This PDF file contains the front matter associated with SPIE Proceedings Volume 8948, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

Adaptive optics have been introduced to compensate the effects of aberrations in high resolution microscopy. Adaptive elements, such as deformable mirrors or spatial light modulators enable the dynamic correction of aberrations in a range of applications. These methods have proved particularly useful in three dimensional imaging of tissue specimens. We review the principles behind these methods and their application to high and super resolution microscopy.

Certified clinical multiphoton tomographs for label-free multidimensional high-resolution in vivo imaging have been introduced to the market several years ago. Novel tomographs include a flexible 360° scan head attached to a mechanooptical arm for autofluorescence and SHG imaging as well as a CARS module. Non-fluorescent lipids and water, mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen can be imaged in vivo with submicron resolution in human skin. Sensitive and rapid detectors allow single photon counting and the construction of 3D maps where the number of detected photons per voxel is depicted. Intratissue concentration profiles from endogenous as well exogenous substances can be generated when the number of detected photons can be correlated with the number of molecules with respect to binding and scattering behavior. Furthermore, the skin ageing index SAAID based on the ratio elastin/collagen as well as the epidermis depth based on the onset of SHG generation can be determined.

Atherosclerosis is among the most widespread cardiovascular diseases and one of the leading cause of death in the Western World. Characterization of arterial tissue in atherosclerotic condition is extremely interesting from the diagnostic point of view. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires a morpho-functional approach. Multimodal non-linear microscopy has the potential to bridge this gap by providing morpho-functional information on the examined tissues in a label-free way. Here we employed multiple non-linear microscopy techniques, including CARS, TPF, and SHG to provide intrinsic optical contrast from various tissue components in both arterial wall and atherosclerotic plaques. CARS and TPF microscopy were used to respectively image lipid depositions within plaques and elastin in the arterial wall. Cholesterol deposition in the lumen and collagen in the arterial wall were selectively imaged by SHG microscopy and distinguished by forward-backward SHG ratio. Image pattern analysis allowed characterizing collagen organization in different tissue regions. Different values of fiber mean size, distribution and anisotropy are calculated for lumen and media prospectively allowing for automated classification of atherosclerotic lesions. The presented method represents a promising diagnostic tool for evaluating atherosclerotic tissue and has the potential to find a stable place in clinical setting as well as to be applied in vivo in the near future.

Second Harmonic Generation (SHG) of collagen signals allows for the analysis of collagen structural changes throughout metastatic progression. The directionality of coherent SHG signals, measured through the ratio of the forward-propagating to backward propagating signal (F/B ratio), is affected by fibril diameter, spacing, and order versus disorder of fibril packing within a fiber. As tumors interact with their microenvironment and metastasize, it causes changes in these parameters, and concurrent changes in the F/B ratio. Specifically, the F/B ratio of breast tumors that are highly metastatic to the lymph nodes is significantly higher than those in tumors with restricted lymph node involvement. We utilized in vitro analysis of tumor cell motility through collagen gels of different microstructures, and hence different F/B ratios, to explore the relationship between collagen microstructures and metastatic capabilities of the tumor. By manipulating environmental factors of fibrillogenesis and biochemical factors of fiber composition we created methods of varying the average F/B ratio of the gel, with significant changes in fiber structure occurring as a result of alterations in incubation temperature and increasing type III collagen presence. A migration assay was performed using simultaneous SHG and fluorescent imaging to measure average penetration depth of human tumor cells into the gels of significantly different F/B ratios, with preliminary data demonstrating that cells penetrate deeper into gels of higher F/B ratio caused by lower type III collagen concentration. Determining the role of collagen structure in tumor cell motility will aid in the future prediction metastatic capabilities of a primary tumor.

Adverse events in normal tissues after irradiation of malignant tumors are of great importance in modern radiation oncology. Second harmonic generation (SHG) microscopy allows observe the structure of collagen fibers and bundles without additional staining. The study objective was evaluation the dose-time dependences of the structural changes occurring in collagen of rat rectum and bladder after gamma-irradiation. Animals were irradiated by a local field at single doses of 10 Gy and 40 Gy. The study of collagen state was carried out in a week and a month after radiation exposure. Paraffin-embedded material was sectioned on the slices 10 mkm thick and SHG-imaging was performed by LSM 510 Meta (Carl Zeiss, Germany). Excitation was implemented with a pulsed (100-fs) titanium-sapphire laser at a wavelength of 800 nm and a pulse repetition frequency of 80 MHz, registration was performed at two wavelengths: 362-415 nm according to collagen fluorescence and 512-576 nm according to myoglobin fluorescence. In a week after irradiation, sings of epithelial damage and edema of submucosal layer, more significant after the dose of 40 Gy were observed on LSM-images. The SHG signal decreased at this time reflecting the processes of collagen degradation independently either in bladder or in rectum. In a month after radiation the increase of size and number of collagen-bearing structures was observed, more essential after irradiation in a dose of 40 Gy. LSM microscopy with SHG allows evaluate changes of normal tissues after ionizing radiation and get information in addition to standard and special histological staining.

