This PDF file contains the front matter associated with SPIE Proceedings Volume 11244, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

This talk will present a historical perspective of how multiphoton microscopy has revolutionized bioimaging and is continuing to opening up new modalities of imaging as well as the prospect of image-guided and light activated therapy .Major impetus has been provided by the development in three areas: 1) New biocompatible organic chromophores and their nanoformulations as well as inorganic nanocrystals with large optical nonlinearity; 2) Compact, user friendly and significantly lower cost ultra-short pulse lasers; 3) New designs of multiphoton microscope . They are described with examples from our work . Some Promising future directions will be discussed

None of the methods used for hyperspectral imaging work for strongly scattering tissues. In this presentation we show that using the spectral phasor approach we could obtain the spectral phasor representation at each pixel of the image using two-photon excitation and a special filter in the emission that reproduce the sine and cosine transform that is needed to measure the spectral phasor in highly scattering tissues.

This talk reviews some early applications of multiphoton microscopy at Cornell during the first decade and a half of its development. During this period, numerous collaborative imaging experiments were carried out at Cornell and by other early adopters which defined the strengths (and weaknesses) of MPM for biological imaging and led to the commercialization of MPM by BioRad Microscience and later Zeiss. In addition to imaging applications, I will also discuss the use of nonlinear excitation for 3D localized photochemical activation in femtoliter volumes, a truly unique attribute of laser scanned multiphoton microscopy.

Two-photon excitation facilitates fluorescence fluctuation spectroscopy experiments by providing a well-defined observation volume that is identical across multiple emitting species. We have developed a multi-color approach, spatial cumulant analysis (SpCA), which provides images of molecular stoichiometries. We have used SpCA to determine the dynamics of dopamine receptors (DR) on the insulin secreting β-cells. There are two inhibitory DRs in the β-cell, D2 and D3, which differentially impact cAMP production, Ca2+ influx, and K+ currents. We show how multicolor spatial cumulant analysis of labeled proteins of interest can accurately measure the stoichiometry of receptors, G-proteins, and downstream signaling targets.

Brain research is a multi-disciplinary endeavor, and inspires the development of innovative measurement tools. Multiphoton microscopy is the go-to technique for high spatial resolution, deep imaging in scattering biological tissue, and relies heavily on the new development of ultrafast lasers that deliver high pulse energy, flexible repetition rate, and wide wavelength coverage. By pushing the boundaries of imaging depth and speed, multiphoton microscopy enables large-scale, non-invasive monitoring of brain activity in live animals, and is poised to play a major role in understanding how brains work.

Current methods to assess T cell function use labels that prevent non-destructive quality control of T cell infusions. Here, we use autofluorescence imaging of NAD(P)H and FAD, co-enzymes of metabolism, in quiescent and activated T cells for label-free, non-destructive determination of T cell activation state and subtype. Logistic regression models achieved 97-99% accuracy for classification of T cell activation, and random forest models of achieved >97% accuracy for four-group classification of quiescent and activated CD3+CD8+ and CD3+CD4+ T cells. These results indicate that NAD(P)H and FAD imaging is a powerful method for label-free, non-destructive quality control of T cell infusions.

Recently, we have developed Image Scanning Microscopy (ISM) that doubles the resolution of a conventional confocal microscope by replacing the confocal pinhole with an imaging detector. Here, we describe theory and realization of a new fully optical non-linear ISM suitable for two-photon excited fluorescence and second-harmonic generation. It provides excellent sensitivity and high frame-rate in combination with two-times improved lateral resolution compared to a conventional two-photon laser-scanning microscope. We demonstrate the performance using fixed and living specimen, as well as hydrogels. The modular design allows straight-forward implementation into existing microscopes. We also present a cost-efficient FLIM-ISM detector providing super-resolved fluorescence lifetime images using two-photon excitation and can be implemented into any confocal microscope.

Quantum biology posits that non-trivial coherent macromolecular interactions can influence biological behavior. If and how nature can maintain coherence in a hot wet environment is unknown. Two-photon time-resolved fluorescence anisotropy, photon-antibunching, fluorescence correlation spectroscopy, and one-photon circular dichroism was used to demonstrate that homodimers of a yellow fluorescent protein (FP) behave coherently (excitonic coupling) at room temperature, and this coupling alters their ability to emit photons independently. This supports the hypothesis that FPs have evolved mechanisms to allow coherent interactions under physiological conditions. Since FPs are experimentally tractable, they are ideally suited for studying how nature can enable biological coherence.

