This PDF file contains the front matter associated with SPIE Proceedings Volume 10498, including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations such as the bulky fluorescent tags and limited multiplexing ability in the era of “omics”. Here I will present two chemical imaging strategies, respectively. First, we devised a live-cell Bioorthogonal Chemical Imaging platform suited for probing the dynamics of small bio-molecules, which cannot be effectively labeled by bulky fluorophores. This scheme couples the emerging stimulated Raman scattering microscopy with tiny and Raman-active vibrational probes (e.g., alkynes, nitriles and stable isotopes including 2H and 13C). Exciting biomedical applications such as imaging fatty acid metabolism related to lipotoxicity, glucose uptake and metabolism, drug trafficking, protein synthesis in brain, DNA replication, protein degradation, RNA synthesis and tumor metabolism will be presented. Second, we invented a super-multiplex optical imaging technique. We developed electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy, achieving exquisite vibrational selectivity with high versatility and sensitivity. Chemically, we created a unique vibrational palette consisting of novel dyes bearing conjugated and isotopically-edited triple bonds, each displaying a single epr-SRS peak in the cell-silent spectra window. Up to 24 resolvable colors are currently achieved with great potential for further expansion. Using this approach, we monitored DNA and protein metabolism in neuronal co-cultures and brain tissues. This super-multiplex optical imaging approach promises to facilitate untangling the intricate interactions in complex biological systems, and can also find broad applications in photonics and biotechnology in general.

Fluorescence lifetime imaging has become a common technique in microscopy. The information contained in the fluorescence decay curve can be obtained with different methods. A major issue is that the fluorescence decay must be measured at many pixels either sequentially or in parallel, which generally results in relatively noisy curves. Another major issue is data analysis of the decay at each point of an image and the visualization of the results. Nonetheless, methods of data analysis and visualization have become relatively common and today we can analyze an image for different molecular species and molecular processes. In this talk I will discuss a method, called the phasor that was developed in my lab and that is now used in several commercial instruments. Using the phasor method applied to cell autofluorescence it has become possible to construct quantitative indices of cell metabolism and to identify molecular species characteristic to some diseases.

Nonlinear optical microscopy combined with fluorescence lifetime imaging is a non-invasive imaging technique, based on the study of fluorescence decay times of naturally occurring fluorescent molecules, enabling a noninvasive investigation of the biological tissue with subcellular resolution. Cancer exhibits altered cellular metabolism, which affects the autofluorescence of metabolic cofactors NAD(P)H and FAD. In this study features of tumor metabolism in different systems of organization (from cell culture to patient lesion) was showed. The observed differences in the relative contributions of free NAD(P)H and FAD testify to an increased a glycolytic metabolism in cancer cells compare to fibroblasts. In 3D spheroids, the cells of the proliferating zone had greater a1 and lower tm values than the cells of the quiescent zone, which likely is a consequence of their higher glycolytic rate. During the growth of colorectal cancer in the experimental mouse model, the contribution of the free component of NAD(P)H was increased. Dysplastic nevus and melanoma is characterized by raised contribution of free NADH compare to healthy skin. Therefore, melanoma cells had very short value of τ1.

Photoacoustic tomography (PAT), combining optical and ultrasonic waves via the photoacoustic effect, provides in vivo functional, metabolic, molecular, and histologic imaging. PAT has the unique strength of high-resolution imaging across the length scales of organelles, cells, tissues, and organs with consistent contrast. PAT has the potential to empower multiscale biology research and accelerate translation from microscopic laboratory discoveries to macroscopic clinical practice. PAT may also hold the key to the earliest detection of cancer by in vivo label-free quantification of hypermetabolism, the quintessential hallmark of cancer. Broad applications include imaging of the breast, brain, skin, esophagus, colon, vascular system, and lymphatic system in both humans and animals.

Metabolic imaging by NAD(P)H FLIM requires the decay functions in the individual pixels to be resolved into the decay components of bound and unbound NAD(P)H. Metabolic information is contained in the lifetime and relative amplitudes of the components. The separation of the decay components and the accuracy of the amplitudes and lifetimes improves substantially by using ultra-fast HPM-100-06 and HPM-100-07 hybrid detectors. The IRF width in combination with the Becker & Hickl SPC-150N and SPC-150NX TCSPC modules is less than 20 ps. An IRF this fast does not interfere with the fluorescence decay. The usual deconvolution process in the data analysis then virtually becomes a simple curve fitting, and the parameters of the NAD(P)H decay components are obtained at unprecedented accuracy.

Cisplatin is an effective anticancer drug commonly used in the treatment of solid tumors. Although DNA is considered as the primary target, the cisplatin action at the cellular level remains unknown. Advanced fluorescence microscopy techniques allow probing various physiological and physicochemical parameters in living cells and tissues with unsurpassed sensitivity in real time. This study was focused on the investigation of cellular bioenergetics and cytosolic pH in colorectal cancer cells during chemotherapy with cisplatin. Special attention was given to the changes in cisplatininduced apoptosis that was identified using genetically encoded FLIM/FRET sensor of caspase-3 activity. Metabolic measurements using FLIM of the metabolic cofactor NAD(P)H showed decreased contribution from free NAD(P)H (a1, %) in all treated cells with more pronounced alterations in the cells undergoing apoptosis. Analysis of cytosolic pH using genetically encoded fluorescent sensor SypHer1 revealed a rapid increase of the pH value upon cisplatin exposure irrespective of the induction of apoptosis. To the best of our knowledge, a simultaneous assessment of metabolic state, cytosolic pH and caspase-3 activity after treatment with cisplatin was performed for the first time. These findings improve our understanding of the cell response to chemotherapy and mechanisms of cisplatin action.

The evaluation of complex metabolic changes of individual live cells and heterogeneous cell cultures is not feasible using traditional methods due to their destructive behavior and lack of spatial information. Two-photon excited fluorescence of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) facilitate a label-free and non-destructive evaluation of metabolic activity. This study explores the phasor approach in combination with two-photon fluorescence lifetime imaging microscopy (FLIM) as a potential method to evaluate pharmacologically induced metabolic changes that occur during the browning of adipocytes. The possibility of browning of white adipose cells is a desirable prospect for the treatment of obesity and related disorders. Here, we compared the results obtained by Fourier-based phasor analysis with the traditional exponential FLIM analysis as well as results of an extracellular flux analyzer. The alteration of glycolytic function and oxidative phosphorylation after treatment with pharmacological reagents significantly shift the contribution of each of the fluorescence lifetime components to the total fluorescence intensity. Further, we showed that the ratios of the lifetime components obtained by the phasor approach reflect the shift in mitochondrial and cytosolic NADH concentration. The phasor analysis agrees with traditional assessments, such as the optical free-to-bound NADH ratio as well as the oxygen consumption rate and extracellular acidification rate as determined by the extracellular flux analyzer. Our results support the concept that non-invasive sensing of fat metabolism and browning of fat may be possible by analyzing the fluorescence lifetime of NADH using the phasor approach.

Pancreatic cancer has the worst prognosis of all cancers (5-year survival rate of 7%). Patients that are eligible for surgery often receive adjuvant chemotherapy to improve survival. There is a critical need for a tool to match individual patients with optimal drugs for their cancer. The goal of this work is to validate Optical Metabolic Imaging (OMI) of patient-derived pancreatic tumor organoids as a high-throughput predictive drug screen for patients. Three-dimensional organoids were successfully generated from surgically resected pancreatic tumors. These organoids were treated with the patient’s prescribed adjuvant therapy, and early metabolic changes were measured using multiphoton fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzymes NAD(P)H and FAD. Changes at the single-cell level were quantified using the OMI Index, a linear combination of the optical redox ratio (ratio of the fluorescence intensities of NAD(P)H to FAD), and the mean NAD(P)H and FAD fluorescence lifetimes. Population density modeling on the OMI Index was used to evaluate cell-level heterogeneities in drug responses. Additionally, mass spectrometry imaging (MSI) was used to map metabolites in organoids. Combining multiphoton OMI images and MSI images provides a detailed map of the complex metabolic changes that occur in response to treatment, which may be used to identify additional drug targets. Patient survival data after surgery was used as validation of drug response in organoids. This platform shows promise for predicting long-term response to therapy in pancreatic cancer patients.

A common property during tumor development is altered energy metabolism, which could lead to a switch from oxidative phosphorylation and glycolysis. The impact of this switch for theranostic applications could be significant. Interestingly altered metabolism could be correlated with a change in the fluorescence lifetimes of both NAD(P)H and FAD. However, as observed in a variety of investigations, the situation is complex and the result is influenced by parameters like oxidative stress, pH or viscosity. Besides metabolism, oxygen levels and consumption has to be taken into account in order to understand treatment responses. For this, correlated imaging of phosphorescence and fluorescence lifetime parameters has been investigated by us and used to observe metabolic markers simultaneously with oxygen concentrations.

