This PDF file contains the front matter associated with SPIE Proceedings Volume 10069 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

This talk will focus on design and applications of nanomaterials exhibiting strong multiphoton upconversion for multiphoton microscopy as well as for image-guided and light activated therapy .1-3 Such processes can occur by truly nonlinear optical interactions proceeding through virtual intermediate states or by stepwise coupled linear excitations through real intermediate states. Multiphoton processes in biocompatible multifunctional nanoparticles allow for 3D deep tissue imaging. In addition, they can produce in-situ photon conversion of deep tissue penetrating near IR light into a needed shorter wavelength light for photo-activated therapy at a targeted site, thus overcoming the limited penetration of UV or visible light into biological media. We are using near IR emitters such as silicon quantum dots which also exhibit strong multiphoton excitation for multiphoton microscopy. Another approach involves nonlinear nanocrystals such as ZnO which can produce four wave mixing, sum frequency generation as well as second harmonic generation to convert a deep tissue penetrating Near IR light at the targeted biological site to a desired shorter wavelength light suitable for bio imaging or activation of a therapy. We have utilized this approach to activate a photosensitizer for photodynamic therapy. Yet another type of upconversion materials is rare-earth ion doped optical nanotransformers which transform a Near IR (NIR) light from an external source by sequential single photon absorption, in situ and on demand, to a needed wavelength. Applications of these nanotransformers in multiphoton photoacoustic imaging will also be presented. An exciting direction pursued by us using these multiphoton nanoparticles, is functional imaging of brain. Simultaneously, they can effect optogenetics for regioselective stimulation of neurons for providing an effective intervention/augmentation strategy to enhance the cognitive state and lead to a foundation for futuristic vision of super human capabilities. Challenges and opportunities will be discussed.

Harmonic generation microscopy is a noninvasive repetitive imaging technique that provides real-time 3D microscopic images of human skin with a sub-femtoliter resolution and high penetration down to the reticular dermis. In this talk, we show that with a strong resonance effect, the third-harmonic-generation (THG) modality provides enhanced contrast on melanin and allows not only differential diagnosis of various pigmented skin lesions but also quantitative imaging for longterm tracking. This unique capability makes THG microscopy the only label-free technique capable of identifying the active melanocytes in human skin and to image their different dendriticity patterns. In this talk, we will review our recent efforts to in vivo image melanin distribution and quantitatively diagnose pigmented skin lesions using label-free harmonic generation biopsy. This talk will first cover the spectroscopic study on the melanin enhanced THG effect in human cells and the calibration strategy inside human skin for quantitative imaging. We will then review our recent clinical trials including: differential diagnosis capability study on pigmented skin tumors; as well as quantitative virtual biopsy study on pre- and post- treatment evaluation on melasma and solar lentigo. Our study indicates the unmatched capability of harmonic generation microscopy to perform virtual biopsy for noninvasive histopathological diagnosis of various pigmented skin tumors, as well as its unsurpassed capability to noninvasively reveal the pathological origin of different hyperpigmentary diseases on human face as well as to monitor the efficacy of laser depigmentation treatments. This work is sponsored by National Health Research Institutes.

Multiphoton tomography (MPT) is a novel label-free clinical imaging method for non-invasive tissue imaging with high spatial (300 nm) and temporal (100 ps) resolutions. In vivo optical histology can be realized due to the nonlinear excitation of endogenous fluorophores and second-harmonic generation (SHG) of collagen. Furthermore, optical metabolic imaging (OMI) is performed by two-photon autofluorescence lifetime imaging (FLIM). So far, applications of the multiphoton tomographs DermaInspect and MPTflex were limited to dermatology. Novel applications include intraoperative brain tumor imaging as well as cornea imaging. In this work we describe two-photon imaging of ex vivo human corneas unsuitable for transplantation. Furthermore, the cross-linking (CXL) process of corneal collagen based on UVA exposure and 0.1 % riboflavin was studied. The pharmacokinetics of the photosensitizer could be detected with high spatial resolution. Interestingly, an increase in the stromal autofluorescence intensity and modifications of the autofluorescence lifetimes were observed in the human corneal samples within a few days following CXL.

Remodeling of the extracellular matrix in human ovarian cancer, can be reflected in increased collagen concentration, changes in alignment and/or up-regulation of different collagen isoforms, including Col III. Using fibrillar gel models, we demonstrate that Col I and Col III can be quantitatively distinguished by 3 distinct SHG polarization specific metrics: i) determination of helical pitch angle via the single axis molecular model, ii) dipole alignment via anisotropy, and iii) chirality via SHG circular dichroism (SHG-CD). These sub-resolution differentiations are possible due to differences in the α helix angles of the two isoforms, which co-mingle in the same fibrils. We also investigated the mechanism of the SHG-CD response and show that unlike conventional CD, it is dominated by electric dipole interactions and is consistent with the two state SHG model. We further applied these 3 polarization resolved analyses to human normal, high risk, benign tumors, and malignant human ovarian tissues. We found that these tissues could all be differentiated by these metrics, where high grade tissues had analogous α-helical pitch angles to the in the Col I/Col III gel model. This confirms literature suggestions based on immunofluorescence and gene expression that Col III is up-regulated in high grade ovarian cancers. The different tissues also displayed differing anisotropies, indicating the fibril assemblies are distinct and likely do not result from remodeling of existing collagen but synthesis of new collagen. Importantly, these SHG polarization methods provide structural information not otherwise possible and can serve as label-free biomarkers for ovarian and other cancers.

Second-harmonic generation (SHG) microscopy is a label-free imaging technique to study collagenous materials in extracellular matrix environment with high resolution and contrast. However, like many other microscopy techniques, the actual spatial resolution achievable by SHG microscopy is reduced by out-of-focus blur and optical aberrations that degrade particularly the amplitude of the detectable higher spatial frequencies. Being a two-photon scattering process, it is challenging to define a point spread function (PSF) for the SHG imaging modality. As a result, in comparison with other two-photon imaging systems like two-photon fluorescence, it is difficult to apply any PSF-engineering techniques to enhance the experimental spatial resolution closer to the diffraction limit. Here, we present a method to improve the spatial resolution in SHG microscopy using an advanced maximum likelihood estimation (AdvMLE) algorithm to recover the otherwise degraded higher spatial frequencies in an SHG image. Through adaptation and iteration, the AdvMLE algorithm calculates an improved PSF for an SHG image and enhances the spatial resolution by decreasing the full-width-at-halfmaximum (FWHM) by ~20%. Similar results are consistently observed for biological tissues with varying SHG sources, such as gold nanoparticles and collagen in porcine feet tendons. By obtaining an experimental transverse spatial resolution of ~400 nm, we show that the AdvMLE algorithm brings the practical spatial resolution closer to the theoretical diffraction limit. Our approach is suitable for adaptation in micro-nano CT and MRI imaging, which has the potential to impact diagnosis and treatment of human diseases.

Tendon rupture is a trauma that is difficult to fully recover from. Therefore, non-destructive and non-invasive evaluation method for the tendon healing is strongly required. In this study, we performed the orientation analysis of collagen fiber in healing tendon by two-dimensional Fourier transform (2D-FT) of SHG image. The extracted 2D-FT power spectra imply the correlation with the degree of the tendon healing. These results indicate that SHG microscopy has a unique potential as a non-destructive and non-invasive indicator of tendon healing.

Rett Syndrome (RTT) is a pervasive, X-linked neurodevelopmental disorder that predominantly affects girls. It is mostly caused by a sporadic mutation in the gene encoding methyl CpG-binding protein 2 (MeCP2).The clinical features of RTT are most commonly reported to emerge between the ages of 6-18 months and implicating RTT as a disorder of postnatal development. However, a variety of recent evidence from our lab and others demonstrates that RTT phenotypes are present at the earliest stages of brain development including neurogenesis, migration, and patterning in addition to stages of synaptic and circuit development and plasticity. We have used RTT patient-derived induced pluripotent stem cells to generate 3D human cerebral organoids that can serve as a model for human neurogenesis in vitro. We aim to expand on our existing findings in order to determine aberrancies at individual stages of neurogenesis by performing structural and immunocytochemical staining in isogenic control and MeCP2-deficient organoids. In addition, we aim to use Third Harmonic Generation (THG) microscopy as a label-free, nondestructive 3D tissue visualization method in order to gain a complete understanding of the structural complexity that underlies human neurogenesis. As a proof of concept, we have performed THG imaging in healthy intact human cerebral organoids cleared with SWITCH. We acquired an intrinsic THG signal with the following laser configurations: 400 kHz repetition rate, 65 fs pulse width laser at 1350 nm wavelength. In these THG images, nuclei are clearly delineated and cross sections demonstrate the depth penetration capacity (< 1mm) that extends throughout the organoid. Imaging control and MeCP2-deficient human cerebral organoids in 2D sections reveals structural and protein expression-based alterations that we expect will be clearly elucidated via both THG and three-photon fluorescence microscopy.

