This PDF file contains the front matter associated with SPIE Proceedings Volume 10882, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

Multiphoton microscopy was more than a revolution in optical microscopy and more in general in optical bioimaging. In the last 25 years has enabled unprecedented exploration of biological systems including living organisms. Two-photon excitation of fluorescence is only one of the aspects that made multiphoton microscopy a key method for deciphering the biological machinery. In fact, it also opened an important window on a label-free approach by allowing intrinsic fluorescence microscopy outside the ultraviolet window and second/ third harmonic generation imaging. SHG is highly sensitive to the structure of ordered aggregates, and therefore, the control of polarisation in illumination and detection can provide additional structural information about the organisation of the investigated molecules. At the very same time, 4D (x-y-z-t) imaging at high-spatiotemporal resolutions is allowed. Recently, we introduced some intriguing schemes like single wavelength two-photon STED (SW-SPE-STED) microscopy; intensity weighted subtraction (IWS) and image scanning microscopy (ISM) to shift the effective spatial frequency cutoff to higher frequencies than the one's diffraction limited. Enhanced volumetric imaging in two-photon microscopy via acoustic lens beam shaping has been achieved, too. However, a recent important realisation in the realm of label-free approaches is given by the coupling with Mueller matrix microscopy and ptycography. The very same red laser source can be used to prime both linear and non-linear imaging. This fact makes the multiphoton microscope an architecture able to capture different messages related to the occurring light-matter interactions at the very same time. We show some examples related to Mueller matrix elements correlated with fluorescence like in the case of the chromatin-DNA organisation in intact nuclei. Here, DAPI is used for two-photon excitation fluorescence and the parameter (1,4) of the Mueller matrix, also known as circular intensity differential scattering (CIDS) element, is correlated for painting chromatin distribution in interphase nuclei. Multiphoton microscopy turns in Multi messenger multiphoton microscopy with the potential of taking advantage of machine learning approaches.

Multiphoton vibrational microscopy is opening a new window to elucidating the rules of life at molecular level. CARS and SRS microscopy has enabled high-speed vibrational imaging of living cells till video rate. However, the ultimate speed and imaging sensitivity are limited by the ultralow cross section of Raman scattering. We report a highly sensitive, high-speed chemical imaging platform based on visible beam sensing of the thermo-refractive effect of infrared absorption. A lab-built resonant circuit is used to extract the photothermal signal at the IR modulation frequency. A virtual lock-in camera is further developed to enable superfast IR-based wide-field chemical imaging at 1250 frames per second. Structural illumination is deployed to further break the diffraction limit of the visible probing beam. With molecular fingerprint information, micro-molar sensitivity and nanoscale spatial resolution, this platform is able to map drug distribution in a pharmaceutical formulation, metabolic activities inside a living cell and further brings IR spectroscopy to in vivo settings.

Molecular native fluorescence (autofluorescence) can be used as a research tool to understand the underlying mechanisms of molecular interactions and cellular processes under physiological conditions in cells and tissues. Autofluorescent NAD(P)H, and flavins (FAD) are widely utilized as biomarkers for cellular energy metabolism. Tryptophan (Trp) is another endogenous fluorescent biomarker in cancer investigations. Understanding of the intricacies of an array of diseases, from cancer to diabetes to Alzheimer’s disease requires high-resolution imaging techniques such as FLIM, FRET and FLIRR (Fluorescence Lifetime Redox Ratio) for gaining insights to cellular behavior at the molecular level. Fluorescence lifetime imaging (FLIM) is a sensitive technique to investigate NAD(P)H, FAD and Trp in living cancer cells and tissues. The characterization of these endogenous molecules help us to understand the heterogeneous distribution of the metabolic signals or mapping of the metabolic signals in these pathologies. The traditional intensity-based redox ratio includes intensity artefacts, particularly in tissues, due to differential absorption and scattering in tissues and usage of various average excitation intensity levels at different depths. We developed the FLIRR assay, measured by discrete ROIs (1 or 2x2 pixels), mapping cellular morphology and analyzing the heterogeneous environment of the lifetime distribution in prostate cancer cells. Increasing FLIRR levels serve as indicators of changing metabolic states and increased E% between Trp and NAD(P)H was seen at various time durations upon treatment with doxorubicin.

We report on a fast-acquisition FLIM system comprising a single detector, four parallel TCSPC channels, and a device that distributes the photon pulses into the four recording channels. The system features an electrical IRF width of less than 7 ps (FWHM), and a time channel width down to 820 fs. The optical time resolution with an HPM-100-06 hybrid detector is shorter than 25 ps (FWHM). The system is virtually free of pile-up effects and has drastically reduced counting loss. FLIM data can be recorded at acquisition times down to the fastest frame times of the commonly used galvanometer scanners. Fast recording does not compromise the time resolution; the data can be recorded with the TCSPC-typical number of time-channels numbers of up to 1024 or even 4096. Pixel numbers can be increased to 1024 x 1024 or 2048 x 2048 pixels. The system is therefore equally suitable for fast FLIM and precision FLIM applications.

Altered bioenergetic metabolism is a common property during tumor development and neurological disorders. It is often accompanied by a switch from oxidative phosphorylation (OXPHOS) to glycolysis and vice versa. Depending on the energy metabolism of the cells they become more oxidized (when there is more OXPHOS) or more reduced (more glycolysis). Various algorithms have been developed to correlate the metabolic state of the cells with the fluorescence lifetimes of coenzymes such as NAD(P)H and FAD and with the relative contributions of their decaying components. 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, influencing the lifetime of NAD(P)H. Also for FAD the situation is not unambiguous, taking into account that both FMN and FAD are involved in the complexes of the mitochondrial respiratory system, leading to different lifetime results. Besides, oxygen concentration and consumption has to be taken into account. Within this talk the various aspects of bioenergetic alterations will be discussed and new sophisticated methods will be presented such as correlated FLIM (fluorescence lifetime imaging) and PLIM (phosphorescence lifetime imaging) to follow up disease development and responses, for example during photodynamic therapy (PDT). Correlated FLIM/PLIM aims to provide important insights in new theranostic procedures.

This paper presents ex-vivo investigation of the brain tumors, namely glioblastoma and anaplastic astrocytoma, by macroscopic fluorescence lifetime imaging (FLIM) of endogenous metabolic cofactor nicotine amide dinucleotide (phosphate), NAD(P)H. The results of the study on the rat models indicate that the metabolism in brain tumors differs between tumor types and differs from normal brain tissue. It was also found that the brain tumors have specific optical metabolic signatures that differ them from most solid tumors, underling the complexity of glioma metabolism. The approach used in the experiments shows prospects to determine the surgical margins of gliomas and to investigate metabolic heterogeneity of the tumors on a macroscale. However, one has to be careful in the interpretation of the data obtained by FLIM.

Human skin is constantly exposed to environmental stresses such as UV light and pollution. These agents cause oxidative stress associated with reactive oxygen species (ROS) generation, that will interfere with the normal cellular redox equilibrium. As ROS are mainly produced within mitochondria, the cellular metabolic activity could be impacted by UV light. We dynamically assessed UVA light (representing the majority of solar UV rays reaching Earth surface) effects on cellular metabolic activity of reconstructed human skin using multiphoton fluorescence lifetime imaging microscopy (FLIM). Multiphoton FLIM offers non-invasive, label-free quantitative functional information on cellular metabolic activity based on the endogenous two-photon excited fluorescence (2PEF) of NADH (reduced form of nicotinamide adenine dinucleotide) and FAD (flavine adenine dinucleotide) metabolic coenzymes. The experiments were performed in both stratum granulosum and spinosum layers (T-Skin™ model, Episkin™), before and after (30 min and 2 h) UVA exposure (20 J/cm²; 20 min exposure; 320 – 400 nm). We observed quasi similar effects in both epidermal layers after UVA exposure: • Decrease of RedOx ratio NADH / (NADH + FAD) at 30 min and 2 h; • Increase in the proportion of protein-bound NADH at 2 h, and in the proportion of free FAD as early as 30 min after UVA exposure; This study shows that the effects of UVA light on epidermis, can be non-invasively evidenced and followed overtime using NADH/FAD multiphoton FLIM imaging method. Altogether, these data suggest that epidermal cells respond to UVA light by promoting oxidative phosphorylation, the most efficient metabolic pathway for ATP production.