The technique is based on time correlated single photon counting to detect the fluorescence lifetime of NAD(P)H and FAD by FLIM and the phosphorescence lifetime of newly developed phosphors and photosensitizers by PLIM. For this, the photosensitizer TLD1433 from Theralase, which is based on a ruthenium (II) coordination complex, was used. TLD1433 which acts as a redox indicator was mainly found in cytoplasmatic organelles. The most important observation was that TLD1433 can be used as a phosphor to follow up local oxygen concentration and consumption during photodynamic therapy. Oxygen consumption was accompanied by a change in cell metabolism, observed by simultaneous FLIM/PLIM. The combination of autofluorescence-FLIM and phosphor-PLIM in luminescence lifetime microscopy provides new insights in light induced reactions.

Cancer cells are known to display a variety of metabolic reprogramming strategies to fulfill their own growth and proliferative agenda. With the advent of high resolution imaging strategies, metabolomics techniques etc., there is an increasing appreciation of critical role that tumor cell metabolism plays in the overall breast cancer (BC) growth. A recent study from our laboratory demonstrated that the development of invasive cancers could be causally connected to deficits in mitochondrial function. Using this study as a rationale, we hypothesize that the widely accepted multistep tumor growth model might have a strong metabolic component as well. In this study, we explore the possibility of targeting mitochondrial Complex I enzyme system for not only metabolic detection of cancer-associated redox changes but also for modulating breast cancer cell growth characteristics. As a proof-of-principle, we demonstrate two approaches (pharmacological and genetic) for modulating mitochondrial Complex I function so as to achieve breast cancer control.

Skin cancer is associated with abnormal cellular metabolism which if identified early introduces the possibility of intervention to prevent its progress to a deadly metastatic stage. This study combines multiphoton microscopy with fluorescence lifetime imaging (FLIM) using an orthotopic melanoma mouse model, to detect changes in redox states of single epidermal cells as a metabolic marker to monitor the progress of tumor growth. This method utilizes imaging of the ratio of the amounts of the free and protein-bound forms of the intracellular autofluorescent metabolic co-enzyme nicotinamide adenine dinucleotide (NADH). Here we investigate the impact of the primary tumor lesion on the epidermal layers at three different growth stages of melanoma lesion compared to normal skin as a control. We show a significant increase in the free-to-bound NADH ratio with the growth of the solid melanoma tumor, while concurrently the short and the long lifetime components of NADH remained constant. These results demonstrate the potential of FLIM for rapid, non-invasive and sensitive assessment of melanoma progression revealing its potential as a diagnostic tool for melanoma detection and as an aid for melanoma staging.

Current technologies for cellular metabolomics are complex, costly, and lack single-cell resolution. We propose optical metabolic imaging (OMI) as a tool to noninvasively monitor cellular metabolism on a single-cell level. OMI uses two-photon microscopy and time correlated single photon counting to measure the fluorescence lifetime and intensity of endogenous metabolic coenzymes NAD(P)H and FAD. However, the biochemical basis of changes in OMI parameters such as redox ratio (NAD(P)H intensity divided by FAD intensity), NAD(P)H lifetime, and FAD lifetime remains unclear. A thorough understanding of these biochemical sources of contrast is needed to interpret OMI studies of drug efficacy, and thus leverage OMI as a tool for drug screening and metabolic research. To address this issue, we profiled 11 CRISPR-mediated knockout human cell lines with single gene deletions corresponding to mitochondrial proteins involved in characterized pathways, and complexes ranging from oxidative phosphorylation to mitochondrial structure. The wild-type and knockout cells were plated at an equal density on 35mm imaging dishes in parallel with additional plates for other analyses. After 48hrs, cells were imaged or collected for mass spectrometry-based metabolomic, proteomic, and lipidomic analyses. OMI of the wild-type and knockout haploid cells yielded statistically significant differences in redox ratio, NAD(P)H and FAD mean lifetimes (ANOVA, p<0.005) that were specific to the knockout. Comparisons between OMI and parallel multi-omic analysis of each knockout are currently underway. This comprehensive dataset will provide a better understanding of the biochemical basis for OMI measurements, which could enable robust, single-cell monitoring of metabolic activity.

Multimodal optical imaging of suspected tissues is showing to be a promising method for distinguishing suspected cancerous tissues from healthy ones. In particular, the combination of steady-state spectroscopic methods with timeresolved fluorescence provides more precise insight into native metabolism when focused on tissue autofluorescence. Cancer is linked to specific metabolic remodelation detectable spectroscopically. In this work, we evaluate possibilities and limitations of multimodal optical cancer detection in single cells, collagen-based 3D cell cultures and in living organisms (whole mice), as a representation of gradually increasing complexity of model systems.

Taking inspiration from the philosophical and sociological speculation by Zygmunt Bauman (Z. Bauman, Liquid Modernity, Polity Press, Cambridge, 2000) a new paradigm for optical microscopy is proposed regarding design, implementation, and applications. The two-photon excitation microscope of the future, combining different converging technologies (Piazza et al. 2017 J.Biophotonics; Sancataldo et al. 2017 Optica; Lanzanò et al. 2017 Nature Comm.), is a liquid tunable microscope able to compete regarding spatial resolution and analytical capacity with the electron microscope.It is liquid because it overlaps in an efficient and optimised way different mechanisms of contrast and it is tuneable because it offers a real time tuneability regarding spatial and temporal resolution like a radio tuned to the preferred radio station. It is smart because can adapt its architecture to the current scientific question.2PE, SHG, polarised SHG (Bianchini and Diaspro, J.Biophotonics, 2008) and Mueller matrix signature (Mazumder et al. 2017 J.Optics; Sheppard et al. 2017 JOSA A) are combined to form a new liquid image. Super resolved methods, including expansion microscopy, will be integrated. Such a scalable approach will be proposed to address questions related to cell functioning coupling high-order structure organisation of chromatin and its effect at the nanoscale (Diaspro et al. 1990 IEEE Trans.Biomed. Eng.).

Multiphoton microscopy has become an ubiquitous research tool for neuroscience, disease studies, embryology and immunology. Refinements in non-linear imaging techniques have gone hand-in-hand with improvements in tuning range, power, pulse management and other features of femtosecond lasers specifically designed for these applications. While potential clinical applications require lasers with multiple outputs able to support multimodal imaging, recent developments in neuronal imaging like 3-photon microscopy or optogenetic activation require higher energies per pulse and longer wavelengths than conventional multiphoton microscopy. Here we describe the design and characteristics of laser sources able to address all these applications, staring with the more established ultrafast laser technology based on Titanium Sapphire lasers and their OPOs and covering recently developed sources like Ytterbium fiber mode-locked lasers and amplifiers with their tunable wavelength extensions.

We developed a module for dual-output, dual-wavelength lasers that facilitates multiphoton imaging and spectroscopy experiments and enables hyperspectral imaging with spectral resolution up to 5 cm⁻¹. High spectral resolution is achieved by employing spectral focusing. Specifically, two sets of grating pairs are used to control the chirps in each laser beam. In contrast with the approach that uses fixed-length glass rods, grating pairs allow matching the spectral resolution and the linewidths of the Raman lines of interest. To demonstrate the performance of the module, we report the results of spectral focusing CARS and SRS microscopy experiments for various test samples and Raman shifts. The developed module can be used for a variety of multimodal imaging and spectroscopy applications, such as single- and multi-color two-photon fluorescence, second harmonic generation, third harmonic generation, pump-probe, transient absorption, and others.

Single-photon avalanche diode (SPAD) arrays have recently emerged as promising detectors for many wide-field fluorescence lifetime imaging microscopy (FLIM) applications, thanks to their picosecond range temporal resolution, single-photon sensitivity and fast parallel readout. However, ultra-fast SPAD cameras with very large number of pixels (> 100,000) equipped with time-correlated single-photon counting (TCSPC) capabilities for each pixel have not been developed to date, due to the difficulty with implementing compact in-pixel ps-timestamping. Here, we report a time-resolved image sensor for real-time FLIM comprising the largest SPAD array reported so far. Thanks to its in-pixel time-gated architecture, this 512×512 pixels sensor can perform time-resolved photon-counting at a maximum of 97 kfps. The state-of-the-art photon detection probability and dark count rate of the SPAD allow the camera to collect enough photons for lifetime extraction in low light conditions at video rate. To reach the timing performance obtained by conventional time-to-digital converters (TDC) architectures, we used a time-gated technique based on large (5 ns) time-gates shifted by picosecond delays. Using a finite number of overlapping gates, fluorescence lifetime can be accurately recovered. This architecture and the corresponding FLIM data is ideally suited for phasor analysis, a powerful and computationally simple method to extract fluorescence lifetime information, which allows real-time monitoring of FLIM information provided by such large imagers.