SHG microscopy was successfully used to characterize the orientation of sutural lamellae within corneal samples by means of an epi-detection scheme. In particular, the attention was focused on the organization and orientation of corneal collagen lamellae in the first 30 μm of the stromal layer below Bowman’s membrane. In fact, it is thought that the irregular orientation of these specific lamellae, an in particular the existence of so-called sutural lamellae, affects the mechanical properties of the cornea and is responsible of the overall corneal stiffness. The methodology was tested by acquiring image stacks of the central portion of the cornea in ex vivo samples of healthy corneas, keratoconic corneas and keratoconic corneas treated with cross-linking. These samples represent a good benchmark for testing the methodology, considering that keratoconus is an ophthalmic disease in which the cornea loses its stiffness and acquires an abnormal conical shape because of a different organization of sutural lamellae. The samples were first imaged and characterized on the basis of forward/backward SHG ratio, finding a different ratio in keratoconus with respect to both the healthy cornea and cross-linked keratoconus. The inclination of corneal sutural lamellae was then characterized by means of a three-dimensional correlation analysis on SHG images, acquired using a backward detection geometry. Such method provided good discrimination capabilities, demonstrating that this approach can be used not only for diagnosing keratoconus in a very early stage, but also for performing treatment follow-up on cross-linked corneas.

We perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier laser source tunable between λ=1,100 and 1,400 nm. We demonstrate an imaging depth of 1,200 μm in vasculature and 1,160 μm in neurons. We also demonstrate deep-tissue imaging using Indocyanine Green (ICG), which is FDA approved and a promising route to translate multiphoton microscopy to human applications.

Neuroscience applications benefit from recent developments in industrial femtosecond laser technology. New laser sources provide several megawatts of peak power at wavelength of 1040 nm, which enables simultaneous optogenetics photoactivation of tens or even hundreds of neurons using red shifted opsins. Another recent imaging trend is to move towards longer wavelengths, which would enable access to deeper layers of tissue due to lower scattering and lower absorption in the tissue. Femtosecond lasers pumping a non-collinear optical parametric amplifier (NOPA) enable the access to longer wavelengths with high peak powers. High peak powers of >10 MW at 1300 nm and 1700 nm allow effective 3-photon excitation of green and red shifted calcium indicators respectively and access to deeper, sub-cortex layers of the brain. Early results include in vivo detection of spontaneous activity in hippocampus within an intact mouse brain, where neurons express GCaMP6 activated in a 3-photon process at 1320 nm.

Bone marrow transplantation became the standard choice for treatment of many leukemias, tumors and metabolic diseases. Understanding the dynamic behavior of bone marrow niches, especially in case of bone marrow transplantation is critical to improve the efficiency of the treatment. Intravital microscopy was demonstrated to be a powerful tool to study physiological structure of bone marrow niche. However, current method of intravital microscopy has difficulty in longitudinal monitoring the same bone marrow niche site due to the invasion of the prior-imaging surgery. In this study, we introduce a method to improve the bone marrow niche imaging process and enable the longitudinal imaging of murine calvarium bone marrow. Mouse model for calvarium bone marrow imaging was made by attaching cover glass window to the calvarium bone. Longitudinal imaging of whole bone marrow engraftment process was carried out to demonstrate the advantage of our mouse model. Qualitative and quantitative analysis were also executed on the image data. The result provided a dynamic and full visualization of the bone marrow engraftment process. The study was expected to provide helpful tool for bone marrow studies and useful information for bone marrow transplantation in future.

Spontaneous neuronal activity has been measured at cellular resolution in mice, zebrafish, and C. elegans using optical sectioning microscopy techniques, such as light sheet microscopy (LSM) and two photon microscopy (TPM). Recent improvements in these modalities and genetically encoded calcium indicators (GECI’s) have enabled whole brain imaging of calcium dynamics in zebrafish and C. elegans. However, these whole brain microscopy studies have not been extended to mice due to the limited field of view (FOV) of TPM and the cumbersome geometry of LSM. Conventional TPM is restricted to diffraction limited imaging over this small FOV (around 500 x 500 microns) due to the use of high magnification objectives (e.g. 1.0 NA; 20X) and the aberrations introduced by relay optics used in scanning the beam across the sample. To overcome these limitations, we have redesigned the entire optical path of the two photon microscope (scanning optics and objective lens) to support a field of view of Ø7 mm with relatively high spatial resolution (<10 microns). Using optical engineering software Zemax, we designed our system with commercially available optics that minimize astigmatism, field curvature, chromatic focal shift, and vignetting. Performance of the system was also tested experimentally with fluorescent beads in agarose, fixed samples, and in vivo structural imaging. Our large-FOV TPM provides a modality capable of studying distributed brain networks in mice at cellular resolution.

Widely tunable ultrafast lasers have enabled a large number of biological imaging techniques including point scanning multiphoton excited fluorescence (MPEF), SHG/THG and stimulated Raman imaging. Tunable ultrafast lasers offer spectral agility, covering the entire relative transparency window in live tissue (700-1300nnm) and flexibility with multi-color, synchronized outputs to support sophisticated label free techniques (e.g. stimulated Raman modalities). More recently newly available high peak power lasers based on Ytterbium technology drive advances in two-photon light-sheet, 3 photon excited fluorescence and holographic patterning for optogenetics photo-stimulation. These laser platforms offer a unique blend of compactness, ease of use and cost efficiency, and ideally complement tunable platforms typically based on Ti:Sapphire and IR optical parametric oscillators (OPO). We present various types of ultrafast laser architectures, link their optical characteristics to key bio-imaging requirements, and present relevant examples and images illustrating their impact in biological science. In particular we review the use of ultrafast lasers in optogenetics and fast in-vivo Calcium imaging deep in mouse brain.

Due to the subject nature of histopathology, there is a significant inter-observer discordance for the differentiation between low-risk prostate cancer (Gleason score ≤ 6), which can be left without treatment, and high-risk prostate cancer (Gleason score >6), which requires active treatment. Our previous study using Raman spectromicroscopy reveals that cholesteryl ester accumulation underlies human prostate cancer aggressiveness. However, Raman spectromicroscopy could only provide compositional information of certain lipid droplets of interest, which overlooked cell-to-cell variation and hindered translation to accurate automated diagnosis. Here, we demonstrated quantitative mapping of cholesteryl ester molar percentage in human prostate cancer tissues using hyperspectral stimulated Raman scattering microscopy that renders compositional information for every pixel in the image. Specifically, hundreds of SRS images at Raman shift between 2800~3000 cm^-1 were taken, and multivariate curve resolution algorism was used to retrieve concentration images of lipid, lipofuscin, and protein. We found that the height ratio between the prominent cholesterol band at 2870 cm^-1 and the CH₂ stretching band at 2850 cm^-1 was proportional to the molar percentage of cholesteryl ester present in the total lipids. Based on the calibration curve, we were able to quantitatively map cholesteryl ester level in intact prostate cancer tissues. Our data showed that not only the amount of cholesteryl ester-rich lipid droplets, but also the CE molar percentage, was significantly greater in prostate cancer tissues with Gleason score > 6 compared to the ones with Gleason score ≤ 6. Our study offers an opportunity towards more accurate prostate cancer diagnosis.

While melanoma is not the most common form of skin cancer, it represents the vast majority of skin cancer-related deaths. Indeed, while combination therapies such as Dabrafenib and Trametinib have shown great promise in clinical trials for treating metastatic disease, some melanoma subtypes nevertheless develop resistances to front-line treatments. Under in vitro conditions, some metastatic human melanoma cell lines have been observed to evolve resistance to treatment while simultaneously changing color under brightfield microscopy, hinting at perturbations in pigment synthesis. The process known as melanogenesis gives rise to the two forms of melanin found in mammals: eumelanin, a dark brown/black pigment, and pheomelanin, a much more pale red/blond pigment. Interestingly, pheomelanin has been shown to contribute to the onset and development of melanoma in an ultraviolet-radiation-independent manner through a mechanism of oxidative stress. Eumelanin, on the other hand, is a known antioxidant whose chemical properties seem to shield cells against oxidative damage. To study these pigments in closer detail, nonlinear optical microscopy including coherent anti-Stokes Raman scattering (CARS) was used for the specific visualization and quantification of the relative abundance of pheomelanin and eumelanin within these treatment resistant cell lines. These microscopy toolkits provide a means to monitor changes in pigmentation in a noninvasive and non-destructive manner without the use of exogenous dyes to better understand the molecular basis of treatment resistance.