We describe a metabolic-imaging system based on simultaneous recording of lifetime images of NAD(P)H and FAD. The system uses one-photon excitation by ps diode lasers, scanning by galvanometer mirrors, confocal detection, and two parallel TCSPC FLIM recording channels. The two lasers, with wavelengths of 375nm and 405 nm, are multiplexed to alternatingly excite NAD(P)H and FAD. One FLIM channel detects in the emission band of NAD(P)H, the other in the emission band of FAD. The FLIM data are processed by SPCImage data analysis software. For both channels, the data analysis delivers images of the amplitude-weighted lifetime, tm, the component lifetimes, t1 and t2, the amplitudes of the components, a1 and a2, and the amplitude ratio, a1/a2. Moreover, it delivers the fluorescence-lifetime redox ratio (FLIRR), a_2nadh/a_1fad. We demonstrate the performance of the system at the example of human bladder cells. Normal cells and tumor cells were discriminated by the tm images, the a1 images, and the FLIRR images.

Fluorescence lifetime imaging (FLIM) data has consistently revealed a significant difference in mean FAD lifetime between in vivo and in vitro models. We hypothesized that the observed difference in mean FAD lifetime could be a result of environmental differences, such as differing glucose levels, oxygen levels, or pH, between the two models. We investigated the effects of environmental pH on the autofluorescence lifetime of FAD. We adjusted the pH of HEPEScontaining media using sodium hydroxide and hydrochloric acid. We then replaced the normal media of plated BT474 cells with the pH-adjusted media, allowed 20 minutes for cellular changes to occur, and then imaged the cells using time correlated single photon counting FLIM. We found that the mean lifetime of FAD increased with increased pH, resulting in a significant increase between pH 3.9, 6.2, 7.4, 9.1, and 9.5. The mean lifetime of NAD(P)H decreased at pH 3.9, 9.1, and 9.5 relative to a control pH of 7.3, and the optical redox ratio showed no significant changes except at pH 3.9 relative to a control pH of 7.3. These results suggest that the difference in mean FAD lifetime between in vivo and cell culture models could result from pH changes in the cellular environment.

Multiphoton tomography based on tunable femtosecond near infrared 80 MHz laser radiation has been used to map twophoton- excited time-resolved photoluminescence with picosecond temporal resolution from in-bulk thin photovoltaic layers. The time-resolved photoluminescence reflects carrier lifetimes and is therefore an important measure for the efficiency of a solar cell. Conventional one-photon solar cell characterization methods are dominated by surface effects and cannot provide information on subsurface carrier dynamics. In contrast, by using two-photon excitation, subsurface carrier dynamics can be monitored in 3D, providing morphological and spatial information on local defects and crystalline grain boundaries We present results on time-resolved photoluminescence and second harmonic generation imaging in forward and backward directions of CdTe/CdS films by time-resolved single photon counting and false-color photoluminescence mapping. High-resolution two-photon optical sectioning was carried out with a modified multiphoton FLIM tomograph MPTflex employing near-infrared sensitive photodetectors.

The clinical diagnosis of melanocytic lesions is an ongoing medical challenge. Non-invasive tools and technologies can help to distinguish equivocal lesions. The aim of the study was to improve the in vivo diagnostic possibilities for the differentiation of benign and malignant melanocytic lesions based on combination of three imaging label-free modalities (multiphoton tomography, fluorescence lifetime imaging and optical coherence angiography). Thirty-two melanocytic lesions were studied, using multiphoton tomography, fluorescence lifetime imaging and optical coherence angiography. Multiphoton tomography features of benign melanocytic nevi were characterized by the normal morphology of both the keratinocytes and the nevus cell nests surrounded by collagen. Dysplastic nevi were characterized by their increased intercellular distances and enlarged cell nuclei. Melanomas showed the presence of melanocytes and dendritic structures in all layers of the epidermis. Analysis of the metabolic state revealed that melanomas and dysplastic nevi were characterized by enhanced glycolysis. Optical coherence angiography shows that benign nevi had regular vascular networks and equal numbers of thin and thick vessels. Vascular networks of dysplastic nevi were characterized thin curved vessels. Thick irregular spiral vessels formed a dense microvascular network of melanomas.

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of macromolecular complexes and sub-cellular structures with spatial resolution well below the diffraction limit. However, resolution in the double-digit nanometer range can be obtained only using high intensity depletion laser, at the cost of increased photo-damage, which significantly limits STED applications in live specimens. To minimize this, we use the separation by lifetime tuning (SPLIT) technique, in which phasor analysis is used to efficiently distinguish photons emitted from the center and from the periphery of the excitation spot of a STED microscope. Thus, it can be used to improve the resolution without increasing the STED beam intensity. Our approach utilizes a combination of pulsed excitation and pulsed depletion lasers to record the time-resolved photons by FastFLIM. The photons stream are successively analyzed using the SPLIT technique, demonstrating that the resolution improves without increasing the depletion laser intensity.

Light sheet microscopy has become an indispensable tool for fast, low phototoxicity volumetric imaging of biological samples; predominantly providing structural or analyte concentration data in its standard format. Fluorescence lifetime imaging microscopy (FLIM) provides functional contrast, but often at limited acquisition speeds and with complex implementation. We have developed a digitally scanned light sheet microscope for fast 2-colour volumetric imaging with imaging performed in the frequency domain at 20MHz using the PCO.FLIM camera. The camera enables rapid acquisition of two phases simultaneously at 0 and 180 degrees and with a phase shift relative to the modulated square-wave excitation. Whilst this frequency domain method has been well known for decades, application to light-sheet imaging is novel and provides straightforward functional read-out of fluorescence resonance energy transfer for protein interaction studies deep inside intact specimens such as Zebrafish. We demonstrate application of fluorescence lifetime contrast using the fluorescent protein biosensors in both live Zebrafish and organoids in digitally scanned light sheet FLIM. We apply signal processing techniques to improve data analysis and visualization and discuss this alongside practical application of real-time volumetric imaging of live biological specimens.

Increasing the speed of Fluorescence Lifetime Imaging (FLIM) is essential for imaging dynamic processes in life science. The rapidFLIM approach dramatically reduces acquisition times through a combination of fast beam scanning, hybrid photomultiplier detectors, which are capable of handling very high count rates, and TCSPC modules with ultra short dead times. With this hardware combination, excellent photon statistics can be achieved in significantly shorter time spans, allowing fast processes to be measured with the high spatial resolution offered in confocal microscopy. Depending on image size, rapidFLIM enables following dynamic processes like protein interactions, chemical reactions or highly mobile species in live cell imaging with a rate of several frames per second. The separation of overlapping fluorescence emissions in biological samples has been improved in the last years by using spectral confocal microscopy in combination with linear unmixing. However, the separation of multiple labels in biological samples remains challenging, especially when strong tissue autofluorescence (AF) overshadows specifically labeled structures. Combining the spectral approach with fluorescence lifetime measurements based on a simultaneous acquisition of both spectral and lifetime parameters could significantly improve the separation quality between multiple labels and tissue AF. We demonstrate this approach in highly autofluorescent human lung tissue, where the fluorescence signals from specific stainings are sometimes weaker than tissue AF. We use dual color Pulsed Interlevaed Excitation (PIE) in conjunction with a spectral FLIM (sFLIM) detection system featuring eight separate TCSPC timing channels and analyze the data by applying a unique pattern matching technique.