We report on multimodal depth-resolved imaging of unstained living Drosophila Melanogaster larva using sub-50 fs pulses centered at 1060 nm wavelength. Both second harmonic and third harmonic generation imaging modalities are demonstrated.

Wound healing is a process to repair the damaged tissue caused by thermal burn, incised wound, or stab wound. Although the wound healing has many aspects, it is common for dynamics of collagen fiber, such as decomposition, production, or growth, to be closely related with wound healing. If such the healing process can be visualized as a timelapse image of the collagen fiber in the same subject, one may obtain new findings regarding biological repairing mechanisms in the healing process. In this article, to investigate the temporal modoification of dermal collagen fiber in the burn wound healing, we used second-harmonic-generation (SHG) microscopy, showing high selectivity and good image contrast to collagen molecules as well as high spatial resolution, optical three-dimensional sectioning, minimal invasiveness, deep penetration, the absence of interference from background light, and in vivo measurement without additional staining. Since SHG light arises from a non-centrosymmetric triple helix of three polypeptide chains in the collagen molecule, SHG intensity sensitively reflects the structure maturity of collagen molecule and its aggregates. A series of time-lapse SHG images during the wound healing process of 2 weeks clearly indicated that condensation and melting of dermal collagen fibers by the deep dermal burn, decomposition of the damaged collagen fibers in the inflammation phase, production of new collagen fibers in the proliferation phase, and the growth of the new collagen fibers in the remodeling phase. These results show a high potential of SHG microscopy for optical assessment of the wound healing process in vivo.

One of the unique features of the brain is that its activity cannot be framed in a single spatio-temporal scale, but rather spans many orders of magnitude both in space and time. A single imaging technique can reveal only a small part of this complex machinery. To obtain a more comprehensive view of brain functionality, complementary approaches should be combined into a correlative framework. Here, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, taking advantage of blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living thy1-GFP-M mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from the apical portion, the whole pyramidal neuron can then be segmented. The correlative approach presented here allows contextualizing within a three-dimensional anatomic framework the neurons whose dynamics have been observed with high detail in vivo.

Routine procedures in standard histopathology involve laborious steps of tissue processing and staining for final examination. New techniques which can bypass these procedures and thus minimize the tissue handling error would be of great clinical value. Coherent anti-Stokes Raman scattering (CARS) microscopy is an attractive tool for label-free biochemical-specific characterization of biological specimen. However, a vast majority of prior works on CARS (or stimulated Raman scattering (SRS)) bioimaging restricted analyses on a narrowband or well-distinctive Raman spectral signatures. Although hyperspectral SRS/CARS imaging has recently emerged as a better solution to access wider-band spectral information in the image, studies mostly focused on a limited spectral range, e.g. CH-stretching vibration of lipids, or non-biological samples. Hyperspectral image information in the congested fingerprint spectrum generally remains untapped for biological samples. In this regard, we further explore ultrabroadband hyperspectral multiplex (HM-CARS) to perform chemoselective histological imaging with the goal of exploring its utility in stain-free clinical histopathology. Using the supercontinuum Stokes, our system can access the CARS spectral window as wide as >2000cm^-1. In order to unravel the congested CARS spectra particularly in the fingerprint region, we first employ a spectral phase-retrieval algorithm based on Kramers–Kronig (KK) transform to minimize the non-resonant background in the CARS spectrum. We then apply principal component analysis (PCA) to identify and map the spatial distribution of different biochemical components in the tissues. We demonstrate chemoselective HM-CARS imaging of a colon tissue section which displays the key cellular structures that correspond well with standard stained-tissue observation.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder currently without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neurites. In an effort to understand the underlying mechanisms, biochemical analysis (protein immunoblot) of plaque extracts reveals that they consist of amyloid-beta (Aβ) peptides assembled as oligomers, protofibrils and aggregates. Their spatial distribution has been confirmed by Thioflavin-S or immuno-staining with fluorescence microscopy. However, it is increasingly understood that the protein aggregation is only one of several mechanism that causes neuronal dysfunction and death. This raises the need for a more complete biochemical analysis. In this study, we have complemented 2-photon fluorescence microscopy of Thioflavin-S and Aβ immuno-stained human AD plaques with CARS microscopy. We show that the chemical build-up of AD plaques is more complex and that Aβ staining does not provide the complete picture of the spatial distribution or the molecular composition of AD plaques. CARS images provide important complementary information to that obtained by fluorescence microscopy, motivating a broader introduction of CARS microscopy in the AD research field.