The invention of Multiphoton excitation microscopy took place 30 years ago. The tremendous success and proliferation of applications related to this non-linear imaging modality resulted in the development of femtosecond tunable lasers specifically designed to address varieties of probes and optical set-ups. Recent innovations like three-photon imaging, optogenetics activation of many neurons and new approaches towards diagnostic applications fostered an even more rapid pace of innovation resulting in laser sources producing high energy tunable pulses and compact, dedicated and cost-effective sources. We will describe these new sources and how they fit diverse applications within Multiphoton excitation Microscopy

Femtosecond laser-induced plasma generation is used surgically and may also cause photodamage in nonlinear microscopy. Photodamage in multiphoton microscopy already starts at irradiances 1.5 times above the value used for autofluorescence imaging but the cavitation bubble threshold is 20 x higher. We explore the realm of low-density plasma effects between multi-pulse nonlinear imaging and single-pulse surgical regime. We characterize the transition from unchanged tissue (emitting autofluorescence) to slightly changed tissue (hyperfluorescence), drastically changed tissue (plasma luminescence) and disintegrated biomolecules (gas bubble formation). By plotting the threshold values in (irradiance, radiant exposure) space, we identified a “safe” region for nonlinear microscopy.

Antibiotic resistance kills an estimated 700,000 people each year worldwide, and study predicts that this number could rise to 10 million by 2050 if efforts are not made to curtail resistance (Nature, 2017, 543:15). Yet, the pace of resistance acquisition from mutation in pathogens is faster than clinical introduction of new antibiotics. This severe situation calls for an urgent need of developing unconventional ways to combat the resistance. To tackle this challenge, we are developing a novel phototherapy platform for fighting against a broad spectrum of drug-resistant infections. In particular, we have found that many intrinsic chromophores are probe to photobleaching through one-photon and even two-photon absorption. Importantly, these chromophores are virulence factors or essential for bacteria to survive in a stressed condition. Thus, photo-destruction of these intrinsic chromophores sensitizes the bacteria to attack by ROS or conventional antibiotics. Unlike photodynamic therapy,

We describe a metabolic-imaging system based on simultaneous recording of lifetime images of NAD(P)H and FAD. The system uses two-photon excitation by a dual-wavelength femtosecond fibre laser. The two wavelengths of the laser, 780 nm and 880 nm, are multiplexed synchronously with the frames or the lines of the scan. The recording system uses two parallel TCSPC FLIM channels, detecting from 420 to 475 nm and 480 to 600 nm. By using the multiplexing functions of the TCSPC modules, separate images for NAD(P)H and FAD are recorded. A third image is obtained for the SHG of the 880 nm laser wavelength. Data analysis delivers images of the amplitude-weighted lifetime, tm, the component lifetimes, t₁ and t₂, the amplitudes of the components, a₁ and a₂, the amplitude ratio, a₁/a₂, and the fluorescence-lifetime redox ratio (FLIRR), a_2nadh/a_1fad. We demonstrate the performance of the system for metabolic imaging of mammalian skin.

Simultaneous metabolic and oxygen imaging is a promising idea to follow up therapy response, disease development and to determine prognostic factors. A common property during tumor development is altered energy metabolism, which could lead to a switch between oxidative phosphorylation (OXPHOS) and glycolysis. The impact of this switch for theranostic applications is significant. FLIM of metabolic coenzymes, as NAD(P)H, FAD and FMN, is now widely accepted to be the most reliable method to determine cell metabolism and different algorithms are actually proved to get reproducible results. The phosphorescence lifetime of newly developed drugs is able to indicate local oxygen changes. Therefore, simultaneous imaging of phosphorescence and fluorescence lifetime parameters enables analysis of bioenergetic alterations and oxygen consumption. Dyes based on ruthenium (II) and Iridium (III) coordination complexes, were used for PLIM. For example, TLD1433, a Ru(II) complex possess a variety of different triplet states, which enables complex photochemistry and redox reactions. TLD1433 can be used as a phosphor to follow up local oxygen concentration and consumption during treatment. Alternatively ISK-1, an Ir(III) complex seems to be a perfect sensor for oxygen imaging. Within this presentation correlated luminescence lifetime imaging will be presented, applications will be demonstrated and pitfalls discussed. With respect to the last point the different redox pairs involved in cell metabolism (as FAD/FMN) will be revalued.

Traditionally, destructive sampling and analysis are used to determine the fate, kinetics and effects of exogenous materials in the body. Minimally invasive confocal and multiphoton microscopy (MPM) in 3D space over in time to deep tissue depths has enabled us to quantify endogenous fluorescent species in the body as well as exogenous fluorescent molecules, cells and nanoparticles that have been administered into the body and/or are applied to the skin, kidney and liver ex vivo and in vivo. Of particular importance has been the ability to get specificity in drug, metabolite and endogenous solute measurement in tissues in vivo by using specific spectral excitation and emission wavelengths, the use of fluorescence lifetime and the measurement of fluorescence anisotropy. We have applied MPM to characterise physiologically based pharmacokinetics of solutes, mesenchymal stem cells and nanoparticles in various organs. More recently, we have used MPM to examine stem cell and nanoparticle – tissue interactions directly in acute liver and kidney injury models, tumor models and inflammatory models. MPM has also been used to measure changes in the redox state of cells, as well the use of photochemical probes to measure adverse biochemical events such as the formation of reactive oxygen species. Sun-induced skin damage, with its sequelae of photoaging, actinic keratosis and various skin cancers is a particular issue for many of us in subtropical and temperate climates. Our group has therefore also used MPM to quantify the metabolic changes seen in melanoma lesions, the safety of nanoparticle sunscreens, whose use may prevent these lesions, and to aid in the mechanistic and regulatory evaluation of topical product efficacy, bioequivalence and safety. In conclusion, MPM fluorescence lifetime imaging microscopy (FLIM) is a promising technology to aid in product characterization and development as well as in the translational diagnosis of skin related pathologies in the clinic.