Multiphoton fluorescence lifetime imaging (FLIM) is gaining ground as a non-invasive and very sensitive research tool, and even as a method in clinical applications. Skin science is the predestined field for the latter, since skin is optically accessible without surgery. A hot topic is using metabolic imaging to investigate melanoma lesions. 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]. Another important topic which is closely bound up with skin cancer risk is safety aspects of sun screens. Multiphoton FLIM enables tracing of nanoparticle after application on the skin. Furthermore, in case of penetration through the stratum corneum again metabolic imaging can be used to investigate toxicity on skin cells [5]. References 1. O. Warburg, On the origin of cancer cells. Science (1956) 123:309-14 2. L. Pires, M.S. Nogueira, S. Pratavieira, et al. Time-resolved fluorescence lifetime for cutaneous melanoma detection. Biomed Opt Express (2014) 5:3080-9. 3. S. Seidenari, F. Arginelli, M. Manfredini, Multiphoton Laser Microscopy with Fluorescence Lifetime Imaging and Skin Cancer. In: Skin Cancer (Baldi A, Pasquali P, Spugnini EP, eds): (2014) Springer New York, 279-90. 4. M.N. Pastore, H. Studier H, C.S. Bonder, and M.S. Roberts. Non‐invasive metabolic imaging of melanoma progression. Exp Dermatol. (2016) 26:607–614. 5. A. M. Holmes, J. Lim, H. Studier, and M.S. Roberts, Varying the morphology of silver nanoparticles results in differential toxicity against micro-organisms, HaCaT keratinocytes and affects skin deposition. Nanotoxicology (2016) 10:10, 1503-1514

Single-photon avalanche diode (SPAD) imagers can perform fast time-resolved imaging in a compact form factor, by exploiting the processing capability and speed of integrated CMOS electronics. Developments in SPAD imagers have recently made them compatible with widefield microscopy, thanks to array formats approaching one megapixel and sensitivity and noise levels approaching those of established technologies. In this paper, phasor-based FLIM is demonstrated with a gated binary 512×512 SPAD imager, which can operate with a gate length as short as 5.75 ns, a minimum gate step of 17.9 ps, and up to 98 kfps readout rate (1-bit frames). Lifetimes of ATTO 550 and Rhodamine 6G (R6G) solutions were measured across a 472×256 sub-array using phasor analysis, acquiring data by shifting a 13.1 ns gate window across the 50 ns laser period. The measurement accuracy obtained when employing such a scheme based on long, overlapping gates was validated by comparison with TCSPC measurements and fitting analysis results based on a standard Levenberg-Marquardt algorithm (<90% accuracy for the lifetime of R6G and ATTO 550). This demonstrates the ability of the proposed method to measure short lifetimes without minimum gate length requirements. The FLIM frame rate of the overall system can be increased up to a few fps for phasor-based widefield FLIM (moving closer to real-time operation) by FPGA-based parallel computation with continuous acquisition at the full speed of 98 kfps.

Fluorescence lifetime imaging (FLI) is widely regarded as the most robust means to utilize Förster resonance energy transfer (FRET) to study protein-protein interactions. Upon donor excitation, FLI estimates FRET occurrence by determining the reduction of the fluorescence lifetime of the donor when in close proximity (2-10nm) of an acceptor. Recently, macroscopic FLI-FRET (MFLI-FRET) in living mice has been attained by using a near-infrared (NIR)-labeled transferrin (Tf) FRET pair. To harness the potential of multiplexing FLI-FRET in live organisms, it is necessary to employ NIR dark acceptor fluorophores to avoid spectral cross-contamination. IRDye QC-1 (QC-1, LI-COR) is a dark quencher that has a broad absorbance spectrum encompassing the NIR range. Herein, we demonstrate that QC-1 is an effective acceptor for quenching of Alexa Fluor 700 (AF700) via FRET in IgG antibody interactions. Additionally, we characterized the cellular uptake of Tf conjugated to QC-1 using confocal microscopy, NIR FLI microscopy, and wide-field MFLI imaging. The AF700/QC-1 FRET pair exhibits a linear trend in FRET with increasing A:D ratio. In vivo MFLI-FRET imaging was performed under reflectance geometry to compare Tf AF700/AF750 and Tf AF700/QC1 at A:D ratio 2:1 2, 6, and 24h post-injection. FRET was detected in the liver, an important organ for pharmacokinetic studies that shows elevated expression of transferrin receptor (TfR), but not in the bladder, an important organ for drug clearance. Although we observed slightly less FRET using AF700/QC-1 compared to AF700/AF750, both in vitro and in vivo, we found that QC-1 is suitable for FRET imaging and multiplexing approaches.

Molecular oxygen is an important reporter of metabolic and physiological status at the cellular and tissue level, and its concentration is used for the evaluation of many diseases (e.g.: cancer, coronary artery disease). The development of accurate and quantitative methods to measure O2 concentration ([O₂]) in living cells, tissues and organisms is challenging and is subject of intense research. We developed a protein-based, fluorescent oxygen sensor that can be expressed directly in cells to monitor [O₂] in the intracellular environment. We fused Myoglobin (Myo), a physiological oxygen carrier, with mCherry, a fluorescent protein, to build a fluorescence resonance energy transfer (FRET) pair, Myo-mCherry. The changes in the spectral properties of Myoglobin upon oxygen binding result in changes of the FRETdepleted emission intensity of mCherry, and this effect is detected by monitoring the fluorescence lifetime of the probe. We present here the preparation and characterization of a series of Myo-mCherry variants and mutants that show the versatility of our protein-based approach: the dynamic range of the sensor is tunable and adaptable to different [O₂] ranges, as they occur in vitro in different cell lines, the probe is also easily targeted to subcellular compartments. The use of fluorescence overcomes the most common issues of data collection speed and spatial resolution encountered by currently available methods for O₂-monitoring. By using Fluorescence Lifetime Imaging Microscopy (FLIM), we show that we can map the oxygenation level of cells in vitro, providing a quantitative assessment of [O₂].

We present an optical technique, called AB/FCS-fingerprinting, capable of characterizing fluorophore independence in aqueous solution using two-photon excitation microscopy and time-correlated single photon counting. Fluorescence correlation spectroscopy (FCS) is used to monitor fluctuations in the intensity of emissions. Auto- or cross-correlation analysis of these fluctuations can measure the average number of fluorescent molecules in an aqueous sample. Photon antibunching (AB) is observed from single quantum entities, which can emit only one photon at a time. By recording the number of coincident photons detected as a function of time between photon detections, AB analysis is used to determine the number of independent emitters in a sample. In AB/FCS-fingerprinting, the number of independent emitters in a sample is compared with the average number of fluorescent molecules in the same sample using a microscope that can be rapidly reconfigured to measure either AB or FCS from serial dilutions of a florescent sample. Since the number of fluorescent molecules is not necessarily equal to the number of independent emitters, a comparison of these values can provide insight into the independence of fluorophores in molecular assemblies. We validated this technique by measuring AB/FCS-fingerprinting of serial dilutions of mVenus, mNeonGreen, and an organic fluorophore, Alexa-Fluor-488. Experimental results were in good agreement with Monte-Carlo antibunching simulations for a single quantum emitter and with the predictions of a zero-truncated Poisson distribution model for photon antibunching from monomeric fluorophores in solution.