Nonlinear optical microscopy (e.g., higher harmonic (second-/third- harmonic) generation (HHG), simulated Raman scattering (SRS)) has high diagnostic sensitivity and chemical specificity, making it a promising tool for label-free tissue and cell imaging. In this work, we report a development of a simultaneous SRS and HHG imaging technique for characterization of liver disease in a bile-duct-ligation rat-modal. HHG visualizes collagens formation and reveals the cell morphologic changes associated with liver fibrosis; whereas SRS identifies the distributions of hepatic fat cells formed in steatosis liver tissue. This work shows that the co-registration of SRS and HHG images can be an effective means for label-free diagnosis and characterization of liver steatosis/fibrosis at the cellular and molecular levels.

In this work we describe a device that extends capabilities of multiphoton microscopes based on dual wavelength output femtosecond laser sources. CARS with 17cm^-1 spectral resolution is experimentally demonstrated. Our approach is based on spectral focusing CARS. For pulse shaping of the pump and Stokes beams we utilize transmission gratings based stretcher. It allows the dispersion of the stretcher to be continuously adjusted in wide range. The best spectral resolution is achieved when the chirp rates in both pump and Stokes beam are matched. The device is automated. Any change in the beam path lengths due to the stretcher adjustment or wavelength tuning is compensated by the delay line. We incorporated into the device a computer controlled beam pointing stabilization system that compensates the beam pointing deviation due to dispersion in the system. High level of automation and computer control makes the operation of the device easy. We present CARS images of several samples that demonstrate high spectral resolution, high contrast and chemical selectivity.

Label-free multiphoton imaging is promising for replacing biopsy and could offer new strategies for intraoperative or surgical applications. Coherent anti-Stokes Raman scattering (CARS) imaging could provide lipid-band contrast, and second harmonic generation (SHG) imaging is useful for imaging collagen, tendon and muscle fibers. A combination of these two imaging modalities could provide rich information and this combination has been studied by researchers to investigate diseases through microscopy imaging. The combination of these two imaging modalities in endomicroscopy imaging has been rarely investigated. In this research, a fiber bundle consisted of one excitation fiber and 18 collection fibers was developed in our endomicroscopy prototype. The 18 collection fibers were divided into two collection channels with 9 fibers in each channel. These two channels could be used together as one channel for effective signal collection or used separately for simplifying detection part of the system. Differences of collection pattern of these two channels were investigated. Collection difference of central excitation fiber and surrounding 18 fibers was also investigated, which reveals the potential ability of this system to measure forward to backward (F/B) ratio in SHG imaging. CARS imaging of mouse adipocyte and SHG imaging of mouse tail tendon were performed to demonstrate the CARS and SHG tissue imaging performance of this system. Simultaneous CARS and SHG imaging ability of this system was demonstrated by mouse tail imaging. This fiber bundle based endomicroscopy imaging prototype, offers a promising platform for constructing efficient fiber-based CARS and SHG multimodal endomicroscopes for label free intraoperative imaging applications.

A compact, alignment-free, and inexpensive fiber source for coherent Raman spectroscopy would benefit the field considerably. We present a fiber optical parametric oscillator offering the best performance from a fiber-source to date. Pumping the oscillator with amplified pulses from a 1 μm fiber laser, we achieve widely spaced, narrowband pulses suitable for coherent anti-Stokes Raman scattering microscopy. The nearly transform limited, 2 ps signal pulses are generated through the use of normal dispersion four wave mixing in photonic crystal fiber, and can be tuned from 779-808 nm, limited by the tuning range of the seed laser. The average signal power can reach 180 mW (pulse energies up to 4 nJ). The long-wavelength idler field is resonant in the oscillator, and the use of a narrow bandpass filter in the feedback loop is critical for stable operation, as seen in both simulation and experiment. Due to the self-consistent nature of the oscillator, this source provides lower relative intensity noise on its output pulses than parametric amplifiers based on the same frequency conversion process. We present high quality images of mouse tissues taken with this source that exhibit an outstanding signal to noise ratio at top imaging speeds.

In narrow-bandwidth coherent Raman scattering (CRS) microscopy, efficient signal generation is accomplished with two-color laser sources providing synchronized picosecond pulses whose frequency difference and spectral widths match the molecular Raman frequency and bandwidth, respectively. With vibrational bandwidths of typically 10 cm^-1, the optimum laser pulse durations thus correspond to about 2 ps. Here, we present a new light source consisting of an amplified Yb-fiber oscillator providing 2-ps pulses at 1031 nm and a synchronously green-pumped optical parametric oscillator (OPO). The OPO slightly shortens the pulses to < 2 ps while maintaining a bandwidth of 10 cm^-1. Output power levels of 1 W in both the 1031-nm and the OPO-branch with continuously tunable frequency differences between the two beams covering a broad range from 700 to 4500 cm^-1 are achieved. In addition to CARS microscopy, this light source allows for SRS imaging via an integrated electro-optical modulation of the 1031-nm beam at 20 MHz with a depth of >95%, locked to the laser repetition rate of 80 MHz. The OPO noise at 20 MHz was found to be only 60% above the combined detector and laser noise of a conventional Nd:YVO pump source. This represents a significant reduction in laser noise when compared to other fiber-based laser sources previously proposed for SRS microscopy. When SRS imaging with this new light is compared with a Nd:YVO pumped OPO (delivering 7 ps and 5 ps pulses, respectively), a 5- to 6-fold increase in SRS signal strength and signal-to-noise ratio has been achieved. Video-rate SRS and the capability of multi-spectral SRS imaging are demonstrated.