Development of an optimal systemic drug delivery strategy to the brain will require non- invasive or minimally invasive methods to quantify the permeability of the cerebral microvessel wall or blood-brain barrier (BBB) to various therapeutic agents and to mea- sure their transport in the brain tissue.We developed a new drug delivery technique combine with multiphoton fluorescence imaging to quantify the BBB permeability. We then further applied ultrasound sonication combined with microbubble injection to open the BBB for drug delivery. The transient increase of BBB permeability was imaged and quantified by using multiphoton fluorescence microscopy.

The advent of two-photon excitation (2PE) to produce microscopy images is the most important revolution in fluorescence microscopy. Since the recording of fluorescence by Sheppard within a non linear scanning microscopy scheme in 1978 to the effective application in 1990 by Denk and colleagues at the Webb labs at Cornell, 2PE microscopy changed the way of getting information from 3D biological systems in a way that is more powerful than confocal or super resolved fluorescence microscopy. The big jump, in terms of applications, took place with the advent of pulsed and mode-locked Titanium-Sapphire lasers. From the very first attempt to adapt confocal scanning heads like the ultracompact PCM2000 by Nikon to image scanning microscopy using PRISM by Genoa Instruments, a lot of solutions have been implemented. 2PE super resolved microscopy using an image scanning approach and the coupling with label-free Mueller matrix signature are the tip of an iceberg to be explored.

In this work large area reconstruction are obtained using a mesoscale two-photon system for structural analysis and a light sheet two-photon microscope for functional information. The mesoscale methodology developed allows analyzing the cytoarchitecture of the human brain in three dimensions at high resolution. Functional imaging has been used to investigate whole organ, like zebrafish larval brain activity, using standard scanning or light sheet two-photon illumination. Both modalities are capable to sample whole brain with single cell resolution, with light sheet imaging being capable to perform high rate volumetric imaging allowing to map in real time whole-brain calcium dynamics not affected by undesired visual stimulation artefacts, as occurring in one photon excitation fluorescence microscopy. Large area non linear imaging allows in general extended measurements of the neuronal activity in normal conditions under pathological modeling of epilepsy and during visual stimulation.

We propose a straightforward implementation of two-photon image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced single-photon avalanche diode (SPAD) array detector and a novel blind image reconstruction algorithm is shown to dramatically improve the optical resolution of two-photon imaging, in various test samples. We show how our computational ISM approach is able to adapt to changing imaging conditions, thus ensuring optimal image quality. We also show how our recently introduced blind deconvolution approaches can be integrated into the image reconstruction workflow to further improve the image quality.

Our contribution is focused on broadening of the spectrum of available non-linear optical (NLO)-phores (contrast agents for nonlinear optical microscopy) by design and synthesis of new organic dyes with appropriate optical properties. One of the main pre-requisites of microscopy utilizing non-linear excitation is the existence of molecules that are able to provide NLO response for the second-harmonic generation (SHG) or for the two-photon excited fluorescence (TPEF). Many molecules naturally occurring in living tissue such as collagens or NAD(P)H were successfully used in this regard, but there is a natural interest in broadening of the spectrum of available NLO-phores. Gathered results confirm applicability of the newly synthesized dyes as new potential NLO-phores for confocal laser scanning microscopy with nonlinear excitation in rat aorta.

We use SHG microscopy to study the collagen alterations in idiopathic pulmonary fibrosis (IPF) in ex vivo human tissues and in vitro spheroid models. We found the collagen fibril and macromolecular structures are altered in IPF, consistent with decreased organization. Further, machine learning based texture analysis successfully classified the fiber morphology at near 100% accuracy. To gain insight into the underlying mechanisms of these alterations, we grew IPF based spheroids under different crosslinking conditions (increased or inhibited). SHG polarization measurements (linear and SHG-CD) indicated that increased crosslinking altered the collagen helical structure similar to those of the human tissues.

Most of the cornea is organized through a lattice pattern of collagen fibrils which is responsible for its transparency. Developing avian cornea is a highly organized extracellular matrix composed largely of striated collagen fibrils. In this study, we use embryonic corneas from chicks as model animal to study the temporal and spatial variation of corneal stroma. Through the use of Fast Fourier Transform second harmonic generation microscopy, we quantified collagen alignment of the entire corneal thickness during development. Corneal structural variation at different stage of developing embryos was studied. These results indicate that both the rotational pitch and overall rotational angle of corneal stroma is highly involved in the developing process of cornea, and these properties exhibit strong correlation during the development of left and right corneas.