Functional non-linear imaging has become an essential tool to improve our understanding of how the brain works. Progress in neuroscience tools like functional probes and opsins, as well as novel imaging approaches using SLMs or adaptive optics enables to study hundreds or thousands of neurons at speeds matching the typical brain’s activity patterns. Studies in immunology and disease states on the other hand use more conventional lasers parameters and imaging tools, and benefit from a deeper integration of the laser with the microscope. In this presentation we will describe new generation lasers specifically designed to address the most advanced imaging needs in these two all-important areas. We will describe novel sources for functional brain imaging and optogenetics, based on Ytterbium fiber laser media that produce tens of watts of average power, energy per pulse of tens of microjoule at 1035 nm and unparalleled flexibility in repetition rate. Such high average powers/energies are necessary to stimulate large neuron populations when used for time interval shorter than the onset of cell damage; they can also be used to pump one or more wavelength conversion devices like OPAs and OPCPA used for 3-photon imaging and for fast volumetric Ca imaging. Application to stem cell research or disease states are generally less demanding in power and energy but requirements for high throughput and high quality images requires lasers tools that are more deeply integrated with the microscope providing fast dispersion and power management. In this presentation we will describe the state of the art of these different types of laser sources.

Multiphoton microscopy enables imaging deep inside living specimens. High photon flux is required for the nonlinear excitation of fluorescent markers, which confines the excitation to the small volume of the focal spot. This results in intrinsic optical sectioning and enables non-descanned detection of the fluorescence signal. Although tunable spectral detection has been standard in confocal microscopy for many years, it is still common in multiphoton microscopy to manually change the filters in the detection beam path of the microscope to accommodate for different fluorescence markers. We revolutionized the non-descanned detection by implementing a freely tunable spectral detector for four detection channels. This enables the adaptation to new transgenic markers in seconds and separates strongly overlapping spectra without mathematical restoration. Here, we introduce the technological concept and show its application in imaging of biological specimens demonstrating the capability of the spectral detector.

This presentation focuses on ultrafast fiber lasers as excitation light-source for advanced microscopy. We will present our latest innovations and show how multiphoton microscopy can benefit from these developments. This will propel imaging techniques like TPEF, SHG, CARS or STED among others that excel conventional microscopy techniques in a deeper penetration depth, label-free imaging capability or higher spatial resolution. Wavelengths around 900 nm for addressing certain applications with fluorescent proteins and pulse durations as short as 100 fs remain a challenge for fiber lasers. TOPTICA has now launched the third generation of ultrafast fiber lasers overcoming these difficulties. The novel FF ultra laser platform is capable of generating such short pulses at not only the wavelengths 780 nm and 1050 nm but most recently at 920 nm too. This enables the imaging of the popular green fluorescent protein (GFP) and its derivatives with lowest possible power level because of the high efficiency. In the course of this work, we present selected applications proving the suitability of this industrial grade laser family for a broad variety of microscopic applications.

We present an ultra‐low noise, near to mid infrared light source for a variety of multiphoton imaging and spectroscopy techniques. The system is based on an optical parametric oscillator (OPO) pumped by a femtosecond Ytterbium solid state oscillator with tens of megahertz repetition rate. This light source supplies three intrinsically synchronized light beams at wavelengths: 1040 nm, 1400-2000 nm (tunable) and 2200-4200 nm (tunable). Without active stabilization, the OPO preserves the shot-noise limited performance of the Yb-oscillator, along with a high long-term stability and a TEM00 beam profile. While this tuning range is already suitable for two- and three-photon microscopy, it now becomes possible to address vibrational modes and thus molecular specificity by employing further frequency conversion stages. Tailored frequency doubling provides either a narrow linewidth (0.5-1.2 nm) or a broadband (>40 nm) beam, tunable from 750-950 nm. The fixed Stokes beam of the Yb-oscillator can directly be used as a pump source for coherent Raman scattering such as SRS or CARS spectroscopy/microscopy. To this end, we will demonstrate the capability of our system for both SRS imaging at video rate with a spectral precision of 13 cm-1 as well as SRS spectroscopy with more than 400 cm-1 bandwidth in a single shot. By further mixing the two output beams of the OPO, we are able to additionally produce mid-infrared light that is tunable from 4-16 µm. With the help of vibrational sum frequency generation, our system will allow us to cover a spectral range of 700-7000 cm-1.

The human neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor that can be expressed in HEK293T cells by stable transfection. Its ligand is a 13-amino-acid peptide that binds with nanomolar affinity from the extracellular side to NTSR1. Ligand binding induces conformational changes that trigger the intracellular signaling processes. Recent single-molecule studies revealed a dynamic monomer – dimer equilibrium of the receptor in vitro. Here we report on the oligomerization state of the human NTSR1 in the plasma membrane of HEK293T cells in vivo. We fused different fluorescent marker proteins mRuby3 or mNeonGreen to the C-terminus of NTSR1 and mutated a tetracysteine motif into intracellular loop 3 (ICL3) for subsequent FlAsH labeling. Oligomerization of NTSR1 was monitored before and after stimulation of the receptor with its ligand by FLIM and homoFRET microscopy (i.e. Förster resonance energy transfer between identical fluorophores detected by fluorescence anisotropy), by colocalization microscopy and by time-lapse imaging using structured illumination microscopy (SIM).

Ophthalmic imaging by fluorescence techniques is a tool which gets more and more established in eye disease diagnosis and research. All type of clinical imaging is usually restricted to the use of endogenous fluorophores present in the tissue. The excitation and emission spectra of these fluorophores are overlapping and poorly defined. Moreover, the apparent spectra are changed by variation in the relative concentration of fluorophores and by absorbers present in the tissue. Intensity images, even those with spectral resolution, therefore deliver very limited information on the state of the tissue. A considerable improvement in the field of retinal imaging is obtained by using fluorescence lifetime imaging ophthalmoscopy (FLIO). The fluorescence lifetime measured by TCSPC is independent of the concentration, and enables the possibility to measure even the weak retinal autofluorescence. Moreover, it delivers direct information on the configuration of endogenous fluorophores, on binding to proteins or lipids, on the redox state, and on other metabolic parameters. We will describe the technical problems of FLIO data and their solutions, demonstrate the performance of existing systems in ophthalmology and present some results.

Developing large arrays of single-photon avalanche diodes (SPADs) with on-chip time-correlated single-photon counting (TCSPC) capabilities continues to be a difficult task due to stringent silicon real estate constraints, high data rates and system complexity. As an alternative to TCSPC, time-gated architectures have been proposed, where the numbers of photons detected within different time gates are used as a replacement to the usual time-resolved luminescence decay. However, because of technological limitations, the minimum gate length implement is on the order of nanoseconds, longer than most fluorophore lifetimes of interest. However, recent FLIM measurements have shown that it is mainly the gate step and rise/fall time, rather than its length, which determine lifetime resolution. In addition, the large number of photons captured by longer gates results in higher SNR. In this paper, we study the effects of using long, overlapping gates on lifetime extraction by phasor analysis, using a recently developed 512×512 time-gated SPAD array. The experiments used Cy3B, Rhodamine 6G and Atto550 dyes as test samples. The gate window length was varied between 11.3 ns and 23 ns while the gate step was varied between 17.86 ps and 3 ns. We validated the results with a standard TCSPC setup and investigated the case of multi-exponential samples through simulations. Results indicate that lifetime extraction is not degraded by the use of longer gates, nor is the ability to resolve multi-exponential decays.