Genetically expressed Förster resonance energy transfer (FRET) biosensors are a powerful tool to study the activation of signalling proteins and pathways in a live cell and in vivo setting. The prototypical RhoGTPases Rac1 and RhoA are temporally and spatially synchronized during cell protrusion; this tight control and dynamic regulation of activity is necessary for coordinated cell migration. We demonstrate an approach to simultaneous imaging of Rac1 and and RhoA biosensors in live tissue to study the spatiotemporal relationship between these two molecules. We use a custom multichannel fluorescence lifetime imaging (FLIM) system and employ a spectral-lifetime fitting approach to overcome the spectral overlap between the biosensors by exploiting the difference in lifetimes of the biosensor fluorophores. We fit our data to an analytical model of the lifetime and intensity of each biosensor to determine the abundance and activity of each biosensor across the field of view. We have crossed a recently published ECFP-YFP Rac1 FRET biosensor reporter mouse with a EGFP-RFP RhoA FRET mouse to produce a dual reporter mouse. We demonstrate that we can readout the activation of Rac1 and RhoA in primary cells and tissues derived from the dual biosensor mouse. This approach allows us to directly visualise the mutually inhibitory response of RhoA and Rac1 on a subcellular level to treatment with small molecule inhibitors and activators.

Stem cells and 'induced pluripotent stem cells' (iPS cells) attract considerable attention in medicine. So far, iPS cells which morphologically and functionally resemble embryonic stem cells have been generated by means of viruses through the reprogramming of somatic cells for research purposes. Typically, optical techniques such as light microscopy in combination with fluorescent markers are applied, sometimes accompanied with fluorescence-activated cell sorting (FACS), for the characterization and isolation of both tissue-specific stem and iPS cells. Nowadays, modern laser-based techniques such as nonlinear multiphoton microscopy and optical transfection using femtosecond lasers are used for iPS and stem cell imaging as well as for virus-free optical reprogramming.

Photon upconversion is a nonlinear process in which the sequential of absorption of two or more photons leads to the anti-stoke emission. Different than the conventional multiphoton excitation process, upconversion can be efficiently performed at low excitation densities. Recent developments in lanthanide-doped upconversion nanoparticles (UCNPs) have led to a diversity of applications, including detecting and sensing of biomolecules, imaging of live cells, tissues and animals, cancer diagnostic and therapy, etc. Measuring the upconversion lifetime provides a new dimension of its imaging and opens a new window for its applications. Due to the long metastable intermediate excited state, UCNP typically has a long excited state lifetime ranging from sub-microseconds to milliseconds. Here, we present a novel development using the FastFLIM technique to measure UCNP lifetime by laser scanning confocal microscopy. FastFLIM is capable of measuring lifetime from 100 ps to 100 ms and features the high data collection efficiency (up to 140-million counts per second). Other than the traditional nonlinear least-square fitting analysis, the raw data acquired by FastFLIM can be directly processed by the model-free phasor plots approach for instant and unbiased lifetime results, providing the ideal routine for the UCNP photoluminescence lifetime microscopy imaging.

Monitoring the binding of protein ligands and therapeutic antibodies to their respective receptors (target engagement) is crucial to compound prioritization in anti-cancer targeted drug screening. However, current in vivo optical imaging techniques cannot distinguish between co-localization and actual receptor-ligand binding at the tumor region. Since transferrin receptor (TfR) level is significantly elevated in cancer cells compared to non-cancerous cells, transferrin (Tf) has been successfully used in molecular imaging and targeted anti-cancer drug delivery. The homodimeric nature of TfR allows for measuring fluorescence lifetime FRET (FLI-FRET) to quantitate the TfR-Tf binding and internalization into cancer cells, based on the reduction of donor fluorophore lifetime. Near infrared (NIR) FLI-FRET has been used to directly visualize and quantitate TfR-Tf binding and internalization by providing the fraction of donor-labeled entity that is interacting with its respective receptor. NIR FLI-FRET has been validated at multiscale, using both in vitro microscopy as well as in vivo macroscopy whole-body deep imaging assays using different NIR FRET pairs. Accuracy of NIR FLI-FRET quantitation has been compared between fluorescence intensity and lifetime measurements using both microscopy and macroscopy fluorescence imaging. NIR FLI-FRET employs well-characterized quantitative lifetime-based metrics, standard in FRET microscopy, but with the additional benefit of a seamless multiscale technological platform. In summary, we have successfully demonstrated quantitative imaging of receptor-mediated binding and uptake of Tf using NIR FLI-FRET microscopy and macroscopy imaging in vitro and in vivo, respectively. This novel approach can be extended to other receptors, currently targeted in oncology. Hence, NIR FLI-FRET can find numerous applications in pre-clinical drug delivery and targeted therapy assessment and optimization.

Neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor that is important for signaling in the brain and the gut. Its agonist ligand neurotensin (NTS), a 13-amino-acid peptide, binds with nanomolar affinity from the extracellular side to NTSR1 and induces conformational changes that trigger intracellular signaling processes. Our goal is to monitor the conformational dynamics of single fluorescently labeled NTSR1. For this, we fused the fluorescent protein mNeonGreen to the C terminus of NTSR1, purified the receptor fusion protein from E. coli membranes, and reconstituted NTSR1 into liposomes with E. coli polar lipids. Using single-molecule anisotropy measurements, NTSR1 was found to be monomeric in liposomes, with a small fraction being dimeric and oligomeric, showing homoFRET. Similar results were obtained for NTSR1 in detergent solution. Furthermore, we demonstrated agonist binding to NTSR1 by time-resolved single-molecule Förster resonance energy transfer (smFRET), using neurotensin labeled with the fluorophore ATTO594.

Two-photon fluorescence lifetime imaging microscopy (FLIM) is a technique that not only probes the intensity of fluorophores, but also provides the temporal decay trace of said fluorophores on a pixel-by-pixel basis. These traces can then be transformed into the frequency domain for subsequent analysis, resulting in a scatterplot of phasor coordinates where each phasor corresponds to a single image pixel. With this in mind, it follows that individual fluorophores result in distinct clusters in the phasor plot, and a mixture of two fluorophores results in phasors that fall somewhere along a line linking the two clusters depending on the relative fluorophore concentrations. Until now, distinction of fluorescent species has relied mainly on computing the Euclidean distance between a given phasor and the mean coordinates of reference phasor clusters. However, this approach becomes inadequate in cases where one fluorophore has a much wider lifetime distribution than the other. As such, we propose the use of the Mahalanobis distance as an alternative to the Euclidean distance, as this metric additionally factors in the relative spread of each reference phasor cluster. This method has been applied to studying the oxidative response of ex vivo human skin via endogenous NADH fluorescence as it is exposed to chemical sun filters, the active ingredients in sunscreens. Given that both NADH and sun filters are fluorescent under the same excitation and emission conditions, the proposed Mahalanobis distance approach was used to distinguish the source of fluorescence in images of human skin. This allowed for the assessment of oxidative response as well as the tracking and monitoring of the sun filter formulation as it permeated throughout the skin.

This presentation describes the development of the optical macroscanner and its application for metabolic imaging of large areas of tumors in mice. The scanner allows to interrogate areas as large as 15x15mm with the lateral resolution on the order of 15 micrometers. Acquisition times range from a few seconds for low pixel numbers to several minutes for high-resolution images. We present data for NAD(P)H imaging of tumor with genetically encoded mKate2. In addition, using macroscanner we demonstrated the possibility of visualizing caspase-3 activity using the FRET-biosensor TR23K, which is based on a pair of proteins - a red fluorescent protein as a donor and a chromoprotein as an acceptor. The in vivo assay was noninvasive and could be applied in strongly and weakly fluorescent subcutaneous xenografts in mice using the FLIM-FRET method.

Photoluminescence (PL) refers to light emission initiated by any form of photon excitation. PL spectroscopy and microscopy imaging has been widely applied in material, chemical and life sciences. Measuring its lifetime yields a new dimension of the PL imaging and opens new opportunities for many PL applications. In solar cell research, quantification of the PL lifetime has become an important evaluation for the characteristics of the Perovskite thin film. Depending upon the PL process (fluorescence, phosphorescence, photon upconversion, etc.), the PL lifetimes to be measured can vary in a wide timescale range (e.g. from sub-nanoseconds to microseconds or even milliseconds) – it is challenging to cover this wide range of lifetime measurements by a single technique efficiently. Here, we present a novel digital frequency domain (DFD) technique named FastFLIM, capable of measuring the PL lifetime from 100 ps to 100 ms at the high data collection efficiency (up to 140-million counts per second). Other than the traditional nonlinear leastsquare fitting analysis, the raw data acquired by FastFLIM can be directly processed by the model-free phasor plots approach for instant and unbiased lifetime results, providing the ideal routine for the PL lifetime microscopy imaging.