Molecular oxygen plays a unique role in cellular energy metabolism by serving as the terminal electron acceptor in the mitochondrial respiratory chain. Quantitative imaging of oxygen can provide invaluable information about metabolism in normal and diseased states. Two-photon phosphorescence lifetime microscopy (2PLM) enables imaging of oxygen concentration gradients in 3D with micron-scale resolution. The method has found use in neuroscience, stem cell and tumor biology. We will discuss the principles of 2PLM and focus on the new phosphorescent probes, which enable imaging of oxygen simultaneously with imaging of temperature.

Fluorescence lifetime imaging (FLIM) provides a means to contrast different molecular species and to map variations in the local fluorophore molecular environment, including to read out Förster resonant energy transfer (FRET), e.g. to assay protein interactions or genetically expressed FRET biosensors. We have implemented wide-field time-gated FLIM in a modular open automated microscopy platform for high content analysis (HCA). To demonstrate its relevance to drug discovery, we have demonstrated the capability of our openFLIM HCA platform to assay interactions of low copy number endogenous proteins in yeast cells labelled with fluorescent proteins. We have also demonstrated the capability of multiwell plate FLIM assays to provide readouts of a FRET biosensor in 2-D and 3-D cell cultures.

To overcome limitations of indirect immunofluorescence, a new method is presented to employ the ostensible disadvantage of cross-labeling secondary antibodies by separation of the fluorescence signals via spectral FLIM-FRET. The undesirable cross-labeling among secondary antibodies leads to the generation of new characteristic FRET emission spectra including a change in the donor lifetime. We used a spectrally resolved FLIM detection system with pulse interleaved excitation. The combined spectral FLIM-FRET and pattern-matching analysis forms an excellent tool for use in indirect immunofluorescence that overcomes the undesirable effect of secondary antibody cross-labeling by assigning separate color channels to cross-labeled fluorescent antibodies.

Quantification of ligand-receptor engagement in human breast cancer cells and tumor xenografts has been performed using fluorescence lifetime Forster resonance energy transfer (FLI-FRET) imaging at multiscale, from in vitro microscopy to in vivo macroscopy and across visible to near-infrared wavelengths. We have developed a 3D convolutional neural network architecture, named FLI-Network (FLI-Net), to process fluorescence lifetime decays acquired by either Time-Correlated Single-Photon Counting (TCSCP)- or gated ICCD- based instruments. FLI-FRET ability to measure target engagement across different imaging platforms as well as post-processing analysis approaches can find numerous applications in pre-clinical drug delivery and targeted therapy assessment and optimization.

We report the development of a novel massively-parallelised high-speed multifocal FLIM platform with the ability to acquire data 1024 times faster than a conventional TCSPC system. We demonstrate the system performanceFRET imaging of the fluorescent protein biosensor PercivalHR in iPSC derived neurons to measure the dynamic concentration of ADP/ATP in live cells. The advantages and performance envelope of the system will be shown and the potential for further applications explored.

Metabolic imaging of live cell may allow in understanding the molecular level changes in cells under various diseased state, including cancer. The intrinsic fluorophores, Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are crucial for electron transfer in the oxidation-reduction reactions in the cell. Metabolic imaging based on fluorescence polarization enables to analyze both biochemical distribution and their conformation. In this study, multiphoton fluorescence polarization imaging of NADH and FAD from cancer and normal cell lines of epithelial origin were carried out. Spectral deconvolution method was adopted to isolate fluorescence emission from different coenzymes NADH and FAD. The observed heterogeneity of the multiphoton autofluorescence in live cells was used in intensity-toconcentration image conversion. The multiphoton autofluorescence exhibits anisotropy features at the cellular level, that directly indicate the presence of NADH and FAD in two differing conformation states viz; free and protein-bound. Mapping of anisotropy of cellular autofluorescence enables to probe the distribution of population fractions of free and bound forms of NADH and FAD. Further, the redox ratio between normal and cancer cell lines confirms the changes in the metabolic activities between them. These molecular-level studies demonstrate the potential of probing cellular metabolism associated with cancer, without the need for cell destruction as in the case of conventional biochemical assays.

The mesoscopic scale is between microscopic and macroscopic scales. In life sciences, mesoscopic imaging allows scientists to record, track and study details of biological systems in the context of an organ, body part, or organism. Mesoscopic imaging techniques have been developed for medical and clinical research, such as drug delivery, cancer diagnosis, etc. Especially when combined with novel nanoparticles and organic dyes in the near-infrared spectral regime, the mesoscopic imaging can probe deeper parts of the animal body. Here, we describe a timeresolved mesoscopic imaging approach, which can image deep inside of the whole mouse noninvasively. In addition, it uses the FastFLIM technique to measure the lifetime of the fluorescent probe. Since the lifetime carries information about the probe’s local microenvironment such as temperature, pH, ion concentration, etc., the lifetime imaging map obtained by the FastFLIM-mesoscope allows tracking quantitative dynamics of the probes in the whole animal body. The technique can also be used for quantitative intrinsic NADH metabolism mapping for real time monitoring of mitochondrial function. Here, we will show mesoscopic-scale NADH imaging in an oral cancer model.