Kinesin-1 is a kind of motor protein responsible for intracellular transportation and has been studied in a variety of tissues. However, its roles in cartilage development are not clear. In this study, a kinesin-1 heavy chain (Kif5b) knockout mouse model is used to study the functions of kinesin-1 in the cartilage development. We developed a multimodal nonlinear optical (NLO) microscope system integrating stimulated Raman scattering (SRS), second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) to investigate the morphological and biomedical characteristics of fresh tibial cartilage from normal and mutant mice at different developmental stages. The combined forward and backward SHG imaging resolved the fine structure of collagen fibrils in the extracellular matrix of cartilage. Meanwhile, the chondrocyte morphology in different zones of cartilage was visualized by label-free SRS and TPEF images. The results show that the fibrillar collagen in the superficial zone of cartilage in postnatal day 10 and 15 (P10 and P15) knockout mice was significantly less than that of control mice. Moreover, we observed distorted morphology and disorganization of columnar arrangement of chondrocytes in the growth plate cartilage of mutant mice. This study reveals the significant roles of kinesin-1 in collagen formation and chondrocyte morphogenesis.

We will report on application of broadband coherent anti-Stokes Raman scattering microscopy1 to chemical mapping and characterization of resected prostate sections. While incidence of prostate cancer is very high, only a small fraction of prostate tumors will progress to advanced, metastatic disease and become dangerous, but prostatectomy and follow-on treatment have many undesirable potential side effects. Thus, it is important to predict which tumors will progress and which should be removed, but there is currently no highly reliable way to make such predictions. We will present a retrospective coherent Raman imaging study resected prostate sections focusing on locating tissue regions that present the highest diagnostic value with respect to lethal vs indolent disease. We intend that this provide a guide to optimal spectral sampling of these tissues to address this important clinical problem.

Stimulated Raman scattering (SRS) microscopy has been used for rapid label-free imaging of various biomolecules and drugs in living cells and tissues (Science, doi:10.1126/science.aaa8870). Our recent work has demonstrated that lipid and protein mapping of cancer tissue renders pathology-like images, providing essential histopathological information with subcellular resolution of the entire specimen (Cancer Research, doi: 10.1158/0008-5472.CAN-16-027). We have also established the first SRS imaging Atlas of human brain tumors (Harvard Dataverse, doi: (doi:10.7910/DVN/EZW4EK). SRS imaging of tissue could provide invaluable information for cancer diagnosis and surgical guidance in two aspects: rapid surgical pathology and quantitative biomolecular characterization. In this work, we present the use of SRS microscopy for characterization of a few essential biomolecules in breast cancer. Human breast cancer tissue specimens at the tumor core, tumor margin and normal area (5 cm away from the tumor) from surgical cases will be imaged with SRS at multiple Raman shifts, including the peaks for lipid, protein, blood (absorption), collagen, microcalcification (calcium phosphates and calcium oxalate) and carotenoids. Most of these Raman shifts have relatively strong Raman cross sections, which ensures high-quality and fast imaging. This proof-of-principle study is sought to demonstrate the feasibility and potential of SRS imaging for ambient diagnosis and surgical guidance of breast cancer.

Candida is the single most important cause of fungal bloodstream infections worldwide causing significant mortality as high as 50%. This high mortality rate is, in part, due to the inability to rapidly diagnose and simultaneously initiate an effective antifungal therapy early in the disease process. Current culture-based diagnostics are often slow, requiring several days to complete, and are only 50% sensitive in diagnosing candidemia (Candida bloodstream infection). For every 12 hours of delay in starting correct antifungal therapy, the risk of death for a given patient with candidemia increases by 200%. To address this unmet need, we explored the potential of employing stimulated Raman Scattering (SRS) imaging to diagnose candidemia and probe metabolic differences between resistant and susceptible strain at a single cell level. Metabolism is integral to pathogenicity; microorganism have very short life cycles, and therefore only a few hours are needed to observe a full metabolic cycle. SRS imaging at C-H vibration frequency at 2850 cm-1 revealed a substantial difference in lipogenesis between the susceptible and resistant C. albicans. Treating the C. albicans with fluconazole, an antimicrobial drug that targets ergosterol biosynthesis only affected the lipogenesis in the susceptible strain. Our results show that single-cell metabolic imaging under a SRS microscope can be used for diagnose candidemia and early detection of antimicrobial susceptibility.

A range of proposed alkyne Raman tags are examined in-silico for activity and then synthesised generating a library of analogues of the natural product, anisomycin. We report the use of bisaryl butadiyne-anisomycin (BADY-anisomycin) in intracellular SRS microscopy studies of uptake and localisation within live and fixed cells. Following rational design and synthesis, BADY-anisomycin was shown to produce an intense Raman band at 2219 cm^-1, that is centrally located within the cellular silent region and is approximately 60 times more Raman active than the corresponding propargylanisomycin. Finally, we demonstrate two-colour imaging utilising EdU, an alkyne-containing proliferation probe and BADY-anisomycin.

We present combined surface-enhanced stimulated Raman scattering (SE-SRS) and surface-enhanced coherent anti-Stokes Raman scattering (SE-CARS) measurements on individual plasmonic antennas dressed with bipyridyl-ethylene molecules. By carefully optimizing the conditions for performing SE-SRS experiments, we have obtained stable and reproducible molecular surface-enhanced SRS spectra from single nano-antennas. Using surface-enhanced Raman scattering (SERS) and transmission electron microscopy of the same antennas, we confirm that the observed SE-SRS signals originate from only one or a few molecules. We highlight the physics of surface enhancement in the context of coherent Raman scattering and derive sensitivity parameters under the relevant conditions. The implications of single molecule SRS measurements are discussed.

We demonstrate a robust, all-fiber, two-wavelength time-lens source for background-free coherent anti-Stokes Raman scattering (CARS) imaging. The time-lens source generates two picosecond pulse trains simultaneously: one at 1064 nm and the other tunable between 1040 nm and 1075 nm (~ 400 mW for each wavelength). When synchronized to a modelocked Ti:Sa laser, the two wavelengths are used to obtain on- and off-resonance CARS images. Real-time subtraction of the nonresonant background in the CARS image is achieved by the synchronization of the pixel clock and the time-lens source. Background-free CARS imaging of sebaceous glands in ex vivo mouse tissue is demonstrated.

Nonlinear optical spectroscopies and microscopies offer a number of advantages compared to their linear counterparts. In a particular example of coherent Raman and Brillouin microscopies, they both afford substantially stronger signals enhanced by coherent excitation and much better sectioning capabilities due to the nonlinear nature of optical interactions. In this report, we demonstrate how both coherent Raman spectroscopy and coherent Brillouin spectroscopy can be combined together for simultaneous assessment of local chemical and physical properties.

We have previously demonstrated a total internal reflection, wide-field CARS microscope, where the signal is enhanced with the aid of a thin gold layer that supports surface plasmon polariton resonances. This surface-enhanced CARS microscope is capable of generating images of lipid structures in close proximity (<100 nm) to the glass substrate at excitation densities that are 4 orders of magnitude lower than in point-scanning CARS microscopy. In this contribution, we demonstrate its application to visualizing lipids in aqueous media, including imaging of cells, with a unique surface-sensitive contrast that cannot be obtained with conventional CARS microscopy.

Stimulated Raman scattering (SRS) microscopy is a promising technique for label-free imaging of living systems. We demonstrate microsecond-scale SRS spectral imaging by tuning two spectrally focused pulses temporally through a resonant delay-line. Our platform acquired an SRS spectrum within 42 microseconds and formed a spectral image composed of 40,000 pixels in real-time.

A hyperspectral image corresponds to a data cube with two spatial dimensions and one spectral dimension. Through linear un-mixing, hyperspectral images can be decomposed into spectral signatures of pure components as well as their concentration maps. Due to this distinct advantage on component identification, hyperspectral imaging becomes a rapidly emerging platform for engineering better medicine and expediting scientific discovery. Among various hyperspectral imaging techniques, hyperspectral stimulated Raman scattering (HSRS) microscopy acquires data in a pixel-by-pixel scanning manner. Nevertheless, current image acquisition speed for HSRS is insufficient to capture the dynamics of freely moving subjects. Instead of reducing the pixel dwell time to achieve speed-up, which would inevitably decrease signal-to-noise ratio (SNR), we propose to reduce the total number of sampled pixels. Location of sampled pixels are carefully engineered with triangular wave Lissajous trajectory. Followed by a model-based image in-painting algorithm, the complete data is recovered for linear unmixing. Simulation results show that by careful selection of trajectory, a fill rate as low as 10% is sufficient to generate accurate linear unmixing results. The proposed framework applies to any hyperspectral beam-scanning imaging platform which demands high acquisition speed.