Imaging viscosity and its spatiotemporal patterns can provide valuable insight into the underlying physical conditions of biochemical reactions and biological processes in cells and tissues. One way to measure viscosity and diffusion is the use of fluorescence recovery after photobleaching (FRAP). We combine FRAP with FLIM and time-resolved fluorescence anisotropy imaging (tr-FAIM), by acquiring time- and polarization-resolved fluorescence images in every frame of a FRAP series. This allows us to simultaneously monitor translational and rotational diffusion. This approach can be applied to measuring diffusion in homogeneous and heterogeneous environments, and in principle also allows the study of homo-FRET. Another way to measure viscosity and diffusion is through specific flexible dyes, e.g. fluorescent molecular rotors, whose fluorescence quantum yield and fluorescence lifetime depend on the viscosity of the environment, in combination with fluorescence lifetime imaging (FLIM). We show that a bodipybased fluorescent molecular rotor targeting mitochondria reports on their viscosity, which changes under physiological stimuli. Both methods can optically measure viscosity and diffusion on the micrometer scale.

Both ovarian cancer and idiopathic pulmonary fibrosis (IPF) are accompanied by significant collagen remodeling in the respective extracellular matrix (ECM). These diseases have similar attributes and collagen alterations can be probed with the same methods. Remodeling can be reflected in increased collagen concentration, changes in alignment within fibrils and fibers and/or up-regulation of different collagen isoforms. We used pixel-based SHG polarization analyses to discriminate the macro/supramolecular collagen structure in human tissues by: i) determination of the helical pitch angle via the single axis molecular model, ii) dipole alignment within fibrils via anisotropy, and iii) chirality via SHG circular dichroism (SHG-CD). For ovarian cancer, the largest differences were between normal stroma and benign tumors, consistent with gene expression showing Col III is up-regulated in the latter. The tissues also displayed differing SHG anisotropies and SHG-CD responses, consistent with randomization of Col I alignment in fibrils in all tumors. These results collectively indicate the fibril assemblies are distinct in all ovarian tissues and likely result from synthesis of new collagen rather than remodeling of existing collagen. For IPF, the largest change was in the SHG-CD response, indicating the fibrotic collagen has different helical structure than that of normal tissues. Interestingly, for both diseases, no increase in Col IIII was found, in contrast to previous reports by immunostaining. We suggest these polarization-based metrics could form the basis of a new classification scheme and complement conventional classification based on genetic profiles and conventional histology for these diseases as well as other cancers and fibroses.

Cornea is one of the collagen-rich connective tissues and plays an important role in vision. While X-ray scattering techniques are able to determine bulk structure of cornea, second harmonic generation microscopy can reveal depthdependent details of the corneal stroma comprised of a layered network of fibrillar collagen. In this work, we used Fast Fourier Transform second harmonic generation microscopy as a tool to determine the directionality of corneal stroma as a function of depth. Our results also display the position dependent difference of corneal stroma architecture.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are an unlimited ex vivo supply of heart cells for cardiac applications. The establishment of pure iPSC-CMs populations is crucial for downstream medical applications such as human disease modeling, patient-specific stem cell therapy, human transplantation, and drug development. However, a significant challenge is the lack of an established purification method to isolate populations of iPSC-CMs by their phenotype, maturity, and subtype due to the lack of specific iPSC-CM markers. The ability to remove potentially teratoma forming pluripotent stem cells, arrhythmia inducing immature and pacemaking cells, and other non-CMs is extremely important for engineering tissues with desired cell compositions that are both safe for human transplantation and that can accurately replicate cardiac functions. Contemporary purification techniques have either low specificity or require genetic modification. We have proposed that second harmonic generation (SHG) signals, which are known to originate from the sarcomeric myosin filaments in cardiomyocytes, can be a highly specific, labelfree marker for identifying iPSC-CMs. Here, we demonstrate the use of SHG microscopy for characterizing iPSC-CMs and their subtypes.

We have developed a system capable of resolving Two-Photon Fluorescence Emission (TPFE) and Second Harmonic Generation (SHG) signals with high spectral resolution for the characterization of biomarkers. In Multiphoton Microscopy, those biomarkers are TPFE and SHG signals that carry valuable information on morphological and functional biological features, such as the presence of Green Fluorescent Protein (GFP) in a Zebrafish during the building of organs, or the ratio of pyridine nucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) in the classification of cancerous tissue. For this purpose, separation of different signals into channels is typically achieved by the use of optical filters. In doing so, signal spectra can be unknown or overlapping, creating a crosstalk in between the channels. Previously the choice of such filters relied on prior knowledge or heuristic testing. Our system allows for the study of biomarkers due to spectrally resolved imaging. It therefore enables the appropriate selection of channels, tailored to the application, when building fast diagnostic systems. Additionally, knowledge of the spectra can be used to avoid the crosstalk in between channels or compensate for it computationally. To demonstrate the capabilities of the system, we recorded spectrally resolved images of tissue and cell samples. Structural and functional biological features were identified and their spectra could be evaluated. Thus, appropriate filter setups for diagnostic imaging can be suggested and confirmed by means of integration over defined virtual channels.

We developed a spectrally-resolved multi-photon imaging system, based on a closed loop piezoelectric sample scanning stage, a transmission grating and an EMCCD. The method allows detailed analysis of the spectrally-resolved signals, including deconvolution of the resulting emission peaks, and precise differentiation of the resulting signals. The system also makes available the possibility of using point spread function analyses, such as pixel reassignment, Airy scan and full three-dimensional, spectral imaging. We present multi-photon induced fluorescence, second harmonic and fluorescence lifetime imaging analysis supporting the development of non-invasive photo-polymerizable scaffolds for treatment of peripheral artery disease. Currently, the technology, developed by Alumend, LLC, is undergoing clinical trials and is licensed by Alucent Medical. In this work we report a comparison of the second harmonic generation and two photon induced fluorescence imaging in drug-infused arteries, and compare these to fluorescence lifetime images obtained using our commercial (Picoquant) fluorescence lifetime imaging system. Primary signals from the endogenous fluorescence from the drug and the second harmonic generation, prevalent in collagen, are compared. Of particular interest, we observe the photo-chemical modification of the drug fluorescence emission energy and lifetime in the adventitia, composed primarily of collagen. The drug aids in cross-linking the compressed collagen fibrils in the arterial wall during the light activation step, which leads to formation of the stent.

A method is demonstrated enabling polarization analysis of second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) measurements of tissue samples driven by partially or wholly depolarized light. Partial depolarization routinely arises in tissue imaging, but is incompatible with standard Jones tensor formulations for polarization analysis of SHG. A more general Mueller tensor mathematical framework supports compatibility with partially or wholly depolarized incident light, but at the expense of significant increase in unknown parameter and additional mathematical complexity. In this work, the Mueller tensor can be cast in terms of the simpler and more intuitive Jones tensor, greatly reducing the number of potentially unique parameters (from 64 in the general Mueller tensor to as few as 2 in SHG of collagen). Using this architecture, local-frame tensor elements in thick, partially depolarizing tissues were recoverable with no substantial increase in mathematical complexity relative to conventional polarizationdependent nonlinear optical imaging. These results provide a relatively simple but mathematically rigorous framework for integrating partial depolarization effects in biological imaging, supporting polarization analyses in broad classes of samples that were otherwise limited to thin tissue sections.