Coherent Raman scattering provides chemical imaging by using molecular vibrational information sensitive to molecular structure. To add another information of martial symmetry, we propose using fourth order coherent Raman scattering for imaging, because the even order nonlinear phenomenon is forbidden for centro-symmetric material. We have developed a multiplex fourth order coherent Raman scattering microscopy system using a femtosecond laser. A narrowband beam of 17 cm^-1 bandwidth and a broadband beam generated by a photonic crystal fiber enables to obtain a spectrum of fourth order coherent Raman scattering at once. We demonstrate the fourth order coherent Raman, hyper-Raman and second harmonics of trans-4'-(dimethylamino)-N-methyl-4- stilbazolium tosylate crystal by using the developed microscope.

We applied multiphoton microscopic imaging to observe freezing and heating effects in plant- and animal cell samples. The experimental setups consisted of a multiphoton imaging system and a heating and cooling stage which allows for precise temperature control from liquid nitrogen temperature (-196°C; 77 K) up to +600°C (873 K) with heating/freezing rates between 0.01 K/min and 150 K/min. Two multiphoton imaging systems were used: a system based on a modified optical microscope and a flexible mobile system. To illustrate the imaging capabilities, plant leafs as well as animal cells were microscopically imaged in vivo during freezing based on autofluorescence lifetime and intensity of intrinsic molecules. The measurements illustrate the usefulness of multiphoton imaging to investigate freezing effects on animal and plant cells.

A new, simple technique for single molecule fluorescence detection has been developed and detection limit of less than 100 aM fluorophores has been demonstrated. The technique, similarly to Fluorescence Correlation Spectroscopy (FCS) and other related techniques, uses confocal optics, but differs in that it detects individual molecules crossing the inside of a scanning confocal volume without using statistical techniques as applied in FCS or similar methods. The scanning speed of the confocal volume is higher than the Brownian motion speed of the molecules. Thus, the time evolution of the light intensity data reflects the confocal volume intensity profile, which clearly shows the crossing of single molecules. The estimated total scanning volume enables the concentration or the density of molecules to be obtained. In addition, information related to the rotational and translational diffusion of the molecule was obtained for the purpose of identifying different molecules. It was shown that utilizing the plural characteristic properties of molecules passing through a confocal volume makes possible the discrimination of different molecules. The proposed technique is based on the simple principle of counting molecules one by one using a scanning confocal volume, and is hereafter referred to as Scanning Single-Molecule Counting (SSMC).

The multiphoton FLIM tomograph MPTflex with its flexible scan head, articulated arm, and the tunable femtosecond laser source was employed to study cell monolayers and 3D cell clusters. FLIM was performed with 250 ps temporal resolution and submicron special resolution using time-correlated single photon counting. The autofluorescence based on NAD(P)H and flavins/flavoproteins has been measured in mouse embryonic fibroblasts, induced pluripotent stem cells (iPS cells) originated from mouse embryonic fibroblasts and non-proliferative mouse embryonic fibroblasts.

Subunit ε is an intrinsic regulator of the bacterial F_oF₁-ATP synthase, the ubiquitous membrane-embedded enzyme that utilizes a proton motive force in most organisms to synthesize adenosine triphosphate (ATP). The C-terminal domain of ε can extend into the central cavity formed by the α and β subunits, as revealed by the recent X-ray structure of the F₁ portion of the Escherichia coli enzyme. This insertion blocks the rotation of the central γ subunit and, thereby, prevents wasteful ATP hydrolysis. Here we aim to develop an experimental system that can reveal conditions under which ε inhibits the holoenzyme F_oF₁-ATP synthase in vitro. Labeling the C-terminal domain of ε and the γ subunit specifically with two different fluorophores for single-molecule Förster resonance energy transfer (smFRET) allowed monitoring of the conformation of ε in the reconstituted enzyme in real time. New mutants were made for future three-color smFRET experiments to unravel the details of regulatory conformational changes in ε.