Stimulated Raman scattering (SRS) is a label-free imaging technique suitable for studying biological systems. Due to stimulated nature by ultrafast laser pulses, SRS microscopy has the advantage of significantly higher sensitivity but often reduced spectroscopic information. In this paper, we present a newly constructed femtosecond SRS microscope with a high-speed dynamic micromirror device based pulse shaper to achieve flexible and rapid frequency selection within the C-H stretch region near 2800 to 3100 cm^-1 with spectral width of 30 cm^-1. This technique is applicable to lipid profiling such as cell activity mapping, lipid distribution mapping and distinction among subclasses.

The capabilities of a Coherent anti-Stokes Raman scattering (CARS) microscopy technique that utilizes a commercially available microstructured-fibre for the generation of a Stokes supercontinuum, largely depend on the spectral characteristics of the fibre itself. Such fibres, however, have notoriously limited spectral powers and wavelength coverage, thus limiting the excitation efficiency of fibre-based CARS. We demonstrate that through the combination of time-gating and systematic power-tuning of the input pulse into a supercontinuum-generating fibre—a process we term “spectral surfing”—we are able to extend and bolster the excitation efficiencies of spectral-focusing CARS hypermicroscopy across the vibrational spectrum from ~300 cm^-1 to >3500 cm^-1.

We report on a technique that records and displays fluorescence lifetime images at a rate of 10 images per second. Data acquisition is based on multi-dimensional TCSPC in combination with confocal or two-photon laser scanning. The image are calculated from the TCSPC FLIM data via the first moment of the decay data in the pixels of the images. The first-moment technique combines near-ideal photon efficiency with calculation times shorter than the frame times of the commonly used galvanometer scanners. The image rate is thus not slowed down by the lifetime calculation process. Potential applications are clinical FLIM, where suspicious areas can be identified for subsequent high-accuracy imaging, and standard FLIM microscopy, where the technique it helps the user select interesting cells in a large field of view for detailed analysis.

To guide the development of targeted therapies with improved efficacy and accelerated clinical acceptance, novel imaging methodologies need to be established. Toward this goal, fluorescence lifetime Förster resonance energy transfer (FLIM-FRET) imaging assays capitalize on the ability of antibodies or protein ligands to bind dimerized membrane bound receptors to measure their target engagement levels in cancer cells. Conventional FLIM FRET microscopy has been widely applied at visible wavelengths to detect protein-protein interactions in vitro. However, operation at these wavelengths restricts imaging quality and ability to quantitate lifetime changes in in vivo small animal optical imaging due to high auto-fluorescence and light scattering. Here, we have analyzed the uptake of iron-bound transferrin (Tf) probes into human breast cancer cells using FLIM-FRET microscopy in the visible and near-infrared (NIR) range. The development of NIR FLIM FRET microscopy allows for the use of quantitative lifetime-based molecular assays to measure drug-target engagement levels at multiple scales: from in vitro microscopy to in vivo small animal optical imaging (macroscopy). This novel approach can be extended to other receptors, currently targeted in oncology. Hence, lifetime-based molecular imaging can find numerous applications in drug delivery and targeted therapy assessment and optimization.

Phosphorescence probes can have significantly long lifetimes, on the order of micro- to milli-seconds or longer. In addition, environmental changes can affect the lifetimes of these phosphorescence probes. Thus, Phosphorescence Lifetime Imaging Microscopy (PLIM) is a very useful tool to localize the phosphorescence probes based on their lifetimes to study the variance in the lifetimes due to the micro environmental changes. Since the probes respond to the biologically relevant parameters like oxygen concentration, they can be used to study various biologically relevant processes like cellular metabolism, protein interaction etc. In this case, we study the effects of oxygen on Oxyphor G4 with PLIM. Since The Oxyphor G4 can be quenched by O₂, it is a good example of such a probe and has a lifetime around 250us. Here we present the digital frequency domain PLIM technique and study the lifetime of the Oxyphor G4 as a function of the O2 concentration. The lifetime data are successfully presented in a phasor plot for various O₂ concentrations and are consistent with the time domain data. Overall, we can analyze the oxygen consumption of varying cells using this technique.

Over the last two decades, time-resolved fluorescence microscopy has become an essential tool in Life Sciences thanks to measurement procedures such as Fluorescence Lifetime Imaging (FLIM), lifetime based Foerster Resonance Energy Transfer (FRET), and Fluorescence (Lifetime) Correlation Spectroscopy (F(L)CS) down to the single molecule level. Today, complete turn-key systems are available either as stand-alone units or as upgrades for confocal laser scanning microscopes (CLSM). Data acquisition on such systems is typically based on Time-Correlated Single Photon Counting (TCSPC) electronics along with picosecond pulsed diode lasers as excitation sources and highly sensitive, single photon counting detectors. Up to now, TCSPC data acquisition is considered a somewhat slow process as a large number of photons per pixel is required for reliable data analysis, making it difficult to use FLIM for following fast FRET processes, such as signal transduction pathways in cells or fast moving sub-cellular structures. We present here a novel and elegant solution to tackle this challenge. Our approach, named rapidFLIM, exploits recent hardware developments such as TCSPC modules with ultra short dead times and hybrid photomultiplier detector assemblies enabling significantly higher detection count rates. Thanks to these improved components, it is possible to achieve much better photon statistics in significantly shorter time spans while being able to perform FLIM imaging for fast processes in a qualitative manner and with high optical resolution. FLIM imaging can now be performed with up to several frames per second making it possible to study fast processes such as protein interactions involved in endosome trafficking.

Pulsed infrared light can excite action potentials in neurons; yet, the fundamental mechanism underlying this phenomenon is unknown. Previous work has observed a rise in intracellular calcium concentration following infrared exposure, but the source of the calcium and mechanism of release is unknown. Here, we used fluorescence lifetime imaging of Oregon Green BAPTA-1 to study intracellular calcium dynamics in primary rat hippocampal neurons in response to infrared light exposure. The fluorescence lifetime of Oregon Green BAPTA-1 is longer when bound to calcium, and allows robust measurement of intracellular free calcium concentrations. First, a fluorescence lifetime calcium calibration curve for Oregon Green BAPTA-1 was determined in solutions. The normalized amplitude of the short and long lifetimes was calibrated to calcium concentration. Then, neurons were incubated in Oregon Green BAPTA-1 and exposed to pulses of infrared light (0-1 J/cm2; 0-5 ms; 1869 nm). Fluorescence lifetime images were acquired prior to, during, and after the infrared exposure. Fluorescence lifetime images, 64x64 pixels, were acquired at 12 or 24 ms for frame rates of 83 and 42 Hz, respectively. Accurate α₁ approximations were achieved in images with low photon counts by computing an α₁ index value from the relative probability of the observed decay events. Results show infrared light exposure increases intracellular calcium in neurons. Altogether, this study demonstrates accurate fluorescence lifetime component analysis from low-photon count data for improved imaging speed.

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of macromolecular complexes and sub-cellular structures with spatial resolution below the diffraction limit. The spatial resolution of STED is limited by power of the depletion laser at the specimen plane. Higher depletion laser power will improve resolution, but at the cost of increased photo-bleaching, photo-toxicity, and anti-stoke emission background. This degrades the signal-to-noise ratio, and can significantly limit STED applications in living specimens. Here, we present an efficient multi-color STED microscopy method based on the digital frequency domain fluorescence lifetime imaging (FastFLIM) and the phasor plots. Our approach utilizes a combination of pulsed excitation and pulsed depletion lasers to record the time-resolved photons by FastFLIM. We demonstrate that the resolution is improved without increasing the depletion laser power by digital separation of the depleted species from the partially depleted species based on their different decay kinetics. We show the utility of this novel STED method applied in both fixed and live cellular samples, and also show its application to fluorescence lifetime correlation spectroscopy (FLCS) measurements. By combining fluorophores with different fluorescence lifetimes, we simultaneously record two-color STED images of cells labeled with Atto655 and Alexa647 in a single scan by using a single pair of excitation and depletion lasers. This novel approach shortens the data acquisition time while minimizing the photo-toxicity caused when using two separate depletion lasers.

Application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of skin diseases. By means of multiphoton excitation, endogenous biomolecules like NADH, collagen or elastin show autofluorescence or second harmonic generation. Thus, these molecules provide information about the subcellular morphology, epidermal architecture and physiological condition of the skin. To gain a deeper understanding of the linkage between cellular structure and physiological processes, non-invasive multiphotonbased intravital tomography (MPT) and fluorescence lifetime imaging (FLIM) were combined within the scopes of inflammatory skin, chronic wounds and drug delivery in clinical application. The optical biopsies generated via MPT were morphologically analyzed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Independent morphometric algorithms reliably showed a perinuclear accumulation in lesional skin in contrast to an even distribution in healthy skin. Confirmatively, MPT-FLIM showed an obvious metabolic shift in lesions. Moreover, detection of the onset and progression of inflammatory processes could be achieved. The feasibility of primary in vivo tracking of applied therapeutic agents further broadened our scope: We examined the permeation and subsequent distribution of agents directly visualized in patients´ skin in short-term repetitive measurements. Furthermore, we performed MPT-FLIM follow-up investigations in the long-term course of therapy. Therefore, clinical MPT-FLIM application offers new insights into the pathophysiology and the individual therapeutic course of skin diseases, facilitating a better understanding of the processes of inflammation and wound healing.