Neurovascular coupling (NVC) is defined as a local increase in cerebral blood flow in response to neuronal activity, it forms the basis of functional brain imaging and is altered during epilepsy. Because astrocytic calcium signaling (Ca²⁺) has been involved in the response of parenchymal vessels, this study investigates the role of this pathway during epilepsy. We exploit 4-Aminopyridine (4-AP) induced epileptic seizures to show that absolute Ca²⁺ concentration in astrocytic endfeet correlates with the changes in diameter of parenchymal vessels during neural activity in vivo. A two-photon laser scanning fluorescence lifetime microscopy was developed to simultaneously monitor free Ca²⁺ concentration in astrocytic endfeet with the calcium-sensitive indicator Oregon Green 488 BAPTA-1 (OGB-1) and the diameter of parenchymal vessels in the somatosensory cortex of mice following 4-AP injection. Our results reveal that the resting Ca²⁺ concentration in glial cells was spatially heterogeneous and that resting Ca²⁺ concentration in somatic regions was significantly higher than in endfoot regions. Moreover, following 4-AP injection in the somatosensory cortex of mice, we observed increases of Ca²⁺ in astrocytic endfeet associated with vasodilation of parenchymal vessels for each individual ictal event in the epileptic focus. However, vasodilation was seen to be inhibited by increase in absolute resting Ca²⁺ concentration. Our results suggest a role for baseline astrocytic Ca²⁺ concentration in vasodilation.

Second Harmonic Generation (SHG) microscopy provides high-resolution structural imaging of the corneal stroma without the need of labelling techniques. This powerful tool has never been applied to living human eyes so far. Here, we present a new compact SHG microscope specifically developed to image the structural organization of the corneal lamellae in living healthy human volunteers. The research prototype incorporates a long-working distance dry objective that allows non-contact three-dimensional SHG imaging of the cornea. Safety assessment and effectiveness of the system were firstly tested in ex-vivo fresh eyes. The maximum average power of the used illumination laser was 20 mW, more than 10 times below the maximum permissible exposure (according to ANSI Z136.1-2000). The instrument was successfully employed to obtain non-contact and non-invasive SHG of the living human eye within well-established light safety limits. This represents the first recording of in vivo SHG images of the human cornea using a compact multiphoton microscope. This might become an important tool in Ophthalmology for early diagnosis and tracking ocular pathologies.

A conventional tool in the pathological field is histology which involves the analysis of thin sections of tissue in which specific cellular structures are stained with different dyes. The process to obtain these stained tissue sections is time consuming and invasive as it requires tissue removal, fixation, sectioning, and staining. Moreover, imaging of live tissue is not possible. We demonstrate that multiphoton tomography can provide within seconds, non-invasive, label-free, vertical images of live tissue which are in quality similar to conventional light micrographs of histologic stained specimen. In contrast to conventional setups based on laser scanning which image horizontally sections, the vertical in vivo images are directly recorded by combined line scanning and timed adjustments of the height of the focusing optics. In addition, multiphoton tomography provides autofluorescence lifetimes which can be used to determine the metabolic states of cells.

We demonstrate three-photon microscopy (3PM) of mouse cerebellum at 1 mm depth by imaging both blood vessels and neurons. We compared 3PM and 2PM in the mouse cerebellum for imaging green (using excitation sources at 1300 nm and 920 nm, respectively) and red fluorescence (using excitation sources at 1680 nm and 1064 nm, respectively). 3PM enabled deeper imaging than 2PM because the use of longer excitation wavelength reduces the scattering in biological tissue and the higher order nonlinear excitation provides better 3D localization. To illustrate these two advantages quantitatively, we measured the signal decay as well as the signal-to-background ratio (SBR) as a function of depth. We performed 2-photon imaging from the brain surface all the way down to the area where the SBR reaches ~ 1, while at the same depth, 3PM still has SBR above 30. The segmented decay curve shows that the mouse cerebellum has different effective attenuation lengths at different depths, indicating heterogeneous tissue property for this brain region. We compared the third harmonic generation (THG) signal, which is used to visualize myelinated fibers, with the decay curve. We found that the regions with shorter effective attenuation lengths correspond to the regions with more fibers. Our results indicate that the widespread, non-uniformly distributed myelinated fibers adds heterogeneity to mouse cerebellum, which poses additional challenges in deep imaging of this brain region.

We introduce a home-built laser-scanning nonlinear optical microscope, combining two-photon excitation fluorescence (TPEF), stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS). Narrowband pump and tunable Stokes pulses at 40-MHz are delivered by an Erbium-fiber laser source, thus greatly simplifying the excitation scheme and reducing the costs and maintenance with respect to standard bulk free-space lasers. After the sample, a dichroic beam splitter transmits the Stokes beam for SRS imaging and reflects the CARS or TPEF (at shorter wavelengths). Signal-to-noise ratio in SRS imaging is greatly enhanced (by up to 30 dB, reaching shot-noise-limited detection without the need of any electronic auto-balancing) thanks to the use of an innovative scheme that we call In-line Balanced Detection (IBD). IBD-SRS not only completely removes high-frequency laser fluctuations but also passively and automatically balances the low-frequency signal variation due to spatially varying sample transmission. We record SRS/CARS spectra in the 2800-3100 cm-1 Raman vibrational spectrum, thus providing a detailed chemical information on the sample in the C-H stretching region. We report various bioimaging applications of our instrument: the study of breast tumour cells using CARS, three-dimensional visualization of lipid distribution in HuH7 and in HepaRG hepatic cells using SRS and a combined TPEF/SRS study of plant cells. Microscopy in scattering media such as a bovine liver tissue is as well demonstrated.

Coherent Raman hyperspectral imaging technologies have progressed dramatically in recent years, collecting 100’s to 10,000’s of spectra per second with the spectra breadth of traditional spontaneous Raman spectroscopy. There is, however, a lack in available analysis and processing capabilities to bridge the gap between spectroscopy and chemical imaging, in which the end-user is interacting with molecular targets of interest. In this talk we will discuss our latest developments towards this goal, in particular: spectral unmixing/endmember extraction methods and high-speed, high-throughput peak characterization (peak-finding and fitting). Spectral unmixing methods aim to uncover pure species spectra. Certain demonstrated methods, such as vertex component analysis (VCA) require at least 1 pure pixel per chemical is present in the image. Other methods rely on statistical or geometric methods to estimate the pure spectra when no pure pixels are present. In this presentation, we will quantitatively compare results using several state-of-the-art techniques (internally and externally-developed). To autonomously examine retrieved pure spectra, we have developed a high-speed peak finding and fitting algorithm capable of characterizing spectra in micro- to milliseconds, in order to interface with our developed database and data mining methods. Collectively, these developments enable high-speed, high throughput analysis of 1 or many images. Numerical and experimental demonstrations will be presented on an open-source numerical tissue phantom and ~900-color BCARS imagery of murine tissue and clinical specimens.

Over the past decade the range of available excitation wavelengths used in nonlinear microscopy has continuously extended within the near infrared window. Nowadays, excitation wavelengths ranging from 750 nm to 1300 nm are routinely used to perform multi-parametric imaging [1,2] and multiple wavelength excitation are used in many techniques, including multicolor 2-photon excited fluorescence imaging, Coherent Anti-stokes Raman Scattering (CARS), Stimulated Raman Scattering (SRS) or sum-frequency generation. While this trend opens new perspectives and applications in the biomedical sciences, it also raises new technical issues. For instance, it calls for new standards for quantifying and comparing the performances of nonlinear microscopes over a broad range of wavelengths. In particular, microscopes equipped with multiple femtosecond sources spanning the entire near-infrared wavelength range are often problematic to characterize with current approaches based on fluorescent probes. In this study, we present a new and straightforward method to quantify the imaging properties of nonlinear microscopes over a broad range of excitation wavelengths [3]. We show that harmonic generation nanoprobes are a unique tool to map the spatial resolution, field curvature and chromatic aberrations of nonlinear microscopes with a precision below the diffraction limit, across the whole field of view, and with a single calibration sample. We analyze and compare measurements obtained with several microscope objectives designed for multiphoton microscopy over the 850-1100nm wavelength range. Finally, we discuss strategies to minimize the impact of chromatic aberrations during multicolor imaging and we show how our metrology can be used for the post-acquisition correction of chromatic aberrations. [1] Mahou et al. Nat. Methods 9, 815-18 (2012). [2] Alexander et al. Curr. Opin. Cell Biol. 25 , 659-71 (2013) [3] Mahou et al., submitted.