Deconvolution, pixel reassignment or adaptive optics-based strategies utilize information about the detection profile in improving the resolution of optical microscopy. Here, we show a novel method which allows us to obtain the single-photon detection volume of a laser scanning confocal microscope at any desired location of the object. It can create a stationary, virtual ‘guide star’ at the chosen location while the excitation beam is scanning the sample, by using an optical fiber placed in the non-descanned path of the microscope. Our experimental results are verified by diffraction theory-based calculations. The major advantages of our method are that it is alignment free, affordable, sensitive and applicable to many different modes of confocal imaging.

In this work, we have demonstrated a stimulated emission (SE) based pump-probe microscopy with subharmonic modulation and synchronization on the pump and the probe lasers. Critically, a high frequency divider circuit divides the repetition frequency (76 MHz) of the probe laser (Ti:sapphire) to the half repetition frequency (38 MHz), which in turn drives the pump laser (pulsed gain-switched diode laser) synchronously and provides the reference signal for lock-in detection. In this way, the highest possible modulation frequency can be achieved for lock-in detection with shot noise limited sensitivity. The greatly shortened time constant (< 0.1 ms) further improved imaging speed. Over an order of magnitude improvement in signal-to-noise ratio is achieved, when compared with conventional lock-in detection implement.

3-photon excitation enables in vivo fluorescence microscopy deep in densely labeled and highly scattering samples, while maintaining high resolution and contrast. We designed and characterized a dual-plane 3-photon microscope with temporal multiplexing and remote focusing, and performed simultaneous in vivo calcium imaging of two planes deep in the cortex of a transgenic mouse expressing GCaMP6s in nearly all excitatory neurons.

We will overview our initial developments of two, three, and four-photon excitation that started in early nineties with parallel developments in two-color two photon excitation to applications of light quenching (light stimulated emission) as a process to shape the excited state population. We will summarize our basic spectroscopy and time-resolved work in a cuvette system to total internal reflection applications. We will present more recent application of multi-photon processes in the case of metal enhanced fluorescence (MAF) and surface plasmons coupled emission (SPCE). Finally we will present the most recent observation of directly excited phosphorescence (S0→T1 excitation) and discuss potential applications in protein studies and cellular imaging.

Femtosecond-laser pulses can create transient holes in the membrane of a cell, making it briefly permeable to genetic macromolecules. This is a highly effective method for cell transfection and reprogramming. For sufficiently high irradiance values, the laser radiation leads to plasma formation through multiphoton ionization and a subsequent formation of gas-filled bubbles which are shortly visible after the irradiation. While this bubble formation is well known, the underlying microscopic processes, the optimal bubble size and duration which indicate the transient hole formation are less clear. The correspondence between bubble formation and successful optoporation is further complicated by the fact that the formation greatly depends on the irradiated cell position and laser irradiation parameters (power, exposure time). We have investigated the formation of bubbles resulting from short pulse irradiation with two commercial Ti:sapphire lasers using a high-speed camera. Higher laser powers and longer exposure times yielded bigger bubbles which took longer to collapse. Additionally, a correlation between the bubble characteristics and the cell’s metabolism and post-optoporation viability was found. These results can help to optimize the laser parameters for efficient optoporation and high post-treatment cell viability as well as to shine light on the microscopic excitation processes behind the bubble formation.

We will present our latest innovations about ultrafast fiber lasers and show how multiphoton microscopy can benefit from these developments. Wavelengths around 900 nm and pulse durations as short as 100 fs remain a challenge for fiber lasers. Here we present a two-color femtosecond fiber laser system with synchronous outputs. One arm emits pulses at a central wavelength of 780 nm and the novel second laser arm is continuously tunable in its central wavelength between 810 nm and 950 nm. This allows the independent excitation of NADH and FAD and therefore enables optical metabolism and oxygen imaging of cells via FLIM and PLIM measurements.

High-resolution fluorescence imaging using moxifloxacin as a clinically compatible cell labeling agent is described. Moxifloxacin is an antibiotic with good pharmacokinetic properties for tissue penetration and it has intrinsic fluorescence under ultraviolet (UV) excitation. Alternative usage of moxifloxacin as the cell labeling agent was demonstrated in two-photon tissue imaging of various tissues. Cells within tissues were visualized in enhanced contrasts with moxifloxacin. Moxifloxacin based tissue imaging was explored not only in two-photon excitation but also in both single-photon and three-photon excitations. Moxifloxacin based fluorescence imaging is clinically compatible and has potentials for clinical applications where the cellular examination is needed.

Multiphoton fluorescence lifetime imaging (FLIM) is gaining ground as a non-invasive and very sensitive method in life sciences, and even as a clinical tool. First clinical devices employing FLIM are on the market, e.g. MPTflex. A hot topic is using metabolic imaging to investigate melanoma lesions (Fig.1). 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) [1,2,3,4]. In another study, we investigated safety aspects of nanoparticle based sun screens. Multiphoton FLIM enables tracing of nanoparticles after application on the skin [5]. Furthermore, in case of penetration into the viable epidermis metabolic imaging can be employed to investigate toxicity on skin cells [6].