Two-photon excited fluorescence (TPEF) microscopy and fluorescence lifetime imaging (FLIM) are powerful imaging techniques in bio-molecular science. The need for elaborate light sources for TPEF and speed limitations for FLIM, however, hinder an even wider application. We present a way to overcome this limitations by combining a robust and inexpensive fiber laser for nonlinear excitation with a fast analog digitization method for rapid FLIM imaging. The applied sub nanosecond pulsed laser source is synchronized to a high analog bandwidth signal detection for single shot TPEF- and single shot FLIM imaging. The actively modulated pulses at 1064nm from the fiber laser are adjustable from 50ps to 5ns with kW of peak power. At a typically applied pulse lengths and repetition rates, the duty cycle is comparable to typically used femtosecond pulses and thus the peak power is also comparable at same cw-power. Hence, both types of excitation should yield the same number of fluorescence photons per time on average when used for TPEF imaging. However, in the 100ps configuration, a thousand times more fluorescence photons are generated per pulse. In this paper, we now show that the higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to not only use a single pulse per pixel for TPEF imaging but also to resolve the exponential time decay for FLIM. To evaluate the performance of our system, we acquired FLIM images of a Convallaria sample with pixel rates of 1 MHz where the lifetime information is directly measured with a fast real time digitizer. With the presented results, we show that longer pulses in the many-10ps to nanosecond regime can be readily applied for TPEF imaging and enable new imaging modalities like single pulse FLIM.

Fluorescence lifetime imaging microscopy (FLIM) represents a powerful tool for biological studies. Endoscopic FLIM applied to the intracellular native biomarker NADH and FAD represents a promising mean for in vivo in situ malignant tissue diagnosis in the medical field. Else, 2-photon-excited fluorescence (2PEF) provides increased 3D resolution and imaging depth. But very few demonstrations about 2PEF lifetime measurement through a fiber have been reported and none about endoscopic 2P-FLIM through a practical fiber length (< 3m). Our group has recently demonstrated the possibility to efficiently deliver through a very long optical fiber the short and intense excitation pulses required for 2P-FLIM. Our goal is now to check that collecting fluorescence through the same endoscopic fiber does not deteriorate the lifetime measurement. Relying on the basis previously published in case of 1PEF by P. French and co-workers (J. Biophotonics, 2015), we have experimentally quantitatively evaluated the influence on the lifetime measurement of the fiber chromatic and intermodal dispersions. The main result is that the fiber contribution to the system impulse response function, even in the case of a 3-meter long double-clad optical fiber, does not hinder the separation between free and bound NADH states using FLIM. Related calibrations and measurements will be detailed. Ongoing experiments about the development of a 2P-FLIM endomicroscope on the basis of an previously reported 2P-endomicroscope (Ducourthial et al., Sc. Reports, 2015), used under various configurations (i.e. point measurement in the center of the 2P-endomicroscope image, averaged lifetime, binned endoscopic 2P-FLIM image), will be also presented.

Correlated imaging of phosphorescence and fluorescence lifetime parameters of metabolic markers is a challenge for direct investigating mechanisms related to cell metabolism and oxygen tension. A large variety of clinical phenotypes is associated with mitochondrial defects accomplished with changes in cell metabolism. In many cases the hypoxic microenvironment of cancer cells shifts metabolism from oxidative phosphorylation (OXPHOS) to anaerobic or aerobic glycolysis, a process known as “Warburg” effect. Also during stem cell differentiation a switch in cell metabolism is observed. Mitochondrial dysfunction associated with hypoxia has been invoked in many complex disorders such as type 2 diabetes, Alzheimer´s disease, cardiac ischemia/reperfusion injury, tissue inflammation and cancer. Cellular responses to oxygen tension have been studied extensively, optical imaging techniques based on time correlated single photon counting (TCSPC) to detect oxygen concentration and distribution are therefore of prominent interest. Moreover, they offer the possibility by inspecting fluorescence decay characteristics of intrinsic coenzymes to directly image metabolic pathways, whereas oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescent probe. The combination of both fluorescence lifetime imaging (FLIM) of coenzymes like NAD(P)H and FAD and phosphorescence lifetime (PLIM) of phosphorescent dyes could provide valuable information about correlation of metabolic pathways and oxygen tension.

Liver performs xenobiotic excretion out of hepatocytes with metabolic function. However, hepatocellular metabolism was non-uniform in hepatocyte. Hepatocellular metabolism could be different in nucleus and cytoplasm. In this study, we use the molecular probe 6-carboxyfluorescein diacetate (6-CFDA) to simulate xenobiotic metabolism in hepatocytes with multi-photon fluorescence microscopy in vivo. 6-CFDA was processed by intracellular esterase to 6- carboxyfluorescein (6-CF) with green fluorescence. And this probe was used to study differences in cytoplasmic and nuclear metabolism of hepatocytes.

Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities

Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to identify changes in metabolic activity during cancer progression and upon anti-cancer drug treatment. Prostate cancer (PCa) is one of the leading cancers in men in the USA. This research focusses on understanding the lifetime changes of endogenous biomarkers: NAD(P)H, FAD and Trp in LNCaP cells upon treatment with doxorubicin using our 3-channel FLIM approach. The LNCaP cells were treated with doxorubicin for 24hr. Images using FLIM of LNCaP control and treated cells were acquired on Zeiss 780 multiphoton confocal microscope coupled with B and H TCSPC FLIM board. After FLIM data fitting and processing we observed increase in the mean fluorescence lifetime of Trp, NAD(P)H and FAD with doxorubicin treatment. Additionally, we saw reduction in the NAD(P)H/FAD redox ratio with doxorubicin treatment. Our results identify the changes in the lifetime of these endogenous biomarkers and in the cellular redox state as a metabolic response with doxorubicin treatment in prostate cancer cells.

NADH and FAD are important endogenous fluorescent coenzymes participating in key enzymatic reactions of cellular metabolism. While fluorescence intensities of NADH and FAD have been used to determine the redox state of cells and tissues, this simple approach breaks down in the case of deep-tissue intravital imaging due to depth- and wavelength-dependent light absorption and scattering. To circumvent this limitation, our research focuses on fluorescence lifetimes of two-photon excited NADH and FAD emission to study the metabolic state of live tissues. In our custom-built scanning microscope we combine tunable femtosecond Ti:sapphire laser (operating at 740 nm for NADH excitation and 890 nm for FAD excitation), two GaAsP hybrid detectors for registering individual fluorescence photons and two Becker and Hickl time correlator boards for high precision lifetime measurements. Together with our rigorous FLIM analysis approach (including image segmentation, multi-exponential decay fitting and detailed statistical analysis) we are able to detect metabolic changes in cancer xenografts (human pancreatic cancer MPanc96 cells injected subcutaneously into the ear of an immunodeficient nude mouse), relative to surrounding healthy tissue. Advantageously, with the same instrumentation we can also take high-resolution and high-contrast images of second harmonic signal (SHG) originating from collagen fibers of both the healthy skin and the growing tumor. The combination of metabolic measurements (NADH and FAD lifetime) and morphological information (collagen SHG) allows us to follow the tumor growth in live mouse model and the changes in tumor microenvironment.

Multiphoton microscopy is a cutting edge imaging modality leading to increasing advances in biology and also in the clinical field. To use it at its full potential and at the very heart of clinical practice, there have been several developments of fiber-based multiphoton microendoscopes. The application for those probes is now limited by few major restrictions, such as the difficulty to collect autofluorescence signals from tissues and cells theses being inherently weak (e.g. the ones from intracellular NADH or FAD metabolites). This limitation reduces the usefulness of microendoscopy in general, effectively restraining it to morphological imaging modality requiring staining of the tissues. Our aim is to go beyond this limitation, showing for the first time label-free cellular metabolism monitoring, in vivo in situ in real time. The experimental setup is an upgrade of a recently published one (Ducourthial et.al, Scientific Reports, 2016) where femtosecond pulse fiber delivery is further optimized thank’s to a new transmissive-GRISM-based pulse stretcher permitting high energy throughput and wide bandwidth. This device allows fast sequential operation with two different excitation wavelengths for efficient two-photon excited NADH and FAD autofluorescence endoscopic detection (i.e. 860 nm for FAD and 760 nm for NADH), enabling cellular optical redox ratio quantification at 8 frames/s. The obtained results on cell models in vitro and also on animal models in vivo (e.g. neurons of a living mouse) prove that we accurately assess the level of NADH and FAD at subcellular resolution through a 3-meters-long fiber with our miniaturized probe (O.D. =2.2 mm).