In coherent Raman scattering microscopy, the sample is examined through the Raman-active modes. Although many molecular modes display a nonzero Raman cross-section, some modes are intrinsically weak and are better viewed through their dipole-allowed (IR-active) transition. A more complete spectroscopic assessment of the sample would thus include a mechanism to prove both Raman and IR-active modes. In this contribution, we combine coherent Raman microscopy with second- and third-order sum-frequency generation microscopy, providing sensitivity to all optically accessible vibrational modes. All techniques can be carried out on the same laser-scanning imaging platform, generating images at fast acquisition rates and with high spatial resolution. We show examples where the combination of these techniques reveal new information about the tissue.

In this work, we developed a label-free imaging and spectroscopy method to assess the metabolism and thermogenesis of mouse adipose tissues in vivo. An optical redox ratio based on the endogenous fluorescence of mitochondrial coenzymes was used as a biomarker to determine the metabolic state of adipocytes during thermogenesis. The morphological and functional characteristics of different types of adipocytes were assessed in vivo and their thermogenic activities were monitored in real time with a robust spectroscope system.

Vibrationally sensitive sum-frequency generation (SFG) microscopy is a chemically selective imaging technique sensitive to non-centrosymmetric molecular arrangements in biological samples. The routine use of SFG microscopy has been hampered by the difficulty of integrating the required mid-infrared excitation light into a conventional, laser-scanning nonlinear optical (NLO) microscope. In this work, we describe minor modifications to a regular laser-scanning microscope to accommodate SFG microscopy as an imaging modality. We achieve vibrationally sensitive SFG imaging of biological samples with sub-um resolution at image acquisition rates of 1 frame/s, almost two orders of magnitude faster than attained with previous point-scanning SFG microscopes. Using the fast scanning capability, we demonstrate hyperspectral SFG imaging in the CH-stretching vibrational range and point out its use in the study of molecular orientation and arrangement in biologically relevant samples. We also show multimodal imaging by combining SFG microscopy with second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) on the same imaging platfrom. This development underlines that SFG microscopy is a unique modality with a spatial resolution and image acquisition time comparable to that of other NLO imaging techniques, making point-scanning SFG microscopy a valuable member of the NLO imaging family.

For its label-free imaging capability with chemical specificity, Raman microscopy has found various applications in biology, medicine, pharmacology, and material science. In imaging of moving samples and/or a large number of objects, however, spontaneous Raman imaging falls short due to its slow signal acquisition time. Multicolor stimulated Raman scattering (SRS) microscopy is a promising approach to this end because it can probe the vibrational signatures of molecules at multiple vibrational frequencies with orders of magnitude higher sensitivity than spontaneous Raman scattering. Here we report our two recent achievements that boost the chemical imaging speed of multicolor SRS microscopy. The first one is multicolor SRS imaging that operates at 110 frames per second by frame-by-frame wavenumber tuning. Using this method, we demonstrated video-rate single-cell imaging of intracellular metabolites in live Euglena gracilis, a highly motile microalgal species. We revealed large cell-to-cell variations in the amounts of metabolites, which is effective for efficient biomaterial engineering. The second one is four-color SRS imaging that runs at a pixel dwell time of 0.2 µs per pixel, enabled by fast wavelength switching of laser pulses. We used it to demonstrate SRS-based chemical imaging of polymer beads and living cells. Our SRS system’s fast imaging capability made high-throughput Raman imaging possible as well as blur-free Raman imaging, which are highly useful in diverse applications including cell screening and intraoperative diagnostics.

Two-color Stimulated Raman scattering (SRS) microscopy has shown great potential in label-free digital histology with diagnostic results similar to H&E stain. However, achieving real-time two-color SRS imaging is challenging. We have precisely engineered the pulse profiles of the Stokes beams, and fully utilized the in-phase (X) and quadrature (Y) outputs of a phase sensitive lock-in amplifier to realize simultaneous two-color SRS imaging. We have demonstrated its robustness and advantages in rapid histology, as well as real-time in vivo imaging of live animals, both in transmission and epi modes. Moreover, we have also adapted this method to other pump-probe based microscopes, such as transient absorption (TA) microscopy.

We have visualized living cells and tissues using an ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopic system by using a sub-nanosecond supercontinuum (SC) light source. Owing to the ultrabroadband spectral profile of the SC, we can generate multiplex CARS signals in the spectral range of 500–3800 cm⁻¹, which covers the whole molecular fingerprint region, as well as the C-H and O-H stretching regions. Through the combination of the ultrabroadband multiplex CARS method with second harmonic generation (SHG) and third harmonic generation (THG) processes, we have successfully performed selective imaging of ciliary rootlet-composing Rootletin filaments in rat retina.

To understand the mechanisms of important lipid-related biological processes and diseases, it is highly demanded to study the dynamics of lipids in living biological system with high spatiotemporal resolution. However, in vivo quantitative analysis of lipid synthesis and lipolysis has been technically difficult to achieve by conventional lipid extraction and fluorescent staining methods. Recently, SRS microscopy has emerged as a powerful tool to probe small molecules with alkyne (C≡C) or deuterium (C-D) labeling in cell-silent region. The Raman tags have been used for the quantitative study of lipids in cells. In this study, we investigated metabolic dynamics of lipid droplets (LDs) by tracing the alkyne-tagged fatty acid 17-ODYA and deuterium-labeled saturated and unsaturated fatty acids PA-D₃₁ & OA-D₃₄ in living C. elegans. Specifically, we developed a hyperspectral SRS microscope system for LDs characterization. The system can sequentially excite and probe the stimulated Raman scattering-induced CH₂ stretching of endogenous lipids information (2863 cm-1), C≡C stretching from 17-ODYA (2125 cm^-1) and C-D stretching from deuterium-labeled fatty acids (2117 cm^-1). We first examined the concentration levels of fatty acids in E. coli OP50. Two major lipid metabolic processes, namely uptake and turnover, were further studied in adult C. elegans. We imaged alkyne-tagged and deuterated fatty acids using SRS and traced their uptake, transportation, incorporation and turnover over time. Additionally, several other treatments including starvation were also conducted to study their effects on metabolic dynamics of pulse labeled 17-ODYA, PA-D₃₁ and OA-D₃₄.

Resonant coherent Raman spectroscopy based on electronic transitions is known for over 40 years to provide chemically specific information from molecules at micromolar concentrations. Unfortunately, live-cell and tissue imaging becomes challenging at shorter wavelength due to reduced threshold for cell damage and significantly increased light scattering. Thus, we evaluate resonant enhancement of vibrational transitions, which can be selectively excited to promote either stronger signal or to provide better selectivity of molecular excitation. We experimentally validate several new excitation schemes for coherent anti-Stokes Raman scattering imaging and discuss their pros and cons.

Multiphoton microscopy is an essential tool for detailed study of neurovascular structure and function. Wavelength mixing of synchronized laser sources—two-color multiphoton microscopy—increases the spectral window of excitable fluorophores without the need for wavelength tuning. However, implementation of two-color microscopy requires a dual output laser source, which is typically costly and complicated. We have developed a relatively simple and low-cost diamond Raman laser pumped with a ytterbium fiber amplifier. The dual output system generates excitation light at both 1060 nm (pump wavelength) and 1250 nm (first Stokes emission of diamond laser) which, when temporally and spatially overlapped, yield an effective two-color excitation wavelength of 1160 nm. This source provides an almost complete coverage of fluorophores excitable within the range of 1000-1300 nm. When compared with 1060 nm excitation, twocolor excitation at 1160 nm offers a 90% increase in signal for many far-red emitting fluorescent proteins (e.g. tdKatushka2). We demonstrate multicolor imaging of tdKatushka2 and Hoechst 33342 via simultaneous two-color twophoton, and two-color three-photon microscopy in engineered 3-D multicellular spheroids. Additionally, we show that this laser system is capable of in vivo imaging in mouse cortex to nearly 1 mm in depth with two-color excitation. This system can also be used to excite genetically encoded calcium indicators (e.g. RCaMP and GCaMP), which will be paramount in studying neuronal activity.

Adaptive Optics (AO) has revealed as a very promising technique for high-resolution microscopy, where the presence of optical aberrations can easily compromise the image quality. Typical AO systems however, are almost impossible to implement on commercial microscopes. We propose a simple approach by using a Multi-actuator Adaptive Lens (MAL) that can be inserted right after the objective and works in conjunction with an image optimization software allowing for a wavefront sensorless correction. We presented the results obtained on several commercial microscopes among which a confocal microscope, a fluorescence microscope, a light sheet microscope and a multiphoton microscope.