Multiphoton imaging has advanced the traditional fluorescence microscopy with non-invasive label-free 3D-imaging capability and high penetration depth. However, currently available systems are often bulky and employ non-flexible optical setups due to the need of a free-beam delivery with hard-to-exchange light sources. The use of an optical fiber for beam delivery offers the possibility to locate bulky and expensive components away from the point of measurement and therefore allows imaging devices to become more compact and flexible. We report on the use of a kagome hollow core photonic-crystal fiber to deliver laser pulses between laser and imaging system which enables high flexibility regarding the type of laser excitation source and its positioning. The fiber can deliver ultrashort pulses within a broadband range in the near infrared region without inducing significant spectral or temporal pulse distortions. The fiber-delivery capabilities are demonstrated with four different lasers and by employing fiber-delivered pulses for fluorescence intensity as well as lifetime imaging of human skin with a modified clinical multiphoton tomograph.

Fluorescence lifetime imaging (FLIM) of Myoglobin (Myo)-mCherry, is used for sensing oxygen partial pressure (pO2) in the intracellular environment. Herein, we present the potential sources of lifetime error such as sample oversaturation or dimeric Myo-mCherry configurations resulting in self-quenching fluorophores. We also provide a correction protocol for Myo-mCherry expression, adjusting parameters to account for second harmonic generation (SHG) components and dark counts that result in accurate mean lifetime values and pO2 in the cellular environment.

Endoscopes and other optical, non-invasive diagnostic instruments require measurable parameters (biomarkers) that reliably represent early signs of cancer. These biomarkers are challenging to identify in complex tissues due to their dependence on environmental and disease specific influences. In Multiphoton Microscopy (MPM), signals are commonly separated into channels using optical filters. The choice of channels typically relies on generalized prior knowledge. In order to establish more disease specific biomarkers, a reliable cancer model is desired. We present a method to study biomarkers using spheroids as a cancer model. The spheroid development and harvesting are monitored using Optical Coherence Tomography (OCT). We further introduce a hyperspectral MPM system to investigate biomarkers in the autofluorescence of cancerous and normal cell lines. To improve the detection of the selected biomarkers, an algorithm suggests corresponding filters for diagnostic or research purposes.

The formation and accumulation of advanced glycation end products (AGEs) contribute to diabetic complications such as retinopathy, neuropathy, nephropathy, and cardiovascular diseases. It is clear that the development of effective technique in AGEs detection and the establishing the correlation of measured AGE parameters to diabetic pathogenesis are invaluable in the monitoring of disease progression and drug discovery of anti-AGE compounds. Since some AGE are fluorescent, we propose to investigate the degree of tissue glycation in forming fluorescent AGEs (fAGEs). In this preliminary study, we will investigate the effects of glucose, fructose, and galactose, three of the most abundant dietary simple sugars, in fAGEs production. Excised tissues will be treated in solutions containing the three sugar types; multiphoton autofluorescence imaging will then be performed on the treated tissues to determine their autofluroescence levels.

A tunable acoustic gradient lens and a resonant mirror are integrated into a multiphoton excited microscope that is rapidly and precisely controlled by an embedded field programmable gate array to acquire images at 8 kHz for x-z plane. The pulse train signal of a femtosecond laser provides up to 95 MHz voxel rate, and one pulse per pixel. A volumetric image rate at up to 30 Hz can be realized according to the precise focal position and the excited signal, and deep learning. Biological images can be demonstrated at high-speed volumetric imaging rate.

Bleed-through is a common problem in multi-channel fluorescence microscopy when simultaneously imaging multiple organelles tagged with different fluorophores. This is majorly due to the large emission spectra of fluorophores. The correction of bleed-through in a multi-channel fluorescence microscopy system is essential to accurately identify or track the location of multiple fluorophores simultaneously. This paper presents a method for eliminating the bleed-through in a dual-channel fluorescence microscopy system using highly sensitive photomultiplier tubes. Fluorescein and Alexa fluor 594 dyes that were diluted in phosphate buffer solution at different dilution ratios were used to establish the relationship between the intensities of both channels. The bleed-through intensity in the red channel was quantified using a nonlinear polynomial model. Bleed-through correction was performed using the derived polynomial coefficients and the detected intensity in the green channel. The approach was experimentally validated on a mixed solution of fluorescein and Alexa fluor 594 using the scanning mode dual-channel fluorescence microscopy system.

We demonstrate the use of an all-PM fiber laser, delivering 35 fs pulses at 800 nm and a 40-MHz repetition rate, for twophoton excited fluorescence (2PEF) and Second Harmonic Generation (SHG) nonlinear microscopy. The laser has been combined to a compact group delay dispersion pre-compensation set-up to ensure the shortest pulse and so the highest peak power on the sample, minimizing the risk to damage it. We carried out measurements on vegetal samples like vine shoot or cleaning paper as well as on the forefinger of a volunteer, for current biocompatible powers under 10 mW. To the best of our knowledge, the use of an all-PM fiber laser delivering 35 fs pulses for microscopy applications has never been reported. Due to its compactness and cost-efficiency, this laser is a very attractive alternative to Ti:Sapphire modelocked lasers.