Traditional assessments of cellular metabolism are often destructive, time consuming and without visual information. Fluorescence lifetime imaging microscopy (FLIM) provides a highly sensitive, non-invasive, and label-free alternative. This study uses FLIM in combination with two-photon microscopy to investigate pharmacological induced metabolic changes of adipocytes via changes in the fluorescence of the metabolic co-factors NADH and FAD. In agreement with recent publications NADH fluorescence suggests the presence of four distinct lifetimes in cell culture and tissue with two unbound and two protein bound states which show different responses to treatment with metabolic modifiers. We evaluated the effects on NADH fluorescence lifetime after systematic manipulations to change the balance between oxidative and glycolytic metabolism using five pharmacological reagents - Oligomycin, 2-DG, FCCP, Rotenone, and Glucose - which interact with different parts of the metabolic pathway. We established several ratios between the four distinct lifetimes of NADH after treatment and compared the results to oxygen consumption rate and extracellular acidification rate. We demonstrated, for the first time, a correlation between the two unbound fluorescence lifetimes components and glycolytic and oxidative metabolic activity with a significant higher sensitivity compared to the commonly used free-to-bound ratio of NADH. Analyzing all four lifetime components of NADH has the potential to become a powerful tool to evaluate metabolic activity of adipocytes with subcellular resolution.

Current label-free metabolic microscopy techniques are limited to obtaining contrast from fluorescent molecules NAD(P)H and FAD+, and are unable to determine redox state along the mitochondrial respiratory chain itself. The respiratory chain electron carriers do not fluoresce, but some are heme proteins that have redox-dependent absorption spectra. The most prominent of these, cytochrome c, has been extensively characterized by transient absorption spectroscopy, which suggests that pump-probe measurements in the vicinity of 450 - 600 nm can provide strong contrast between its redox states. Motivated by the success of pump-probe microscopy targeting another heme protein, hemoglobin, we seek to extend the technique to the cytochromes, with the ultimate goal of dissecting respiratory chain function of individual cells in live tissue. To that end, we have developed a new optical system producing ultrafast, visible, independently-tunable pulse pairs via sum-frequency generation of nonlinearly broadened pulses in periodically-poled lithium niobate. The system is pumped by a homebuilt fiber-based oscillator/amplifier emitting 1060 nm pulses at 1.3 W (63 MHz repetition rate), and produces tunable pulses in the vicinity of 488 and 532 nm. Pump-probe spectroscopy of cytochrome c with this source reveals differences in excited-state absorption relaxation times between redox states. Though redox contrast is weak with this setup, we argue that this can be improved with a resonant galvo-scanning microscope. Moreover, pump-probe images were acquired of brown adipose tissue (which contains dense mitochondria), demonstrating label-free contrast from excited-state absorption in respiratory chain hemes.

The 1700-nm window has emerged as a promising excitation window for multiphoton microscopy (MPM). On one hand, the combined low tissue absorption and scattering make this window well suited for deep-tissue MPM; on the other hand, the long excitation wavelength makes higher-order MPM in biological tissues feasible, e.g., recently 4-photon fluorescence MPM in mouse brain has been demonstrated. Objective lens is a key optical component in the entire MPM setup. Multiphoton signal levels are largely dependent on the transmittance of objective lens. Here we demonstrate experimental results of transmittance measurement of two water immersion objective lenses commonly used for MPM at the 1700-nm window, covering both the excitation and the signal window. Our target application is MPM of even higher order excited at this window, i.e., 4th harmonic generation (FHG) imaging and 5-photon fluorescence generation. Our results show that, although the customized objective lens offers higher transmittance at the excitation window, it suffers from dramatically degraded transmittance at the signal window, compared with the non-customized objective lens. These results will offer guidelines for selection of proper objective lens for higher-order MPM at the 1700-nm window.

Intramural metastasis (IM) of esophageal cancer is defined as metastasis from a primary lesion to the esophageal wall without intraepithelial cancer extension. Esophageal cancer with IM is more common and such cases indicate a poor prognosis. In esophageal surgery, if curative resection is possible, the complete removal of both primary tumor and associated IMs is required. Therefore, accurate diagnosis of IMs in esophageal cancer prior to surgery is of particular importance. Multiphoton microscopy (MPM) with subcellular resolution is well-suited for deep tissue imaging since many endogenous fluorophores of fresh biological tissues are excited through two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). Here, a study to identify IM in fresh tissue section using MPM is reported. In this study, the morphological and spectral differences between IM and surrounding tissue are described. These results show that MPM has the ability to accurately identify IM in esophageal tissues. With improvement of the penetration depth of MPM and the development of multiphton microendoscope, MPM may be a promising imaging technique for preoperative diagnosis of IMs in esophageal cancer in the future.

SPAD (single-photon avalanche diode) arrays are single-photon imagers that can provide unparalleled timeresolved sensing performance. Since their inception in standard CMOS technologies (2003), a host of architectures and target applications have been explored, ranging from simple pixel arrays, with off-chip data processing electronics, to fully integrated “smart” imagers with in-pixel time-stamping electronics and/or on-chip data processing fabric. Applications include (endoscopic) FLIM, (multi-beam multiphoton) FLIM-FRET, SPIMFCS, time-resolved Raman, NIROT, super-resolution microscopy, and PET, to name a few. We will review some representative sensors and applications, the corresponding challenges, and provide an outlook on the future of this fascinating technology

We present a frequency-multiplexed multi-site two-photon imaging method utilizing amplitude modulation of femtosecond laser pulses in the MHz range to tag each excitation beam and the corresponding fluorescence signals with specific frequencies. The frequency tags are generated with an interferometric setup employing acousto-optic deflectors (AODs) to achieve precise spatial overlap of femtosecond pulses with acoustically-shifted frequencies. Creating matching excitation beam patterns in each interferometer arm using multiple AOD driving frequencies and subsequently overlapping these matching patterns results in multiple excitation beams with unique beat frequencies available for scanning. As a proof-of-concept, we demonstrate multiplexed two-photon image acquisition using test samples, and compare the performance of this approach to conventional two-photon laser scanning microscopy.

A three-dimensional (3D) single fluorescent particle tracking strategy based on temporal focusing multiphoton excitation microscopy (TFMPEM) combined with astigmatism imaging is proposed for delivering nanoscale-level axial information that reveals 3D trajectories of single fluorospheres in the axially-resolved multiphoton excitation volume without z-axis scanning. It provides the dynamical ability by measuring the diffusion coefficient of fluorospheres in glycerol solutions with a position standard deviation of 14 nm and 21 nm in the lateral and axial direction and a frame rate of 100 Hz. Moreover, the optical trapping force based on the TFMPEM is minimized to avoid the interference in the tracing measurements compared to that in the spatial focusing MPE approaches. Therefore, we presented a three dimensional single particle tracking strategy to overcome the limitation of the time resolution of the multiphoton imaging using fast frame rate of TFMPEM, and provide three dimensional locations of multiple particles using an astigmatism method.

The adverse health effects of particulate matter exposure are a generally accepted concern. Dramatic statistical figures suggest that fine dust is a main environmental risk in Europe and can be held accountable for hundreds of thousands of deaths per year [1]. Locating and tracking these nanometer sized particles, however, is not straight forward: In epidemiological and toxicology research only measurements based on labels [2] such as radionuclide markers have been applied. In this paper we present a direct, label-free optical contrast mechanism to detect carbon nanoparticles immersed in aqueous environments [3]. The virtue of this technique is its ability to perform in body fluids such as urine but also in cells and tissues. The mechanism is based on white light (WL) generation upon illumination with femtosecond pulsed near-infrared and is therefore non-incandescence related. We demonstrate the technique in various biological settings with dry and suspended carbon black particles (CB), a widely used model compound for soot [4]. Our approach allows for the unequivocal localization of CB alongside of common fluorophores and markers and can be performed on multiphoton laser-scanning microscopy platforms, a system commonly available in research laboratories. [1] European Environment Agency (2015). Press release. [2] Kong et al. Int. J. Mol. Sci. 2013, 14, (11), 22529-22543 [3] Bové and Steuwe et al. Nano letters, 2016, (16) , pages 3173-3178 [4] Arnal et al. Combust. Sci. Technol. 2012, 184, (7-8), 1191-1206.

PIE FastFLIM microscopy allows the quantitative multi-parameter measurement of single molecule protein folding and dynamics. Using donor-acceptor FRET pair-labeled proteins, we detect changes in protein conformation and dynamics by monitoring FRET efficiency, stoichiometry and lifetime. Together with anisotropy decay information, we acquire rotational relaxation times for single molecules. By applying antibunching, FLCS and burst analysis, multi-parameters (such as copy numbers in protein complexes), diffusion coefficient and molecular brightness can be fitted for deeper understanding of the conformational dynamic behavior of single protein molecules. In this paper, we’ll focus on the multiparameters of FRET efficiency, stoichiometry and lifetime.