Cardiovascular risk factors, such as hypertension, have been associated with cognitive decline, potentially due to their impact on brain tissue oxygenation. In this study, high spatial resolution imaging in three dimensions was used to understand changes in brain oxygenation with hypertension. Experiments were performed on Young (WT_Y, 3-4 months, n=8), Old (WT_O, 6-7 months, n=8), and Old with hypertension (HP_O, 6-7 months, n=8) C57bL/6 awake mice. Two photon phosphorescence lifetime microscopy using an O2-sensitive phosphorescent dye PtPC343 was employed to measure two dimensional grids of PO2 in capillary beds (400um*400um, 25*25 pixels, acquired in 4 mins) and decays from arterioles. Scans were obtained continuously at depths from 50 um to 300 um under the brain surface. Using 3D measurements and a 250 um depth stack, we removed the compounding effects on brain oxygenation diffusion from surrounding brain vessels. The entire measurement of each vasculature stack required less than 30 minutes. This study indicates that among vascular risk factors, hypertension can reduce oxygen delivery and could potentially contribute to cognition decline.

We want to implement two-photon excitation fluorescence microscopy (TPEFM) into endoscopes, since TPEFM can provide relevant biomarkers for cancer staging and grading in hollow organs, endoscopically accessible through natural orifices. However, many obstacles must be overcome, among others the delivery of short laser pulses to the distal end of the endoscope. To this avail, we present imaging results using an all-fibre dispersion management scheme in a TPEFM setup. The scheme has been conceived by Jespersen et al. in 2010¹ and relies on the combination of a single mode fibre with normal and a higher order mode fibre with anomalous dispersion properties, fused in series using a long period grating. We show that using this fibre assembly, a simple and robust pulsed laser delivery system without any free-space optics, which is thus suitable for clinical use, can be realised.

A concept to adjust the output spectral bandwidth of a wavelength-tunable fiber optical parametric oscillator (FOPO) is presented. By adjusting the chirp of the pump pulses relative to the chirp of the resonant pulses, the energy of the output pulses can be reorganized into a wide or narrow spectral bandwidth. We present numerical simulations of a FOPO, which is able to generate pulses with an adjustable bandwidth between 12 and 0.8 nm. Such a FOPO should allow the cost-efficient application within two-photon excited fluorescence microscopy, which benefits from high peak powers, combined with coherent Raman microscopy, which requires spectrally narrow pulses.

We present a femtosecond fiber-based optical parametric oscillator (FOPO) for multiphoton microscopy with wavelength tuning by electronic repetition rate tuning in combination with a dispersive filter in the FOPO cavity. The all-spliced, all-fiber FOPO cavity is based on polarization-maintaining fibers and a broadband output coupler, allowing to get access to the resonant signal pulses as well as the idler pulses simultaneously.

The system was pumped by a gain-switched fiber-coupled laser diode emitting pulses at a central wavelength of 1030 nm and an electronically tunable repetition frequency of about 2 MHz. The pump pulses were amplified in an Ytterbium fiber amplifier system with a pulse duration after amplification of 13 ps. Tuning of the idler (1140 nm - 1300 nm) and signal wavelengths (850 nm - 940 nm) was achieved by changing the repetition frequency of the pump laser by about 4 kHz. The generated signal pulses reached a pulse energy of up to 9.2 nJ at 920 nm and were spectrally broadened to about 6 nm in the FOPO by a combination of self-phase and cross-phase modulation. We showed external compression of the idler pulses at 920 nm to about 430 fs and appleid them to two-photon excitation microscopy with green fluorescent dyes.

The presented system constitutes an important step towards a fully fiber-integrated all-electronically tunable and, thereby, programmable light source and already embodies a versatile and flexible light source for applications, e.g., for smart microscopy.

Ti:Sapphire lasers are powerful tools in the field of scientific research and industry for a wide range of applications such as spectroscopic studies and microscopic imaging where tunable near-infrared light is required. To push the limits of the applicability of Ti:Sapphire lasers, fundamental understanding of the construction and operation is required. This paper presents two projects, (i) dealing with the building and characterization of custom built tunable narrow linewidth Ti:Sapphire laser for fundamental spectroscopy studies; and the second project (ii) the implementation of a fs-pulsed commercial Ti:Sapphire laser in an experimental multiphoton microscopy platform.

For the narrow linewidth laser, a gold-plated diffraction grating with a Littrow geometry was implemented for highresolution wavelength selection. We demonstrate that the laser is tunable between 700 to 950 nm, operating in a pulsed mode with a repetition rate of 1 kHz and maximum average output power around 350 mW. The output linewidth was reduced from 6 GHz to 1.5 GHz by inserting an additional 6 mm thick etalon. The bandwidth was measured by means of a scanning Fabry Perot interferometer.

Future work will focus on using a fs-pulsed commercial Ti:Sapphire laser (Tsunami, Spectra physics), operating at 80 MHz and maximum average output power around 1 W, for implementation in an experimental multiphoton microscopy set up dedicated for biomedical applications. Special focus will be on controlling pulse duration and dispersion in the optical components and biological tissue using pulse compression. Furthermore, time correlated analysis of the biological samples will be performed with the help of time correlated single photon counting module (SPCM, Becker&Hickl) which will give a novel dimension in quantitative biomedical imaging.

Antimicrobial resistance is a serious global threat fueling an accelerated field of research aimed at developing novel antimicrobial therapies. A particular challenge is the treatment of microbial biofilms formed upon bacterial growth and often associated with chronic infections. Biofilms comprise bacteria that have adhered to a surface and formed 3D microcolonies, and demonstrate significantly increased antimicrobial resistance compared to the planktonic counterpart. A challenge in developing novel strategies for fighting these chronic infections is a lack of mechanistic understanding of what primarily contributes to enhanced drug resistance. Tools for noninvasive study of live biofilms are necessary to begin to understand these mechanisms on both a single cell and 3D level.

Herein, a method by which multiphoton microscopy is implemented to study a biofilm model of Staphylococcus epidermidis to noninvasively visualize and measure penetration of compounds in 3D biofilm structure and two photon excitation was exploited for spatially confined photoinactivation and microscopy optimized for evaluation of microbiological viability at a microscopic level. Future studies are aimed at future development of the proposed techniques for detailed studies of, e.g., quorum sensing and mechanisms contributing to antimicrobial resistance.

A cholestatic liver disease presents one of the most common liver diseases and can potentially progress to cirrhosis or even cholangiocarcinoma. Conventional techniques are insufficient to precisely describe the complex internal structure, heterogeneous cell populations and the dynamics of biological processes of the liver. Currently, the methods of multiphoton and fluorescence lifetime imaging microscopy are actively introducing to biomedical research. Those methods are extremely informative and non-destructive that allows studying of a large number of processes occurring inside cells and tissues, analyzing molecular cellular composition, as well as evaluating the state of connective tissue fibers due to their ability to generate a second optical harmonic. Multiphoton and FLIM microscopy do not need additional staining of samples or the incorporation of any markers to study metabolism, lipid composition, microstructure analysis, evaluation of fibrous structures. These parameters have pronounced changes in hepatocytes of liver with common pathological diseases. Thereby in this study we investigated metabolic changes in the healthy and cholestatic liver based on the fluorescence of the metabolic co-factors NAD(P)H and FAD by multiphoton microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH was presented. The data can be used to develop new criteria for the identification of hepatic pathology at the level of hepatocyte changes directed to personalized medicine in the future.

Store-operated calcium (SOC) channels, regulated by intracellular Ca²⁺ store, are the essential pathway of calcium signaling and participate in a wide variety of cellular activities such as gene expression, secretion and immune response¹. However, our understanding and regulation of SOC channels are mainly based on pharmacological methods. Considering the unique advantages of optical control, optogenetic control of SOC channels has been developed². However, the process of genetic engineering to express exogenous light-sensitive protein is complicated, which arouses concerns about ethic difficulties in some research of animal and applications in human. In this report, we demonstrate rapid, robust and reproducible two-photon activation of endogenous SOC channels by femtosecond laser without optogenetics. We present that the short-duration two-photon scanning on subcellular microregion induces slow Ca²⁺ influx from extracellular medium, which can be eliminated by removing extracellular Ca2+. Block of SOC channels using various pharmacological inhibitors or knockdown of SOC channels by RNA interference reduce the probability of two-photon activated Ca²⁺ influx. On the contrary, overexpression of SOC channels can increase the probability of Ca²⁺ influx by two-photon scanning. These results collectively indicate Ca²⁺ influx through two-photon activated SOC channels. Different from classical pathway of SOC entry activated by Ca²⁺ store depletion, STIM1, the sensor protein of Ca²⁺ level in endoplasmic reticulum, does not show any aggregation or migration in this two-photon activated Ca²⁺ influx, which rules out the possibility of intracellular Ca²⁺ store depletion. Thereby, we propose this all-optical method of two-photon activation of SOC channels is of great potential to be widely applied in the research of cell calcium signaling and related biological research.