Traditionally, testing of therapeutic agents uses two-dimensional cell cultures which does not recapitulate the complex, three-dimensional architecture of tissues. In the case of cancer drug development, immunotherapy (IT) emerged as an effective treatment strategy for the patients who respond to therapy. However, the overall patient response rate for immunotherapy is around 25%. Therefore, there is a need to develop more effective drug testing platform for cancer patients. In this work, we develop three-dimensional tissue cultures of human oral cancer slices. We observed the growth dynamics of the tissue sections for up to 28 days. Their viability over extended periods suggest that the tissue sections may be used for personalized drug testing.

Rapid and in-depth labeling biological tissue samples is invaluable for studying tissue organization in three-dimensions. The current strategy in finding optimal labeling parameters for dye molecules through tissues often requires trial and error testing. Difficulty in establishing a standard approach is due to lack of information on the diffusion parameters of dye through tissues. In this study, we investigate the temporal progression of dye penetration in different tissue types. By the combination of multiphoton imaging of labeled tissues and application of the theoretical model of diffusion with well-defined boundary conditions, we aim to arrive at systematic parameters for optimizing dye penetration. Determination of the effective diffusion coefficient of the dyes used will then be useful for further application.

In the current clinical care, Gleason grading system based on the architectural pattern of cancerous epithelium in histological images is the most powerful prognostic predictor for prostate cancers (PCa). However, the standard procedure of histological examination often includes complicated tissue fixation and staining, which are time-consuming and may delay the diagnosis and surgery. In this study, the unstained prostate tissues were investigated with multiphoton microscopy (MPM) to produce subcellular-resolution images. And then, a deep learning network (AlexNet) was introduced for automated Gleason grading. We achieved an average accuracy of Gleason grading of 78.1%±3.4% for classification. And the area under the curve (AUC) in test set achieves 0.943 which indicates that the proposed model is effective in Gleason grading. At the end, the heat map was performed to visualize the Gleason score of tumour. Our results suggested that MPM, combined with deep learning method, holds the potential to be used as a real-time clinical diagnostic tool for PCa diagnosis.

Fourier transform fluorescence recovery after photobleaching (FT-FRAP) is proposed and implemented for quantitatively evaluating diffusion and fractional recovery of proteins in complex matrices. Diffusion characterization of proteins is routinely performed for identification of aggregation and for interrogating molecular interactions with excipients. Conventional FRAP is noninvasive, has low sample volume requirements, and can support short measurement times by performing measurements over distances of only a few micrometers. However, conventional measurements are complicated by the need for precise knowledge of the bleach beam profile and potential errors due to sample inhomogeneity. In FT-FRAP, the time-dependent recovery in fluorescence due to diffusion is measured in the spatial Fourier domain, with substantial improvements in the signal-to-noise ratio, mathematical simplicity, representative sampling, and compatibility with multi-photon excitation. A custom nonlinear-optical beam-scanning microscope enabled patterned illumination for photobleaching a sample through two-photon excitation. The fluorescence recovery produced simple single-exponential decays in the spatial Fourier domain. Measurements in the spatial Fourier domain naturally remove bias from imprecise knowledge of the point spread function and reduce measurement variance from inhomogeneity within samples. Comparison between the fundamental FT frequency and higher harmonics has the potential to yield information about anomalous or spatially dependent diffusion with no increase in measurement time. Initial demonstrations of FT-FRAP using patterned illumination are presented, along with a critical discussion of the figures of merit and future developments.

We demonstrate all-optical sensing and imaging of quasi-DC electric fields using vibrationally-resonant electric field-induced sum-frequency generation (VR-EFSFG). Two femtosecond laser pulses at fixed 1035 and tunable 3500 nm are mixed in a cubically-nonlinear CH-rich sample to which an external field is applied by means of metallic electrodes defined via electron-beam lithography. The 2D images are acquired by raster scanning the lasers in an all-reflective laser scanning microscope. Photon-counting detection is implemented in transmission mode. Volt-level potentials across sub-micrometer gaps are imaged at a rate of approximately 0.5 frames per second. Signal enhancement of up to 50 times due to CH vibrational resonance is typically obtained, as verified by wavelength tuning of the mid-IR laser in the range 2400-3400 wavenumbers. Nearly ideal quadratic dependence of the signal on quasi-DC field amplitude is obtained confirming purely cubic nonlinear interaction in the sample. Using numerical modeling we establish the connection to imaging of transmembrane potential on neuronal axons and derive sensitivity limits of the method.