In obesity, subcutaneous white adipose tissue (scWAT) is often marked by reduced adipogenesis, hypoxia, adipocyte hypertrophy, and impaired lipolysis, all of which contribute to overall metabolic dysfunction. The role of the peripheral nervous system is appreciated in the control of WAT lipolysis; sympathetic denervation in WAT blocks lipolysis to a variety of lipolytic stimuli. Yet, we believe additional processes and work is needed to more fully understand adipose hypertrophy. Obese adipose tissue is fibrotic with excess deposition of collagen, yet there is limited work demonstrating the impact of fibrosis on overall tissues structure and peripheral nerves of the scWAT. Here we present a multiphoton approach to image both the peripheral nerves labeled with Alexa 488 and collagen via Second Harmonic Generation (SHG) imaging to investigate the impact of obesity and related fibrosis. The degree of colocalization between the nerve and collagen tissues was examined using both the Pearson’s coefficient and a recently adapted astrophysics technique, the Metric Space Technique (MST). In preliminary findings, both colocalization approaches demonstrated an increase colocalization of nerve and collagen in the aged and obese mice. The MST technique has multiple output functions that are used to examine morphological features such as structure of the nerve/collagen network as well as the circularity of these structures. Both colocalization techniques showed different in the obese mice and indicated a more diffuse circular nerve/collagen network. Collectively, these metrics provide important quantification of the nerve/collagen interaction and the role of the peripheral nervous function in maintaining appropriate adipose function.

Multiphoton tomography based on near infrared femtosecond laser technology has become a versatile high-resolution clinical- and research-imaging tool. Here, we report on a novel moveable multimodal tomograph, the MPTcompact, based on an ultracompact chiller-free fiber-laser system. The tomograph provides in vivo optical biopsies with subcellular resolution as well as optical metabolic imaging capability based on two-photon autofluorescence, second harmonic generation, fluorescence lifetime imaging, reflectance confocal microscopy, and white light CCD imaging. Applications include cancer detection, skin research, cornea research, and in situ evaluation of anti-ageing drugs and pharmaceuticals.

Intravital multiphoton microscopy was used to study hepatobiliary metabolism in chronic pathologies of the liver. Through the use of the probe molecule 6-carboxyfluorescein diacetate (6-CFDA), the effects of liver fibrosis, fatty liver, and hepatocellular carcinoma on the metabolic capabilities of mouse liver were investigated. After the acquisition of time lapse images, a first order kinetic model was used to calculate rate constant resolved images of various pathologies. It was found the ability of the liver to metabolically process the probe molecules varies among different pathologies, with liver fibrosis and fatty liver disease negatively impacted the uptake, processing, and excretion of molecules.

Two-photon microscopy (TPM) is one of the most important imaging techniques in biological imaging since it was invented in 1990s. Due to its unique capabilities, this technique enables noninvasive study of scattering biological specimens in three dimensions with submicrometer resolution and penetration depth up to a few hundred micrometers. Focal modulation microscopy (FMM) provides sub-cellular spatial resolution at large penetration depths in tissue samples by rejecting out-of-focus signal. Combined with focal modulation techniques, this paper proposes two-photon focal modulation microscopy (TPFMM) to further enhance penetration depth by bringing a spatiotemporal phase modulator (STPM) in the TPM. The STPM is equivalent to a time-dependent phase-only pupil filter that alternates between a homogeneous filter and an inhomogeneous filter. When the STPM is homogeneous, the excitation beam is properly focused into the focal volume by the objective lens. The inhomogeneous filter is so designed that it leads to redistribution of the excitation beam and minimize the focal intensity, which can be binary phase and continuous phase distribution. Using the vectorial diffraction theory, we have theoretically demonstrated that TPFMM with the designed STPM can significantly suppress the background contribution from out-of-focus ballistic excitation and achieve almost the same resolution as TPM. The improved background rejection of this imaging modality, enabled by focal modulation, are quantified with three dimensional imaging data obtained from fluorescent beads and fixed tissue samples using a home-made TPFMM. These investigations have its potential to extend the penetration depth of nonlinear microscopy in imaging multiple-scattering biological tissues, such as mouse brain.

Optical approaches have broadened their impact in recent years with innovations in both wide-field and super- resolution imaging, which now underpin biological and medical sciences. Whilst these advances have been remarkable, to date, the ongoing challenge in optical imaging is to penetrate deeper. TRAFIX is an innovative approach that combines temporal focusing illumination with single-pixel detection to obtain wide-field multi- photon images of fluorescent microscopic samples deep through scattering media without correction. It has been shown that it can image through biological samples such as rat brain or human colon tissue up to a depth of seven scattering mean-free-path lengths. Comparisons of TRAFIX with standard point-scanning two-photon microscopy show that the former can yield a five-fold higher signal-to-background ratio while significantly reducing photobleaching of the specimen. Here, we show the first preliminary demonstration of TRAFIX with three-photon excitation imaging dielectric beads. We discuss the advantages of the TRAFIX approach combined with compressive sensing for biomedicine.

GaAsP hybrid detectors, which is new kind of photodetector, has been known as its excellent performance in time correlated single photon counting technique. We have verified that this detector also shows excellent performance in analog mean-delay method, which is another kind of time-domain FLIM, so one can expect enhancement of performance in time-domain FLIM when using the hybrid detector.

Cytochrome c is a small heme protein located within the inner membrane of the mitochondrion. It is an essential component of the electron transport chain. Its redox state is closely related to mitochondrion functions, such as ATP generation and oxygen utilization. Therefore, distinguishing the redox state of cytochrome c represents a potentially novel method for monitoring mitochondrial function in conditions where alterations in electron transport are implicated, such as diabetes, cancer and mitochondrial disease. In our experiment, a pair of pump and probe ultrafast laser pulses, with duration of 750 fs, have been focused at the same spot in a tissue slice. The difference in arrival time of the two pulses can be adjusted with picosecond resolution. It is known that a pump pulse readily dissociates one of the amino acids attached to the central iron ion, but only in the reduced form of cytochrome. This alters the absorption spectrum of reduced cytochrome until the ligand reattaches in about 5ps. That absorption change can be detected by pump-probe microscopy and allows distinction between redox states. By using 533nm pump and 490 nm probe, our system is able to distinguish redox state of cytochrome c in solution. We also have acquired pump-probe response from live insect muscle, selected for high cytochrome content and absence of hemoglobin and myoglobin, and are progressing towards redox imaging of tissue.

Temporal focusing multiphoton excitation microscopy (TFMPEM) is insufficient to perform efficient excitation of fluorophores with the different two-photon absorption spectra in the multi fluorophores imaging via an illuminated configuration of fixed wavelength and polarization direction of the pulse laser because of the a diffraction device. In this study, we overcome and causes it is not useful for in the practical application and its development. In order to implement the temporal focusing microscope system could be able to sever for the different excitation wavelength range of 700-1000 nm, we work on the optical design of the temporal focusing an integration stage and scanning mirror with automatic tuning system for any excitation wavelength. The novel TFMPEM has successfully provided dynamic spectral absorption, 3D two-photon or SHG images with the original ability of the high frame rate.

More people die from melanoma after a Stage I diagnosis than after a Stage IV diagnosis, because the tools available to clinicians do not readily identify which early-stage cancers will be aggressive. Near-infrared pump-probe microscopy detects fundamental differences in melanin structure between benign human moles and melanoma, and also correlates with metastatic potential. However, the biological mechanisms of these changes have been difficult to quantify, as many different mechanisms can contribute to the pump-probe signal. Here we use model systems (sepia, squid and synthetic eumelanin), cellular uptake studies, and a range of pump and probe wavelengths to demonstrate that the clinically observed effects come from alterations of the aggregated mode from “thick oligomer stacks” to “thin oligomer stacks” (due to changes in monomer composition) and de-aggregation of the assembled eumelanin structure. This provides the opportunity to use pump-probe microscopy for the detection and study of melanin-associated diseases.