The attenuation of excitation power reaching the focus is the main issue that limits the depth penetration of highresolution imaging of biological tissue. The attenuation is caused by a combination of tissue scattering and absorption. Theoretical model of the effective attenuation length for in vivo mouse brain imaging has been built based on the data of the absorption of water and blood and the Mie scattering of a tissue-like phantom. Such a theoretical model has been corroborated at a number of excitation wavelengths, such as 800 nm, 1300 nm , and 1700 nm ; however, the attenuation caused by absorption is negligible when compared to tissue scattering at all these wavelength windows. Here we performed in vivo three-photon imaging of Texas Red-stained vasculature in the same mouse brain with different excitation wavelengths, 1700 nm, 1550 nm, 1500 nm and 1450 nm. In particular, our studies include the wavelength regime where strong water absorption is present (i.e., 1450 nm), and the attenuation by water absorption is predicted to be the dominant contribution in the excitation attenuation. Based on the experimental results, we found that the effective attenuation length at 1450 nm is significantly shorter than those at 1700 nm and 1300 nm. Our results confirm that the theoretical model based on tissue scattering and water absorption is accurate in predicting the effective attenuation lengths for in vivo imaging. The optimum excitation wavelength windows for in vivo mouse brain imaging are at 1300 nm and 1700 nm.

In this work, we have established a double modulation lock-in detection technique using two semiconductor laser diodes in stimulated emission based pump-probe microscopy. By modulating the pump and probe beams at two different frequencies, f₁ and f₂, the signal is then recovered with the sum frequency, (f₁+ f₂), so as to minimize the leak-through noise due to the spontaneous emission caused by the pump beam. In this way, the DC background that is often attributed to the stimulated emission is effectively removed. Our technique has implemented in ATTO647N fluorescent dye which is applicable for many biological applications.

The combination of a tunable acoustic gradient (TAG) lens and a resonant mirror were integrated into a multiphoton excited (MPE) microscope that is rapidly and precisely controlled by an embedded field programmable gate array (FPGA) to acquire images at high frame rate.

The Analog mean delay (AMD) method is a multiphoton detection fluorescence lifetime measurement method. We have investigated the effect of the linearity of a photodetector response on the performance of the AMD method, which is a multiphoton detected fluorescence lifetime measurement method. A Monte-Carlo simulation scheme was adapted to generate various electronic signals for the AMD method with different linearity conditions of a photodetector. It is found that the photon economy is better if a photodetector with better linearity is used especially when the number of detected photons per pulse is low.

Acanthamoeba keratitis is a disease in which amoebae named Acanthamoeba invade the cornea of an eye. To diagnose this disease before it becomes serious, it is important to detect the cyst state of Acanthamoeba in the early stage of infection. In the present study, we explored spectroscopic signitures of the cyst state of Acanthamoeba using multimodal nonlinear optical microscopy with the channels of multiplex coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and third harmonic generation (THG). A sharp band at around 1603 cm^-1 in the CARS (Im[χ⁽³⁾]) spectrum was found at the cyst state of Acanthamoeba, which possibly originates from ergosterol and/or 7-dehydrostigmasterol. It can be used as a maker band of Acanthamoeba for medical treatment. Keyword: Acanthamoeba keratitis, coherent anti-Stokes Raman scattering, CARS, second harmonic generation, SHG, microspectroscopy, multiphoton microscopy

The microscope technology with wider view field, deeper penetration depth, higher spatial resolution and higher imaging speed are required to investigate the intercellular dynamics or interactions of molecules and organs in cells or a tissue in more detail. The two-photon microscope with a near infrared (NIR) femtosecond laser is one of the technique to improve the penetration depth and spatial resolution. However, the video-rate or high-speed imaging with wide view field is difficult to perform with the conventional two-photon microscope. Because point-to-point scanning method is used in conventional one, so it’s difficult to achieve video-rate imaging. In this study, we developed a two-photon microscope with spinning disk beam scanner and femtosecond NIR fiber laser with around 10 W average power for the microscope system to achieve above requirements. The laser is consisted of an oscillator based on mode-locked Yb fiber laser, a two-stage pre-amplifier, a main amplifier based on a Yb-doped photonic crystal fiber (PCF), and a pulse compressor with a pair of gratings. The laser generates a beam with maximally 10 W average power, 300 fs pulse width and 72 MHz repetition rate. And the beam incident to a spinning beam scanner (Yokogawa Electric) optimized for two-photon imaging. By using this system, we achieved to obtain the 3D images with over 1mm-penetration depth and video-rate image with 350 x 350 um view field from the root of Arabidopsis thaliana.

In vivo two-photon microscopy is an advantageous technique for observing living mouse brains at high spatial resolutions. We previously used a 1064 nm high-power light source based on an electrically controllable gain-switched laser diode (maximum power: 4 W, repetition rate: 10 MHz, pulse width: 7.5 picoseconds) and successfully visualized EYFP expressing neurons at deeper regions in H-line mouse brains under living conditions. However, severe damages were frequently observed when the laser power after the objective lens was over 600 mW, suggesting that a higher average power might not be suitable for visualizing neural structures and functions at deep regions. To increase fluorescent signals as a strategy to avoid such invasions, here, we evaluated the effects of the excitation laser parameters such as the repetition rate (5 - 10 MHz), or the peak power, at the moderate average powers (10 - 500 mW), by taking the advantage that this electrically controllable light source could be used to change the repetition rate independently from the average power or the pulse width. The fluorescent signals of EYFP at layer V of the cerebral cortex were increased by approximately twofold when the repetition rate was decreased from 10 MHz to 5 MHz at the same average power. We also confirmed similar effects in the EYFP solution (335 μM) and fixed brain slices. These results suggest that in vivo two-photon microscopic imaging might be improved by increasing the peak power at the same average power while avoiding the severe damages in living brains.

We compare two-dimensional auto-correlation (2D-AC) analysis and two-dimensional Fourier transform (2D-FT) for evaluation of age-dependent structural change of facial dermal collagen fibers caused by intrinsic aging and extrinsic photo-aging. The age-dependent structural change of collagen fibers for female subjects’ cheek skin in their 20s, 40s, and 60s were more noticeably reflected in 2D-AC analysis than in 2D-FT analysis. Furthermore, 2D-AC analysis indicated significantly higher correlation with the skin elasticity measured by Cutometer® than 2D-AC analysis. 2D-AC analysis of SHG image has a high potential for quantitative evaluation of not only age-dependent structural change of collagen fibers but also skin elasticity.

In this study, an automated serial two-photon microscope was used to image a fluorescent gelatin filled rodent’s brain in 3D. A method to compute vascular density using automatic segmentation was combined with coregistration techniques to build group-level vasculature metrics. By studying the medial prefrontal cortex and the hippocampal formation of 3 age groups (2, 4.5 and 8 months old), we compared vascular density for both WT and an Alzheimer model transgenic brain (APP/PS1). We observe a loss of vascular density caused by the ageing process and we propose further analysis to confirm our results.

Stimulated emission depletion (STED) microscopy is a powerful bio-imaging technique since it provides molecular spatial resolution whilst preserving the most important assets of fluorescence microscopy. When combined with twophoton excitation (2PE) microscopy (2PE-STED), the sub-diffraction imaging ability of STED microscopy can be achieved also on thick biological samples. The most straightforward implementation of 2PE-STED microscopy is obtained by introducing a STED beam operating in continuous wave (CW) into a conventional Ti:Sapphire based 2PE microscope (2PE-CW-STED). In this implementation, an effective resolution enhancement is mainly obtained implementing a time-gated detection scheme, which however can drastically reduce the signal-to-noise/background ratio of the final image. Herein, we combine the lifetime tuning (SPLIT) approach with 2PE-CW-STED to overcome this limitation. The SPLIT approach is employed to discard fluorescence photons lacking super-resolution information, by means of a pixel-by-pixel phasor approach. Combining the SPLIT approach with image deconvolution further optimizes the signal-to-noise/background ratio.