Breast cancer, the most common type of cancer in women worldwide, as well as its treatment (e.g. radiation therapy) can affect the human skin. Multiphoton imaging could provide new insights into these skin alterations non-invasively and with high-resolution. As a preparation for a later investigation involving patients, areas of the breast and forearm skin of healthy volunteers were imaged using the clinically certified multiphoton imaging tomograph MPTflex based on endogenous skin autofluorescence and second-harmonic signals. Depth-resolved image stacks were acquired in consecutive weeks to explore the influence of hormonal variations on the skin properties. Both breasts were considered and up to three different areas were imaged per session. Acquisition parameters were optimized to minimize artifacts caused by breathing-motion. As a first result, skin properties, such as the epidermal thickness, appear to be influenced by hormonal variations.

Two-photon absorption (2PA) cross sections for 2-aminopurine (2AP), 7-methyl guanosine (7MG), 6-methyl isoxanthopterin (6MI), isoxanthopterin (IXP), 3-methylindole (3MI) and tryptophan (L-trp) are measured for excitation wavelengths between 550 and 810 nm using two independent methods: femtosecond two-photon excited fluorescence (2PEF) and femtosecond nonlinear transmission (NLT). For all systems we find that in the long wavelength region of the lowest-energy one-photon allowed singlet-singlet transition the 2PA spectral profiles follow the one-photon absorption spectral profiles, thus indicating the strongly dipolar nature of the transition. We use the measured 2PA cross sections to estimate the corresponding permanent electric dipole moment change and find the values to vary in the range 1 to 5 D. These experimental findings are corroborated by quantum-chemical computations.

Fluorescence lifetime imaging (FLIM) has been a powerful tool in life science because it can reveal the interactions of an excited fluorescent molecule and its environment. The combination with two-photon excitation (TPE) and timecorrelated single photon counting (TCSPC) provides it the ability of optical sectioning, high time resolution and detection efficiency. In previous work, we have introduced a two-dimensional acousto-optic deflector (AOD) into TCSPC-based FLIM to achieve fast and flexible FLIM. In this work, we combined the AOD-FLIM system with a single particle tracking (SPT) setup and algorithm and developed an SPT-FLIM system. Using the system, we acquired the trajectory and fluorescence lifetime of a moving particle simultaneously and reconstructed a life-time-marked pseudocolored trajectory, which might reflect dynamic interaction between the moving particle and its local environment along its motion trail. The results indicated the potential of the technique for studying the interaction between specific moving biological macromolecules and the ambient micro-environment in live cells.

Fluorescence induced by 1wo-photon absorption (2PA) and three-photon absorption (3PA) is becoming an increasingly important tool for deep-tissue microscopy, especially in conjunction with genetically-encoded functional probes such as fluorescent proteins (FPs). Unfortunately, the efficacy of the multi-photon excitation of FPs is notoriously low, and because relations between a biological fluorophore’s nonlinear-optical properties and its molecular structure are inherently complex, there are no practical avenues available that would allow boosting the performance of current FPs. Here we describe a novel method, where we apply directed evolution to optimize the 2PA properties of EGFP. Key to the success of this approach consists in high-throughput screening of mutants that would allow selection of variants with promising 2PA and 3PA properties in a broad near-IR excitation range of wavelength. For this purpose, we construct and test a wide field-of-view (FOV), femtosecond imaging system that we then use to quantify the multi-photon excited fluorescence in the 550- 1600 nm range of tens of thousands of E. coli colonies expressing randomly mutated FPs in a standard 10 cm diameter Petri dish configuration. We present a quantitative analysis of different factors that are currently limiting the maximum throughput of the femtosecond multi-photon screening techniques and also report on quantitative measurement of absolute 2PA and 3PA cross sections spectra.

Nonlinear microscopy has already shown its impact in biological research, namely in the fields of neurobiology, immunology, cancer research and embryology. Typically, these microscopes operate under free space propagation, using a dichroic mirror to separate the nonlinear signals from the excitation laser. While powerful such implementations are difficult to translate from the laboratory to a clinical setting where the environment is less controlled. Therefore, we propose an alignment-free all-fiber nonlinear microscopy system at 1550 nm based on double-clad fibers (DCF). As sectioning is performed through nonlinear effects, nonlinear microscopy does not require a detection pinhole, and. the DCF inner cladding can be used for efficient collection of nonlinear signals. The built system allows for multiplexing second harmonic generation (SHG) and two-photon excitation fluorescence (2PEF), collected from the inner cladding; and reflectance confocal microscopy (RCM), detected from the core acting as the confocal pinhole. Finally, an asymmetric double-clad fiber coupler (DCFC) is used to address efficiently both DCF channels. This all-fiber system is more compact and less sensitive to alignment, but requires carefully managing the transmission of the femtosecond pulse in the fiber. This is addressed using dispersion compensation fiber, pulse compression and solitonic propagation. Additionally, with a source centered at 1550 nm, we benefit from reduced sample scattering thus increasing the depth of field in comparison with systems operating at 800 nm. Overall we believe that the developed system could be transferred in clinics to enable in-vivo and in-situ imaging of human patient.

The tissues surrounding the seeds play an important role in the control of germination vigour and in the uptake of active ingredients (AIs) applied as seed dressings. The seeds can reduce costs through more efficient germination rates and have less environmental impact due to more efficient use of AIs. In this study, we use epi-detected stimulated Raman scattering (SRS) microscopy with a fiber laser source for analyzing modes of D₂O into intact seeds and determining uptake kinetics. We demonstrate that SRS microscopy is an ideal tool for visualizing uptake of AIs and worthy of further applications in real field conditions.

Larger imaging depth is the quest of almost all the imaging modalities, including multiphoton microscopy (MPM). Recently, it has been domonstrated that excitation at the 1700-nm helps extending imaging depth in MPM, optical coherence tomography, as well as photoacoustic imaging compared with excitation at other wavelengths. In MPM, immersion objective lenses with high numerical aperture (NA) are typically used to achieve better signal resolution, higer signal collection efficiency, and stronger signal generation. Although physically short (~mm), this extra optical path length traversed by the excitation light inevitably introduces absorption of the excitation light, and as a result leads to a decrease in the signal generation. Here we demonstrate experimental characterization of absorption spectrum of various immersion media at the 1700-nm window, including water (H₂O), deuterium oxide (D₂O), and several brands of immersion oil. Our results identify either the best immersion medium for a specific wavelength, or the best wavelength for a specific immersion medium at the 1700-nm window. Furthermore, through quantitative MPM experiments comparing different immersion media, we show that the MPM signal levels can be enhanced by more than ten fold simply by selecting the proper immersion medium, in good agreement with theoretical expectation based on the absorption measurement. Our results will offer guidelines for signal optimization in MPM at the 1700-nm window.

Second-harmonic-generation (SHG) microscopy is a powerful tool for in situ monitoring of collagen fibers in human skin. However, its practical use is still limited in the dermatological field due to the bulky and complicated setup. In this paper, we constructed a photonic-crystal-fiber-coupled, hand-held SHG microscope for in situ monitoring of collagen fibers in human skin. Fiber delivery of femtosecond pulse light was achieved by a large-mode-area photonic-crystal-fiber whereas the compact SHG microscopy setup was enclosed into a lens tube system. The combination of PCF with the lens tube system largely enhances the flexibility of measurement sites in the human skin.

Vocal fold scarring as a result of injury or disease can lead to voice disorders which can significantly affect the quality of life. During the scarring process, the normally elastic tissue of the vocal fold lamina propria is replaced by a much stiffer collagen-based fibrotic tissue, which impacts the fold’s ability to vibrate. Surgical removal of this tissue is often ineffective and can result in further scarring. Injectable biomaterials, a form of tissue engineering, have been proposed as a potential solution to reduce existing scars or prevent scarring altogether. In order to properly evaluate the effectiveness of these new materials, multiphoton microscopy emerges as an effective tool due to its intrinsic multiple label free contrast mechanisms that highlight extracellular matrix elements. In this study, we evaluate the spatial distribution of collagen and elastin fibers in a rabbit model using second harmonic generation (SHG), third harmonic generation (THG) and two photon autofluorescence (TPAF) applied to unlabeled tissue sections. In comparison to traditional methods that rely on histological staining or immunohistochemistry, SHG, THG and TPAF provide a more reliable detection of these native proteins. The evaluation of collagen levels allows us to follow the extent of scarring, while the presence of elastin fibers is thought to be indicative of the level of healing of the injured fold. Using these imaging modalities, we characterize the outcome of injectable biomaterial treatments in order to direct future treatments for tissue engineering.