Altered lipid metabolism is increasingly recognized as a signature of cancer cells. Enabled by label-free spectroscopic imaging, we performed quantitative analysis of lipogenesis at single-cell level in human clear cell renal cell carcinoma (ccRCC), which accounts for about 90% kidney cancers. Our hyperspectral stimulated Raman scattering (SRS) imaging data revealed an aberrant accumulation of lipid droplets in human clear cell renal cell carcinoma (ccRCC), but no detectable lipid droplets in normal or benign kidney tissues. We also found that such lipid accumulation was significantly higher in low grade (Furhman Grade≤2) ccRCC compared that in high grade (Furhman Grade≥3) ccRCC, and was correlated well with the prognosis of ccRCC. Moreover, cholesteryl ester is the dominant form of lipids accumulated in ccRCC. Besides, the unsaturation level of lipids was significantly higher in high grade ccRCC compared to low grade ccRCC. Furthermore, depletion of cholesteryl ester storage significantly reduced cancer proliferation, impaired cancer invasion capability, and suppressed tumor growth and metastasis in mouse xenograft and orthotopic models, with negligible toxicity. These findings herald the potential of using lipid accumulation as a marker for diagnosis of human ccRCC and open a new way of treating aggressive human ccRCC by targeting the altered lipid metabolism.

Solid dosage formulations remain the most important dosage forms for pharmaceuticals. In all solid formulations, the most important properties are the stability and bioavailability of the active pharmaceutical ingredient (API). Different polymorphs of API crystals often differ in the physicochemical properties like chemical and physical stability, solubility and dissolution. These differences leads to variability in drug efficacy, bioavailability, and even toxicity. A thorough understanding of how excipients and physical processing impact the polymorphism, stability, and dissolution rate of API is crucial to accelerate drug development and regulatory approval. However, currently no technology can monitor the dynamic chemical changes of solid formulation in situ at sub micrometer resolution during processing or dissolution. SRS microscopy is a powerful chemical imaging technique that can potentially address these challenges. In particular, it allows label-free imaging of APIs and excipients at high spatial and temporal resolution. Here we present our recent work on using hyperspectral SRS microscopy to resolve polymorphic changes of APIs at low drug loading. In addition, we demonstrate that we can monitor the dissolution of individual entecavir drug crystals in a slow-release implant drug sample. Together, these experiments demonstrate that hyperspectral SRS microscopy can be a valuable technique for resolving details of drug instability and drug dissolution in formulation research.

Quantitative determination of the chemical composition of unstained samples, non-invasively, with high three- dimensional spatio-temporal resolution, will accelerate progress in cell biology. The current state of the art in bioimaging is dominated by either chemically non-specific or invasive methods. In this work, we demonstrate label-free, non-invasive quantitative volumetric imaging of human osteosarcoma cells using coherent anti-Stokes Raman scattering microscopy. A data analysis method developed in-house was applied to represent the chemical composition of the cells as volumetric three-dimensional images indicating water, proteins, DNAP (mixture of DNA and proteins), and lipids, and to determine the dry masses of the organic components with picogram resolution.

Compared with conventional histology, Stimulated Raman scattering (SRS) microscopy provides high specificity, fast speed and label-free histopathological analysis of the lesions by mapping their chemical compositions. However, benchtop SRS microscopy is limited to its bulky size to access the tissues of interest in-vivo inside the human body. To enable SRS in-vivo label-free histology, here, we develop an implantable fiber-scanning SRS endoscope. The endoscope is capable of providing hyperspectral Raman images at C-H and C-D regions. We use a double-clad single-mode fiber to deliver the pump and Stokes femtosecond pulses through the core and collect back-scattering signals through the outer cladding. To remove the nonlinear background induced by the pulse interactions in the fiber, we temporally separate the two pulses by tuning a delay line. We custom-design a micro-objective made of high-dispersive ZnSe glass which enables a simultaneously focusing and recombining the two pulses at spatial and time domains on the sample for excitation. A piezo actuator is designed to resonantly scan the fiber cantilever with spiral patterns. By establishing this technology, we expect the SRS endoscope to have great potential in medical applications such as label-free image-based diagnosis and surgical guidance.

Raman microscopy has the potential of creating a molecular contrast in unstained tissue with high specificity at sub-cellular resolution. To overcome the low signal level in raster-scanned microscopy, commonly non-linear Raman techniques are employed. Imaging is usually performed in the CH-stretch region around 3000cm-1 with Raman contrast by spectral unmixing of the broadband lipid and protein Raman modes. Imaging in the fingerprint region around 1500cm-1 can yield higher information density due to the more specific molecular signatures. However, the problem is that in the fingerprint region the Raman signals are generally lower. We present Raman imaging in the fingerprint region of unstained tumorous tissue samples from human pharynx biopsies employing the time-encoded (TICO) technique. We chose pharynx tissue because this sample location can be accessed by future TICO-Raman endoscopes. In our fiber-based TICO-Raman system the stimulated Raman gain (SRG) signal is encoded in time by a Raman pump laser (1065nm, 600ps pulse length) synchronized to a wavelength-swept Fourier-Domain Mode-Locked (FDML) fiber laser. We sample the fingerprint region between 1300cm-1 – 1900 cm-1 with shot-noise limited sensitivity by employing a dual-balanced, digital and analogue balanced detector. The Stokes laser is a newly developed dispersion compensated FDML laser providing ultra-low noise and long coherence length at 400kHz sweep frequency and 100nm span around 1300nm. We investigate the possibility of applying higher instantaneous powers to increase the signal-to-noise ratio for imaging by lowering the relative shot-noise level suitable for detecting weak, narrowband Raman transitions in the fingerprint region.

Infrared (IR) absorption microscopy is a general method for generating images with vibrational spectroscopic contrast. IR microscopy has been used in a myriad of applications, including imaging of excised tissue samples. One of the major drawbacks of the technique is the limited spatial resolution, which is too low to resolve intracellular details. We developed a new nonlinear optical imaging technique, called third-order sum-frequency generation (TSFG), which addresses this issue. This approach uses an infrared IR pulse to excite fundamental molecular vibrations and a near-infrared (NIR) pulse for two-photon upconversion, producing a visible signal. TSFG is sensitive to the same molecular modes as probed in IR absorption microscopy, but offers a spatial resolution that is one order of magnitude better and enables straightforward detection in the visible range of the spectrum. TSFG is a third-order optical imaging technique, and can be regarded as the IR analogue of coherent anti-Stokes Raman scattering (CARS). We show that TSFG enables fast laser-scanning microscopy of biological samples with a resolution of 0.5 micron or better.

Metabolic dynamics is essential to unraveling the mechanistic basis of many biological processes, and cannot be imaged in vivo by using traditional methods. Here we developed a new method that combines D2O probing and SRS (DO-SRS) to visualize metabolic dynamics in live animals. The enzymatic incorporation of D2O-derived deuterium (D) into biomolecules will generate carbon-deuterium (C-D) bonds in macromolecules. We developed spectral unmixing methods to obtain C-D signals with macromolecular selectivity, and applied DO-SRS to reveal metabolic dynamics in C. elegans, zebrafish, and mice, to study developmental biology, aging, and tissue homeostasis, and to visualize tumor boundaries and metabolic heterogeneity.

I present a newly developed machine intelligence technology called “Intelligent Image-Activated Cell Sorting” [Cell 175, 1 (2018)] that achieves real-time fluorescence-image-based intelligent cell sorting at a high throughput of ~100 events per second. This technology builds on a unique integration of high-throughput cell microscopy, focusing, and sorting techniques on a unique software-hardware platform and hence performs fully automated operation for data acquisition, data processing, decision making, and actuation. Also, I introduce the technology’s broad utility to addressing a fundamental biological question that cannot be answered by conventional technologies – how molecular architectures of cells are connected with their physiological functions.