TCSPC (Time-Correlated Single-Photon Counting) FLIM data with megapixel resolution can be recorded by using bh TCSPC modules in combination with new 64 bit data acquisition software. The large memory space available in the 64 bit environment allows new FLIM procedures to be used. We demonstrate the performance for applications that require imaging of a large number of cells in a single field of view, for multi-wavelength FLIM, for spatial mosaic imaging, and for recording transient changes in the fluorescence decay after a stimulation of the sample. Image quality was further improved by integrating a parallel counter channel that bypasses the timing electronics of the TCSPC module. Photon numbers from this counter are not affected by dead-time effects. Lifetime images are built up by using intensity data from the parallel counter and fluorescence decay data from the TCSPC electronics.

The heterogeneity of genotypes and phenotypes within cancers is correlated with disease progression and drug-resistant cellular sub-populations. Therefore, robust techniques capable of probing majority and minority cell populations are important both for cancer diagnostics and therapy monitoring. Herein, we present a modified CellProfiler routine to isolate cytoplasmic fluorescence signal on a single cell level from high resolution auto-fluorescence microscopic images.

Cell-to-cell contacts are crucial for cell differentiation. The promyogenic cell surface protein, Cdo, functions as a component of multiprotein clusters to mediate cell adhesion signaling. Connexin43, the main connexin forming gap junctions, also plays a key role in myogenesis. At least part of its effects are independent of the intercellular channel function, but the mechanisms underlying are unknown. Here, using multiple optical approaches, we provided the first evidence that Cx43 physically interacts with Cdo to form dynamic complexes during myoblast differentiation, offering clues for considering this interaction a structural basis of the channel-independent function of Cx43.

We propose a method for high precision modulation of the pupil function of a microscope objective lens to improve the performance of multifocal multi-photon microscopy (MMM). To modulate the pupil function, we adopt a spatial light modulator (SLM) and place it at the conjugate position of the objective lens. The SLM can generate an arbitrary number of spots to excite the multiple fluorescence spots (MFS) at the desired positions and intensities by applying an appropriate computer-generated hologram (CGH). This flexibility allows us to control the MFS according to the photobleaching level of a fluorescent protein and phototoxicity of a specimen. However, when a large number of excitation spots are generated, the intensity distribution of the MFS is significantly different from the one originally designed due to misalignment of the optical setup and characteristics of the SLM. As a result, the image of a specimen obtained using laser scanning for the MFS has block noise segments because the SLM could not generate a uniform MFS. To improve the intensity distribution of the MFS, we adaptively redesigned the CGH based on the observed MFS. We experimentally demonstrate an improvement in the uniformity of a 10 × 10 MFS grid using a dye solution. The simplicity of the proposed method will allow it to be applied for calibration of MMM before observing living tissue. After the MMM calibration, we performed laser scanning with two-photon excitation to observe a real specimen without detecting block noise segments.

We report on a flexible nonlinear medical tomograph with multiple miniaturized detectors for simultaneous acquisition of two-photon autofluorescence (AF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) images. The simultaneous visualization of the distribution of endogenous fluorophores NAD(P)H, melanin and elastin, SHG-active collagen and as well as non-fluorescent lipids within human skin in vivo is possible. Furthermore, fluorescence lifetime images (FLIM) can be generated using time-correlated single photon counting.

Fluorescence lifetime imaging microscopy (FLIM) can reveal important biological information and recently stimulated emission (SE) has been applied in FLIM to improve the spatial resolution of micrographs and detect fluorophore over a long working distance. An issue with SE is that the SE signal is much weaker than the probe laser beam that is used to generate the SE, therefore the signal to background ratio is low. Here we demonstrate using interferometric setup to decrease this background laser intensity, thus achieving higher S/N ratio and dye concentration detection sensitivity in SE microscopy.

Multiphoton microscopy was employed to study normal skin wound healing in live rats noninvasively. Wound healing is a process involving series of biochemical events. This study evaluates the regeneration of collagen and change in cellular metabolic activity during wound healing in rats, with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), respectively. In eukaryotic cells ATP is the molecule that holds the energy for cellular functioning. Whereas NADH is an electron donor in the metabolic pathways, required to generate ATP. Fluorescence lifetime of NADH free to protein bound ratio was evaluated to determine the relative metabolic activity. The FLIM data were acquired by a TCSPC system using SPCM software and analyzed by SPCImage software. Additionally, polarization resolved SHG signals were also collected to observe the changes in optical birefringence and hence the anisotropy of regenerated collagens from rat wound biopsy samples. Mat lab programming was used to process the data to construct the anisotropy images. Results indicated that, cells involved in healing had higher metabolic activity during the first week of healing, which decreases gradually and become equivalent to normal skin upon healing completes. A net degradation of collagen during the inflammatory phase and net regeneration starting from day 5 were observed in terms of SHG signal intensity change. Polarization resolved SHG imaging of the wound biopsy sample indicates higher value of anisotropy in proliferative phase, from day 4th to 8th, of wound formation; however the anisotropy decreases upon healing.

