (barley) we compare the two techniques. At 770 nm excitation the cell walls of the embryo exhibited significant autofluorescence, allowing for a subcellular resolution. While details where imaged with an objective of N.A. 1.3, an overview was generated with a N.A. as low as 0.25. The overview image as well as merged images and tomographical data were used to link the high-resolution optical data with the three-dimensional highresolution MR images. There, images of the proton density were acquired using a standard 3D spin-echo imaging pulse sequence. While the optical high-resolution data provides a field of view restricted to only a small part of the embryo, the MR image contains the whole grain. Bridging the scales it might be possible to trace transport of e.g. nutrients from large structure of the plant to the cellular level.

A photo-convertible protein is found in several species of the coral genus Lobophyllia. Its green fluorescence is converted to red by irradiation in the 340-400nm range. It also exhibits a wider range of photo-reactive properties, including a reversible photo-bleaching when in a partially-converted state. We present data on its behaviour under single and multiphoton irradiation.