Industrial nitrogen fertilizers account for nearly 50% of the fossil fuel costs in modern agriculture and contribute to soil and water pollution. Therefore, significant interest exists in understanding and characterizing the efficiency of nitrogen fixation, and the biochemical signaling pathways which orchestrate the plant-microbial symbiosis through which plants fix nitrogen. Legume plant species exhibit a particularly efficient nitrogen uptake mechanism, using root nodules which house nitrogen-fixing rhizobial bacteria. While nodule development has been widely studied, there remain significant gaps in understanding the regulatory hormones’ role in plant development. In this work, we produce 3-dimensional maps of auxin (AX) and cytokinin (CK) hormone concentrations within model plant root tips and nodules with respect to root architecture and cell type. Soybean and Medicago plants were transfected with a two-color fluorescent vector with AXsensitive green fluorescent protein (GFP) and CK-sensitive TdTomato (TdT). 3D images of soybean root nodules were captured using two-photon induced fluorescence microscopy. The resulting images were computationally analyzed using the localization code first developed by Weeks and later adapted by Kilfoil, and analyzed in the context of the root architecture. Statistical analysis of the resulting 3D hormone level maps reproduce-well the known roles of AX and CK in developing plant roots, and are the first quantitative description of these regulatory hormones tied to specific plant architecture. The analytical methods used, and the spatial distribution of these key regulatory hormones in plant roots, nodule primordia and root nodules, and their statistical interpretation are presented.

Deep in vivo imaging of vasculature requires small, bright, and photostable fluorophores suitable for multiphoton microscopy (MPM). Although semiconducting polymer dots (pdots) are an emerging class of highly fluorescent contrast agents with favorable advantages for the next generation of in vivo imaging, their use for deep multiphoton imaging has never before been demonstrated. Here we characterize the multiphoton properties of three pdot variants (CNPPV, PFBT, and PFPV) and demonstrate deep imaging of cortical microvasculature in C57 mice. Specifically, we measure the two- versus three-photon power dependence of these pdots and observe a clear three-photon excitation signature at wavelengths longer than 1300 nm, and a transition from two-photon to three-photon excitation within a 1060 – 1300 nm excitation range. Furthermore, we show that pdots enable in vivo two-photon imaging of cerebrovascular architecture in mice up to 850 μm beneath the pial surface using 800 nm excitation. In contrast with traditional multiphoton probes, we also demonstrate that the broad multiphoton absorption spectrum of pdots permits imaging at longer wavelengths (λ_ex = 1,060 and 1225 nm). These wavelengths approach an ideal biological imaging wavelength near 1,300 nm and confer compatibility with a high-power ytterbium-fiber laser and a high pulse energy optical parametric amplifier, resulting in substantial improvements in signal-to-background ratio (>3.5-fold) and greater cortical imaging depths of 900 μm and 1300 μm. Ultimately, pdots are a versatile tool for MPM due to their extraordinary brightness and broad absorption, which will undoubtedly unlock the ability to interrogate deep structures in vivo.

Reflective objectives (ROs) can reduce chromatic aberration across a wide wavelength range in multiphoton microscopy (MPM). However, a systematic characterization of the performance of ROs has not been carried out. In this paper, we analyze the performance of a 0.5 numerical-aperture (NA) RO and compare it with a 0.55 NA standard glass objective (SO), using two-photon fluorescence (TPF) and second-harmonic generation (SHG). For experiments extending ~1 octave in visible and NIR wavelengths, the SO introduces defocusing errors of ~25% for TPF images of sub-diffraction fluorescent beads and ~10% for SHG images of collagen fibers. For both imaging systems, the RO provides a corresponding error of ~4%. This work highlights the potential usefulness of ROs for multimodal MPM applications.

Fluorescence excited by instantaneous three-photon absorption (3PA) in organic fluorophores is gaining importance as a versatile modality for deep-tissue microscopy and imaging. However, due to technical difficulty of quantifying the higher-order nonlinear absorption cross-section, reliable 3PA cross section values, σ_3PA, covering a broad spectral range have been so far not available. This lack of experimental data hinders us from gaining quantitative understanding of relevant structure-property relationships as well as impedes progress towards developing 3-photon fluorophores optimized for various applications. We report on measurement of the absolute 3PA cross section spectra in the 950 – 1750 nm range in a series of common organic fluorophores in various solvents: (a) Rhodamine 6G in deuterated methanol, (b) Coumarin 153 in DMSO and toluene, (c) Prodan in DMSO and toluene, (d) Fluorescein in pH11 buffer, (e) AF455 in toluene, (f) BDPAS in deuterated methylene chloride. In these experiments, we employ femtosecond wavelength-tunable optical parametric amplifier to excite fluorescence signal that has cubic dependence on the incident photon flux. Absolute values of σ3PA are determined using two complementary methods: (i) calibrating the fluorescence signal relative to one-photon (linear) excitation combined with accurate measurement of the pulse temporal- and spatial profile to determine the excitation photon flux and (ii) calibration of the cubic fluorescence signal relative to quadratic florescence excited in fluorophores with known two-photon absorption cross section. Depending on the method utilized, the peak σ_3PA values have estimated accuracy 50% and vary in the range, σ_3PA = 10^-81 - 10^-79 cm⁶ s² photon^-2 , depending on the system studied, with AF455 showing the most enhanced 3PA efficiency. The 3PA spectral shapes have estimated accuracy of 20% and show some unexpected deviations from corresponding one-photon spectral profiles.

Intravital microscopy using multiphoton processes is the standard tool for deep tissue imaging inside of biological specimens. Usually, near-infrared and infrared light is used to excite the sample, which enables imaging several mean free path inside a scattering tissues. Using longer wavelengths, however, increases the width of the effective multiphoton Point Spread Function (PSF). Many features inside of cells and tissues are smaller than the diffraction limit, and therefore not possible to distinguish using a large PSF. Microscopy using high refractive index microspheres has shown promise to increase the numerical aperture of an imaging system and enhance the resolution. It has been shown that microspheres can image features ~λ/7 using single photon process fluorescence. In this work, we investigate resolution enhancement for Second Harmonic Generation (SHG) and 2-photon fluorescence microscopy. We used Barium Titanate glass microspheres with diameters ∼20–30 μm and refractive index ∼1.9–2.1. We show microsphere-assisted SHG imaging in bone collagen fibers. Since bone is a very dense tissue constructed of bundles of collagen fibers, it is nontrivial to image individual fibers. We placed microspheres on a dense area of the mouse cranial bone, and achieved imaging of individual fibers. We found that microsphere assisted SHG imaging resolves features of the bone fibers that are not readily visible in conventional SHG imaging. We extended this work to 2-photon microscopy of mitochondria in mouse soleus muscle, and with the help of microsphere resolving power, we were able to trace individual mitochondrion from their ensemble.

As plane-polarized light propagates through a turbid medium, scattering alters the phase and polarization differently in different locations. The corresponding depolarization of the beam complicates recovery of the rich information content contained within the polarization-dependence of second harmonic generation (SHG) microscopy. A theoretical framework connecting Jones and Stokes formalisms for describing optical polarization allows prediction of the polarization-dependent SHG produced from “ballistic”, but depolarized incident light. Measurements with collagen-rich tissue sections support the predictions of the framework. Partially polarized SHG produced from a depolarized source enabled recovery of local orientation distribution for collagen and local tensor information. Bridging the gap between SHG instigated by fully depolarized light and partially polarized light more common to practical turbid systems, a method for predicting local nonlinear optical susceptibility tensor elements was developed and applied to collagen in thick sections. Recovered values for the tensor element ratio ρ are in good agreement with previous results for thin tissue and literature reports.

The world’s population is increasing rapidly and higher calorific diets are becoming more common; as a consequence the demand for grain is predicted to increase by more than 50% by 2050 without a significant increase in the available agricultural land. Maximising the productivity of the existing agricultural land is key to maintaining food security and agrochemicals continue to be a key enabler for the efficiency gains required. However, agrochemicals can be susceptible to significant losses and thus often require further chemical to be applied to compensate. Sources of such losses include spray drift, poor spray retention/capture by the target and poor penetration through the plant cuticle. Adjuvants can be used to help mitigate such losses but characterising how they alter the movement of the active ingredients (AIs) can be challenging. In this contribution we demonstrate the use of coherent Raman Scattering (CRS) as a tool to enable in-situ, real-time, label free characterisation of agrochemical AI as they move through wax.

Hyperspectral imaging has emerged as a new technique for the identification and classification of biological tissue1. Benefitting recent developments in sensor technology, the new class of hyperspectral imagers can capture entire hypercubes with single shot operation and it shows great potential for real-time imaging in biomedical sciences. This paper explores the use of a SnapShot imager in fluorescence imaging via microscope for the very first time. Utilizing the latest imaging sensor, the Snapshot imager is both compact and attachable via C-mount to any commercially available light microscope. Using this setup, fluorescence hypercubes of several cells were generated, containing both spatial and spectral information. The fluorescence images were acquired with one shot operation for all the emission range from visible to near infrared (VIS-IR). The paper will present the hypercubes obtained images from example tissues (475-630nm). This study demonstrates the potential of application in cell biology or biomedical applications for real time monitoring.