We report on measurements and characterization of polarization properties of Second Harmonic (SH) signals using a four-channel photon counting based Stokes polarimeter from type I collagen and starch granules. In this way, the critical polarization parameters including the degree of polarization (DOP), the degree of linear polarization (DOLP), and the degree of circular polarization (DOCP), are extracted from the reconstructed Stokes vector based SH images in a pixel-by-pixel manner. The measurements are further extended to determine the molecular structure and orientation of the samples by varying the polarization states of the incident light and recording the resulting Stokes parameters of the SH signal. The combination of SHG microscopy and Stokes polarimeter hence makes a powerful tool to investigate the structural order of starch granules under water and heating environment.

Non-invasive fluorescence retinal imaging in small animals is an important requirement in an array of translational vision applications. Two-photon imaging has the potential for long-term investigation of healthy and diseased retinal function and structure in vivo. Here, we demonstrate that two-photon microscopy through a mouse’s pupil can yield high-quality optically sectioned fundus images. By remotely scanning using an electronically tunable lens we acquire highly-resolved 3D fluorescein angiograms. These results provide an important step towards various applications that will benefit from the use of infrared light, including functional imaging of retinal responses to light stimulation.

Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer.

We report on the development of a compact multiphoton microscopy (MPM) system based on a frequency-doubled, femtosecond erbium-doped fiber laser source at 1.58 μm. By use of periodically poled MgO:LiNbO₃, frequency-doubled pulses at 790 nm with average power of 75 mW and pulse width of 130 fs are applied as the excitation source. The fiber laser is optimized for its parameters along with the dispersive properties of the delivery fiber such that the MPM signal is maximized at the sample location. Micro-electro-mechanical system (MEMS) scanner, miniature objective, and multimode fiber are further used to make the MPM system compact. MPM images are obtained from unstained biological samples. The MPM system with a compact, portable, low-cost fiber laser has a great potential to transform the bench-top MPM system to a portable system for in vivo MPM imaging.

We report two methods for quantitatively determining maximal imaging depth from thick tissue images captured using all–near-infrared (NIR) multiphoton microscopy (MPM). All-NIR MPM is performed using 1550 nm laser excitation with NIR detection. This method enables imaging more than five-fold deep in thick tissues in comparison with other NIR excitation microscopy methods. In this study, we show a correlation between the multiphoton signal along the depth of tissue samples and the shape of the corresponding empirical probability density function (pdf) of the photon counts. Histograms from this analysis become increasingly symmetric with the imaging depth. This distribution transitions toward the background distribution at higher imaging depths. Inspired by these observations, we propose two independent methods based on which one can automatically determine maximal imaging depth in the all-NIR MPM images of thick tissues. At this point, the signal strength is expected to be weak and similar to the background. The first method suggests the maximal imaging depth corresponds to the deepest image plane where the ratio between the mean and median of the empirical photon-count pdf is outside the vicinity of 1. The second method suggests the maximal imaging depth corresponds to the deepest image plane where the squared distance between the empirical photon-count mean obtained from the object and the mean obtained from the background is greater than a threshold. We demonstrate the application of these methods in all-NIR MPM images of mouse kidney tissues to study maximal depth penetration in such tissues.

The purpose of this study is the assembly and characterization of a custom-made non-linear microscope. The microscope allows the adjustment for in vitro, in vivo and ex vivo imaging of biological samples. Two galvanometer mirrors conjugated by two spherical mirrors are used for the lateral scan and for the axial scan a piezoeletric stage is utilized. The excitation is done using a tunable femtosecond Ti: Sapphire laser. The light is focused in tissue by an objective lens 20X, water immersion, numerical aperture of 1.0, and working distance of 2.0 mm. The detection system is composed by a cut off filter that eliminates laser light back reflections and diverse dichroic filters can be chosen to split the emitted signal for the two photomultiplier detector. The calibration and resolution of the microscope was done using a stage micrometer with 10 μm divisions and fluorescent particle slide, respectively. Fluorescence and second harmonic generation images were performed using epithelial and hepatic tissue, the images have a sub-cellular spatial resolution. Further characterization and differentiation of tissue layers can be obtained by performing axial scanning. By means of the microscope it is possible to have a three dimensional reconstruction of tissues with sub-cellular resolution.