Using our lab built two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) hybrid confocal imaging system, we observed, for the first time, the dynamic sarcomeric addition process in a rat cardiomyocyte cell culture system; this culture system expressed in vivo-like myofibril structure and mimicked mechanical overload experienced in a heart muscle tissue. Micro-grooved topographic patterned substrates com- bined with electrical stimulation are used to achieve the in vivo-like myofibril structure. After cardiomyocytes aligned, longitudinal and transverse mechanical stretch was applied to cardiomyocytes in parallel or perpendicu- lar, respectively, to the direction of alignment via stretching the substrates to mimic mechanical overload. Z discs, in which alpha-actinin expressed, have been proposed to involve in the process. TPEF detected alpha-actinin that labeled with enhanced yellow florescent protein via plasmid transfection. SHG is intrinsic to noncentrosym- metric structures, thus was used to detect myosin, a polar molecule expressed in myofibril. Pulse splitter system and synchronized recording system was introduced on TPEF-SHG imaging system to reduce the photodamage during live cell imaging. In our study, TPEF-SHG imaging system was used to study the dynamic process of sarcomeric addition in in vivo-like culture model under mechanical overload. This microscopic technique is ideal for tracking sarcomeric components to successively assemble onto pre-exist myofibrils and for revealing the role of Z discs played in sarcomeric addition. Transition of Z discs from continuous to broadened striation and from broadened to uniform striation under stretch has been observed. We concluded that continuous Z discs is the place of new sarcomeric addition.

The cost of taking a drug to market can exceed $2 billion dollars. The escalating cost of drug discovery is a major motivating factor for seeking new methods to predict the safety and efficacy of new compounds as early as possible in the drug development process to avoid drug attrition during late phases of clinical trials or even the withdrawal of approved drugs. Cardiotoxicity accounts for nearly 30% of US post-marketing drug withdrawal and remains a major concern to the point where the US Food and Drug Administration (FDA) is focused on in vitro cardiotoxicity screening to minimize cardiac risks associated with drugs. A technique that can directly quantify interactions between drugs and cardiomyocytes without the interference from exogenous genetic or chemical labels would be highly beneficial for directly screening these new drugs. Our group has previously shown that second harmonic generation (SHG) signals generated from myosin filaments in cardiomyocytes can be used as a robust label-free optical technique for recording cell shortening dynamics at high spatial and temporal resolution due to the ability of the myosin rod domains in heart muscle cells to emit the frequency-doubled light. The dynamics is recorded without adding any fluorescent labels that may otherwise affect and modify the natural cell contractility of the cell. In this study, we investigated the use of SHG microscopy for measuring drug-induced changes in cardiac cell contractility and discuss its feasibility as a tool for screening drugs and evaluating cardiotoxicity.

Conventional multiphoton microscopy uses periodically pulsed sources as excitation and the sample is illuminated uniformly by the laser. While necessary for structural imaging, monitoring dynamic biological functions such as neuronal activity in the brain typically only requires imaging of the region of interest (ROI), e.g., the neurons. The adaptive excitation source enables imaging of the region of interest only. It reduces the requirement for the output power of the excitation source (by at least an order of magnitude) and simultaneously reduces the excitation power to the sample for obtaining the necessary information (e.g., neuronal activity). We demonstrate three-photon imaging of brain activity in awake transgenic mice (jRGECO1a), with highest speed (30 frames/s), large field-of-view (620x620 μm/512x512 pixels) and deep penetration (750 μm beneath the dura).

Recent studies suggest that cancer cell response to cisplatin can not be fully described in terms of only interaction of the drug with DNA, but can include effects associated with other cellular targets. The study of effects of chemotherapeutic drugs on the viscosity of plasma membrane is important for better understanding the mechanisms of the drug action and evaluating the effectiveness of therapy. The aim of this work was to analyze microviscosity of plasma membrane of cancer cells during chemotherapy with cisplatin. For imaging viscosity at the microscopic level fluorescent molecular rotor BODIPY2 and fluorescence lifetime imaging microscopy (FLIM) were used. We detected a significant increase in membrane viscosity in viable human cervical cancer cells HeLa, both in cell monolayer and tumor spheroids after cisplatin treatment. Measuring viscosity in cisplatin-resistant cell line showed that viscosity increases when cells acquire chemoresistance. These results suggest that microviscosity of membrane plays a role in the cytotoxicity of cisplatin and its mapping may provide a powerful tool for investigation of tumor responses to chemotherapy and mechanisms of drug resistance.

Glioblastoma (GBM) is one of the most aggressive cancer types in nervous system. Due to the limited effectiveness of current treatments, prognosis remains poor for GBM patients. Altered lipid metabolism is a hallmark of GBM. Previous studies show that increased lipid droplets (LDs) could protect cancer cells from chemotherapy and other extracellular stresses. In this study, we apply stimulated Raman scattering (SRS) microscopy to image LDs to monitor uptake of palmitic acid (PA) by U-87 glioma cells. We observe that uptake of free PA rapidly upregulates adipogenesis in glioma cells. We find that glioma cells exhibit distinctive time-dependent and dose-dependent patterns in PA uptake and adipogenesis. Uptake of extra PA significantly decreases the unsaturation degree of LDs. The decreased unsaturation degree and other lipotoxicity effects lead to clear cell death upon PA treatment at a high concentration. The results indicate that inhibition of adipogenesis may have therapeutic effects for GBM utilizing the lipotoxicity effects induced by free fatty acids. This work demonstrates that SRS microscopy for label-free imaging of LDs distribution and their composition is a promising tool for lipid metabolism studies in cancer.