Multiphoton imaging (MPI) is an important fluorescence microscopy technique that allows deep tissue and in-vivo imaging with high selectivity. According to theory, two-photon signal is proportional to the product of the peak power and the average power, allowing optimization of key imaging parameters of the excitation laser, such as average power, repetition rate and pulse duration. Recent progress in compact ultrafast lasers including femtosecond fiber lasers and optically pumped semiconductor lasers makes direct control of these parameters possible. In order to investigate the optimum laser parameters for two photon imaging we experimentally study the effects of repetition rate between 2.85 and 90 MHz and pulse duration between 336 fs and 3.5 ps on two photon signal in SYTOX Green labeled mouse intestine sections at 1030 nm. We found that the optimum repetition rate for this sample is in the range 20 – 40 MHz, depending on average power, and that the pulse duration has no effect on the MPI signal provided that the average power can be adjusted to keep the product of average and peak power constant.

The aim of the study was to estimate energy metabolism in human cervical cancer cells HeLa Kyoto after chemotherapy in vitro and in vivo using two-photon fluorescence lifetime microscopy (FLIM). Cellular metabolism was examined by monitoring of the fluorescence intensities and lifetimes of metabolic cofactors NAD(P)H and FAD. Cancer metabolism was analyzed in dynamics after treatment with cisplatin. Two-photon fluorescence and second harmonic generation microscopies as well as standard histopathology with hematoxylin and eosin were used to characterize cancer tissue structure. We showed an increase of the optical redox ratio FAD/NAD(P)H in cancer cells in vitro and decrease of the relative contribution of free NAD(P)H (ɑ1) in vitro and in vivo, which presumably indicate a shift to more oxidative metabolism after chemotherapy. These data demonstrate the possibility to detect response of cancer cells to chemotherapy using optical metabolic imaging.

We discuss the development and application of a laser-scanning, nonlinear optical microscope capable of generating vibrationally resonant images based on sum-frequency generation (SFG), coherent anti-Stokes Raman scattering (CARS) or third-order sum-frequency generation (TSFG). The combination of these three modalities allows vibrationally sensitive imaging of both χ ⁽²⁾ and χ ⁽³⁾-active structures in biological tissues, addressing both Raman-active as well as IR-allowed vibrational modes. We show the practical utility of these vibrationally sensitive modalities by imaging collagen I rich tissues.

Second harmonic generation (SHG) microscopy is a promising imaging technique for collagenous tissues due to its noninvasiveness and potential for 3D imaging. In tissues with densely packed and thin collagen fibrils, such as cartilage, the focal volume of the laser can comprise multiple fibrils, and the SHG from each of these fibrils can interact and contribute to the detected signal. Coherent amplification is achieved when the fibrils are aligned and oriented in the same direction. The effect of changing the size of the focal volume, as determined by the numerical aperture (NA), is therefore dependent on the length scale at which the fibrils are aligned. This effect on the image contrast is important to examine experimentally before SHG can be used in the clinic. In this study, we measured the SHG intensity and radiation direction as a function of the NA of the excitation and collecting objectives in different areas of the immature articular cartilage of young pigs. The results showed that varying the NA of the excitation objective had more effect on the SHG signals detected in the forward compared to the backward direction and that this effect varied considerably throughout the tissue. The results clearly demonstrated that SHG imaging differs from conventional histology, and the image contrast should only be interpreted in light of the imaging conditions.

Cortical structures in the central nervous system exhibit an ordered laminar organization. Defined cell layers are significant to our understanding of brain structure and function. In this work, multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), which was applied for qualitatively visualizing the structure of cerebral and cerebellar cortex from the fresh, unfixed, and unstained specimen. MPM is able to effectively identify neurons and neurites in cerebral cortex, as well as glial cells, Purkinje cells, and granule cells in cerebellar cortex at subcellular resolution. In addition, the use of automated image processing algorithms can quantify the circularity of neurons and the density distribution of neurites based on the intrinsic nonlinear optical contrast, further providing quantitative characteristics for automatically analyzing the laminar structure of cortical structures. These results suggest that with the development of the feasibility of two-photon fiberscopes and microendoscope probes, the combined MPM and image analysis holds potential to provide supplementary information to augment the diagnostic accuracy of neuropathology and in vivo identification of various neurological illnesses in clinic.

In this work, we present all epi-direction detected images of an unstained mouse retina using multiphoton microscopy with a sub-50 fs Yb-fiber laser centered at 1.07 μm. This wavelength regime is particularly important as the fundamental wavelength is transparent to the eye and the higher harmonics are above the DNA-damaging UV wavelengths. We present a characterization of the multimodal signals emitted from all the retinal layers, as well as from the choroid and the sclera. By characterizing native multiphoton signals from the retina, we move closer to having Yb-fiber considered for in vivo diagnosis of retinal disease through multiphoton microscopy as well as for corrective therapies.

We demonstrate that three-photon microscopy (3PM) with 1300-nm excitation enables functional imaging of GCaMP6s labeled neurons beyond the depth limit of two-photon microscopy (2PM) with 920-nm excitation. We quantitatively compared 2PM and 3PM imaging of calcium indicator GCaMP6s by measuring correlation between activity traces, absolute signal level, excitation attenuation with depth, and signal-to-background ratio (SBR). Compared to 2PM imaging of GCaMP6s-labeled neurons, 3PM imaging has increasingly larger advantages in signal strength and SBR as the imaging depth increases in densely labeled mouse brain, given the same pulse energy, pulse width, and repetition rate at the sample surface. For example, 3PM has comparable signal strength as 2PM and up to two orders of magnitude higher SBR as 2PM in mouse cortex around 700-800um. We also demonstrate 3PM activity recording of 150 neurons in the hippocampal stratum pyramidale (SP) at 1mm depth, which is inaccessible to non-invasive 2PM imaging. Our work establishes 3PM as a powerful tool for calcium imaging at the depth beyond 2PM limits.

Eliminating time-consuming process of conventional biopsy is a practical improvement, as well as increasing the accuracy of tissue diagnoses and patient comfort. We addressed these needs by developing a multimodal nonlinear endomicroscope that allows real-time optical biopsies during surgical procedure. It will provide immediate information for diagnostic use without removal of tissue and will assist the choice of the optimal surgical strategy. This instrument will combine several means of contrast: non-linear fluorescence, second harmonic generation signal, reflectance, fluorescence lifetime and spectral analysis. Multimodality is crucial for reliable and comprehensive analysis of tissue. Parallel to the instrumental development, we currently improve our understanding of the endogeneous fluorescence signal with the different modalities that will be implemented in the stated. This endeavor will allow to create a database on the optical signature of the diseased and control brain tissues. This proceeding will present the preliminary results of this database on three types of tissues: cortex, metastasis and glioblastoma.

Spatial Frequency Modulated Imaging (SPIFI) with single element detection has previously been demonstrated with a time varying amplitude spatial frequency. This has been shown in a variety of modalities (linear, TPEF, SHG) and also with variations on the base design to provide additional dimensions of information. SPIFI is also capable of providing enhanced resolution images. However, the signal-to-noise is a limiting factor in the quality of the resolution enhancement. We present a microscope design which uses a nematic spatial light modulator to provide a time varying amplitude from an amplitude or phase grating. Twophoton excitation fluorescence images of 10-µm fluorescent polystyrene beads are presented using a phase grating. Additionally, the microscope can provide spatial gratings in polarization which provide an alternative means of imaging in third harmonic generation (THG). THG images are provided using an amplitude and polarization-grating modulation pattern.

NAD(P)H is a known biomarker for cellular metabolism; a higher ratio of enzyme-bound NAD(P)H to free/unbound NAD(P)H indicates an increase in metabolic activity. Free NADH has a shorter fluorescence lifetime (τ₁), the bound version (τ₂) a longer lifetime. FLIM’s unique capability to establish inter alia the relative fractions of τ₁ (a₁%) and τ₂ (a₂%) in each pixel, determines the level of metabolic activity. The relative abundances of bound NAD(P)H were analyzed for single cells, confluent and partially confluent cells within 3 Fields-of-View (FoVs). A gradient of increasing a 2% levels of bound NAD(P)H from single, partially confluent to confluent cells was observed.

Femtosecond optical parametric oscillator (OPO) was investigated, the chirped mirror based OPO was tunable by rotating intracavity crystal in the wavelength range of 680-810 nm with maximum output power of 600 mW and pulse duration below 150 fs. The experimental results were compared with numerical modelling while solving coupled electric field equations in spatial and time domain to include the effects of resonator dispersion and self-phase modulation. The results pave a way for a compact femtosecond optical parametric oscillator tunable in a spectrum band limited by resonator mirrors’ spectral phase characteristics.