In this study we present a novel image analysis methodology to quantify and to classify morphological details in tissue collagen fibril organization and lipid deposition. Co-localized collagen (second harmonic, SHG) and lipid (coherent Raman, CARS) images of atherosclerotic artery walls were acquired by a supercontinuum-powered multi-modal nonlinear microscope. Textural features based on the first-order statistics (FOS) and gray level co-occurrence matrix (GLCM) parameters were extracted from the SHG and CARS images. Multi-group classifications based on support vector machine of SHG and CARS images were subsequently performed to investigate the potential of texture analysis in providing quantitative descriptors of structural and compositional changes during disease progression. Using a rabbit model, different collagen remodeling and lipid accumulation patterns in disease tissues can be successfully tracked using these image statistics, thus providing a robust foundation for classification. When the variation of the CARS image features were tracked against the age of the rabbit, it was noticed that older animals (advanced plaques) present a more complex necrotic core containing high-lipid extracellular structures with various shapes and distribution. With combined FOS and GLCM texture statistics, we achieved reliable classification of SHG and CARS images acquired from atherosclerotic arteries with >90% accuracy, sensitivity and specificity. The proposed image analysis methodology can also be applied in a wide range of applications to evaluate conditions involving collagen re-modeling and prominent lipid accumulation.

The feasibility of using reflective surfaces to increase the detection of SHG signals from corneal histological sections in the backward direction was attempted. Three reflective surfaces were tested: aluminum foil, a silver mirror, and a dielectric mirror with specific high reflectivity around 400 nm. To compare the SHG signal detected with and without reflective surfaces, the translation caused by the bending of the sample due to the extra weight on top of the microscope slide was determined. All reflective surfaces resulted in an increase of the detection of SHG signals. Increases of 4%, 11%, and 16% were observed for aluminum foil, dielectric mirror and silver mirror, respectively. A method for increased detection of backward SHG signals when forward detection is not possible was herein demonstrated.

Previous research has shown that the stepwise multi-photon activated fluorescence (SMPAF) of melanin, activated by a continuous-wave (CW) mode near infrared (NIR) laser, is a low cost and reliable method of detecting melanin. SMPAF images of melanin in a mouse hair and a formalin fixed mouse melanoma were compared with conventional multiphoton fluorescence microscopy (MPFM) images and confocal reflectance microscopy (CRM) images, all of which were acquired at an excitation wavelength of 920 nm, to further prove the effectiveness of SMPAF in detecting melanin. SMPAF images add specificity for melanin detection to MPFM images and CRM images. Melanin SMPAF can be a promising technology to enable melanoma imaging for dermatologists.

Basal cell carcinoma (BCC) is the most common type of human skin cancer. The traditional diagnostic procedure of BCC is histological examination with haematoxylin and eosin staining of the tissue biopsy. In order to reduce complexity of the diagnosis procedure, a number of noninvasive optical methods have been applied in skin examination, for example, multiphoton tomography (MPT) and fluorescence lifetime imaging microscopy (FLIM). In this study, we explored two-photon optical tomography of human skin specimens using two-photon excited autofluorescence imaging and FLIM. There are a number of naturally endogenous fluorophores in skin sample, such as keratin, melanin, collagen, elastin, flavin and porphyrin. Confocal microscopy was used to obtain structures of the sample. Properties of epidermic and cancer cells were characterized by fluorescence emission spectra, as well as fluorescence lifetime imaging. Our results show that two-photon autofluorescence lifetime imaging can provide accurate optical biopsies with subcellular resolution and is potentially a quantitative optical diagnostic method in skin cancer diagnosis.

A broad-bandwidth oscillator and pulse shaper with a compensation phase mask applied produce sub-10 fs laser pulses is used to induce selective two-photon excitation in the 380 to 500 nm range. The output is split into two arms with different second order dispersion (SOD). The recombined beams create a train of pulses with phase-shape switching at a rate of 162 MHz. Each pulse induces selective TPEF on the sample at wavelengths determined by the amount of SOD in the beam, which tunes the selective TPEF wavelength. Fluorescence is detected by a single fast photomultiplier tube (PMT) detector; therefore, signal from the PMT detector contains fluorescence signals from two different selectivelyexcited fluorophores. The two separate signals are isolated by quadrature detection using a lock-in amplifier. Images are obtained from the two different fluorophores simultaneously at 81MHz. The wide tunability of the two-photon excitation wavelength, fast switching rate between the selective excitation, and low photodamage (due to low power of laser beam) enables potential application of this method for in vivo dynamic imaging in biological samples

A custom-built intrinsic flow-through dissolution setup was developed and incorporated into a home-built CARS microscope consisting of a synchronously pumped optical parametric oscillator (OPO) and an inverted microscope with a 20X/0.5NA objective. CARS dissolution images (512×512 pixels) were collected every 1.12s for the duration of the dissolution experiment. Hyperspectral CARS images were obtained pre- and postdissolution by rapidly imaging while sweeping the wavelength of the OPO in discrete steps so that each frame in the data stack corresponds to a vibrational frequency. An image-processing routine projects this hyperspectral data into a single image wherein each compound appears with a unique color. Dissolution was conducted using theophylline and cimetidine-naproxen co-amorphous mixture. After 15 minutes of theophylline dissolution, hyperspectral imaging showed a conversion of theophylline anhydrate to the monohydrate, confirmed by a peak shift in the CARS spectra. CARS dissolution images showed that monohydrate crystal growth began immediately and reached a maximum with complete surface coverage at about 300s. This result correlated with the UV dissolution data where surface crystal growth on theophylline compacts resulted in a rapidly reducing dissolution rate during the first 300s. Co-amorphous cimetidinenaproxen didn’t appear to crystallize during dissolution. We observed solid-state conversions on the compact’s surface in situ during dissolution. Hyperspectral CARS imaging allowed visual discrimination between the solid-state forms on the compact’s surface. In the case of theophylline we were able to correlate the solid-state change with a change in dissolution rate.

Multiphoton fluorescence microscopy (MPM) is a method for high resolution, non-invasive investigations of biological tissue. The aim of introducing an annular shaped laser beam is to reduce the ouf-of-focus generated background signal improving imaging of light scattering tissue such as human skin. Simulations show that 50% of the beam radius can be blocked, while preserving the shape of the point spread function. Initial experiments performed on a phantom consisting of fluorescein and fluorescent beads embedded in agar by using a custom built MPM-set up show that by introducing a simple beam blocker to create an annular beam, the background signal is reduced with approximately 5%. Future work will include optimizing the set up, and creating phantoms with more light scattering properties.

We present the development of a fluorescence lifetime imaging microscopy system using a streak camera (SC-FLIM), which uses ultrafast infrared laser for multiphoton excitation and a streak camera for lifetime measurement. A pair of galvo mirrors are employed to accomplish quick time-resolved scanning on a line and 2D fluorescence lifetime imaging. The SC-FLIM system was calibrated using an F-P etalon and several standard fluorescent dyes, and was also used to perform fluorescence lifetime imaging of fluorescent microspheres and a prepared plant stem slide.

We report the implementation of a unique multimodal nonlinear optical microscopy (i.e., coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), third harmonic generation (THG) and two photon excitation fluorescence (TPEF)) platform for label-free imaging of dentin. A picosecond tunable laser together with an OPO is used as the excitation source for simultaneously multimodal imaging. CARS shows similar information as TPEF in dentin, but it has a higher sectioning performance than TPEF and thus it is a good alternative for TPEF. Microtubule structure is revealed nearby dentin enamel junction (DEJ) from the multimodal images. This work demonstrates that combining different nonlinear optical imaging modalities can provide new insights into the understanding of morphological structures and biochemical/biomolecular distributions of the dentine without the need of labeling.

We have developed a new method for quantitative broadband CARS spectral imaging, which uses passive polarisation optics combined with spectrally-resolved balanced homodyne detection to remove the non-resonant background (NRB) in a single exposure. Independent measurement of the Stokes spectrum is not required for NRB removal, and the resulting spectra are amplified by the non-resonant response, vary linearly with concentration, and can be directly related to polarized spontaneous Raman spectra. The technique has relaxed requirements on spectral phase and instrument stability, is suitable for any laser system capable of generating CARS, and has been successfully applied to rapid hyperspectral Raman imaging.

A confocal multiphoton microscopy system with various detection pinholes was used to differentiate backward scattered second harmonic generation (BS-SHG) from backward generated SHG (BG-SHG) based on the fact that BS-SHG is more scattered and therefore has a much bigger spot size than BG-SHG. BS-SHG is quantified from two types of mouse tissues, such as Achilles tendon, and skin, and at various focal depths. It is found that the BS-SHG contributes less to the total backward SHG for the skin than Achilles tendon with thicknesses of around three hundred micrometers. For tissue with larger F/B intensity ratio such as Achilles tendon, increasing the tissue thickness reduces it tremendously. However, for tissue with smaller F/B intensity ratio, tissue thickness increment does not alter it significantly. In addition, larger F/B intensity ratio might be related with a greater scattering coefficient from our Achilles tendon and skin comparison. When the focal point is moved deeper into tissue, the contribution of BS-SHG is found to decrease due to a reduced pass length of the forward propagated photons. On the contrary, when the tissue thickness increases, the contribution of the BS-SHG is increased. These observations for thicker skin tissues are related with our F/B intensity ratio measurement for thin mouse skin sample in terms of that the magnitude of backward generated SHG are dominant among the total backward SHG in mouse skin tissue. Considering the phase mismatching condition in the forward and backward directions, these results may indicate that quasi-phase matching originating from the regular structure of collagen could help with reducing the phase mismatch especially in the backward direction.

Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al. in our two photon excitation fluorescence (TPEF) and SHG hybrid microscope. The system dramatically reduced photodamage to neonatal cardiomyocytes in early stages of culture, greatly increasing cell viability. Thus continuous imaging of live cardiomyocytes was achieved with a stronger laser and for a longer period than has been reported in the literature. The pulse splitter-based TPEF-SHG microscope constructed in this study was demonstrated to be an ideal imaging system for sarcomeric addition-related investigations of neonatal cardiomyocytes in early stages of culture.