Advanced spectroscopic tools are now widely utilized in microscopic optical imaging. Most of those techniques have already reached the level of “quantum limited” detection using classical light. In this report I will explored quantum-optical approaches to microscopic optical imaging. Using two selective examples of Raman and second-harmonic imaging, I will illustrate potential advantages of such approaches and discuss potential challenges and instrumentation developments.

Improving the sensitivity of mammograms in breast cancer screening has increased the detection of suspicious findings such as calcifications and masses at the cost of a high false positive rate (55-85%). Additionally, the diagnostic interpretation of biopsies varies (75.3% concordance), leading to suboptimal treatments and poor patient outcomes. The goal of this pilot study is to investigate whether the chemical composition of breast calcifications, present in more than 80% of mammograms, can be used to improve breast lesion classification. We hypothesized that the spatial and compositional variation of breast calcifications strongly correlates with breast malignancy. To test this hypothesis, we used an advanced Raman imaging technique called hyperspectral stimulated Raman scattering (hsSRS) microscopy to study 12 patient cases (30 calcifications). We characterized unique Raman signatures of type I (calcium oxalate) and type II (calcium hydroxyapatite) calcifications in archival breast tissue at high speed and spatial resolution with hsSRS microscopy. We found that the carbonation level of hydroxyapatite decreases when comparing benign and atypical ductal hyperplasia. However, the average carbonation of hydroxyapatite was highly variable in fibroadenoma cases (3±0.6%) and DCIS (4±1.1%). In the case of DCIS, the carbonation of hydroxyapatite varied relative to the grade and the neoplastic microenvironment (nearby inflammation, necrosis, and more.) In high-grade DCIS, the carbonation was lowest around the periphery where the contact with neoplastic cells was present. Our preliminary results indicate that microcalcifications change with the neoplastic microenvironment. Further studies of neoplastic progression in association with microcalcifications can improve the statistical value of the correlation.

Label free imaging is becoming of increasing interest for medical optics. Two- and Three-photon fluorescence, second harmonic generation (SHG), and coherent anti stokes Raman scattering (CARS) are key modalities for future in–situ diagnosis approaches in diseases such as cancer and neuropathologies. The addition of polarization monitoring provides moreover new elements of information on the way molecules are organized, making use of the coupling between rotating incident polarizations and oriented molecules. Extracting the dependence of nonlinear optical signals to polarization allows to image molecular order beyond the optical diffraction limit size. This parameter provides not only complementary fundamental understanding on molecular interactions, it is also a potential read-out for pathologies that inevitably perturb the architecture of protein assemblies. Whilst polarized nonlinear imaging has been demonstrated in SHG and CARS, it is less explored for two photon fluorescence (2PF) due to the lack of specificity of 2PF signals in biological tissues. In this work we show that label free polarized 2PF (P-2PF) is capable of revealing structure in dense protein fibres in vitro and intact tissues. We show in particular that this approach brings important insights into the origin of 2PF in beta-sheet like architectures such as in elastin or amyloids. We demonstrate moreover the sensitivity of elastin molecular order to the hydrophobicity of its environment and to mechanical forces. At last, we extend this approach to structures containing exogenous fluorescent labels, allowing us to explore the conformational properties of the constructs in cells.

Raman imaging is recognized as a powerful label-free approach to provide contrasts based on chemical selectivity. Nevertheless, Raman-based microspectroscopy still have drawbacks. The first issue is the inherent high data throughput in Raman microspectroscopy, posing challenges for dynamic and large-scale imaging, and its subsequent data storage. The second issue is data presentation: often, Raman microspectroscopy acquires overwhelming data sets, which information is then post-processed to a more useful and straightforward presentation (typically limited to the number of different target chemicals in a system). In parallel, compressive sensing has shown a paradigm shift approach where one can obtain accurate information from fewer samples than assumed by Nyquist-Shannon sampling theorem. A key concept in compressive sensing is to recognize that data sparsity can be exploited to reconstruct data that has been considerably undersampled. Following the compressive sensing spirit, various approaches were proposed to demonstrate compressive Raman microspectroscopy in different flavors, exploiting the fact that both the vibrational spectrum and the chemical components are often sparse. In this contribution, I will discuss different ways of performing compressive Raman, in particular focusing on the challenges that precludes fast imaging of biological specimens, and how we recently tackled them. With these outcomes, compressive Raman imaging soon may be routinely used by non-specialists in vibrational spectroscopy in a “blind” manner.

Type 2 diabetes is an increasingly prevalent disease, with more than 400 million people worldwide diagnosed in 2016. As a stable and accurate biomarker, glycated hemoglobin (HbA1c) is clinically used to diagnose type 2 diabetes with a threshold of 6.5% HbA1c among total hemoglobin (Hb). Current methods such as boronate affinity chromatography or enzymatic assay involve complex processing of large-volume blood samples, which inhibits real-time measurement in clinic. Moreover, these methods cannot measure the HbA1c fraction at single red blood cell level, thus unable to separate the contribution by diabetes from other factors such as diseases related to lifetime of red blood cells. Here, we demonstrate a transient absorption imaging approach that is able to differentiate HbA1c from Hb based on the excited state dynamics measurement. HbA1c fraction inside a single red blood cell is derived quantitatively through phasor analysis. HbA1c fraction distribution for diabetic blood is found apparently different from that for healthy blood. A mathematical model is developed to derive the long-term glucose concentration in the blood. Our technology provides a new way to study heme modification and to derive clinically important information avoid of glucose fluctuation in the bloodstream.

Cryopreservatives like dimethyl sulfoxide and glycerol are common agents that prevent cellular damage upon freezing of tissues or entire organisms. Although the cryopreservation capabilities of these compounds have been known empirically for years, much is unknown about the actual perfusion and distribution of the agents within cells on the microscopic scale. In this contribution, we report on studies that aim to uncover the dynamic distribution of cryopreservatives in the tissue with the aid of stimulated Raman scattering microscopy, enabling a direct and real-time view of the cellular loading and accumulation dynamics of these agents at the micrometer scale.

To avoid the unwanted non-resonant background contribution induced by cross-phase modulation (XPM) in Stimulated Raman Scattering (SRS) microscopy [1], the collection of forward transmitted signal is normally done with a microscope objective having a numerical aperture (NA) higher than the objective used for excitation. However, while high NA microscope objectives are usually bulky and expensive, because a complex design is needed to achieve good optical imaging performances, only a capability to collimate highly divergent beams is needed for forward detection of signal in SRS microscopy. Additionally, because high NA microscope objectives have a short working distance and are bulky, their use as forward collecting optical element is not compatible with tightly closed top-stage incubators, as used in live-cell experiments. Here we show the use of a high NA 3D printed ultra-thin optical lens, composed of micro-reflective and -refractive elements, to replace commercial high NA microscope objectives for forward collection of signal in Stimulated Raman Scattering microscopy. The lens is fabricated on a 170µm thick coverslip with direct laser writing based on Two-Photon Lithography with a commercial system (Nanoscribe) and using the proprietary IP-S photoresist. It has a thickness of 300 µm and a diameter of 1 cm. Thanks to its compactness, this optical element can easily fit inside top-stage microscope incubators. The resulting NA of this catadioptric condenser lens is 1.2 when working in water immersion. We show the complete removal of the non-resonant XPM contribution from SRS spectra of incubated cells. [1] Ji-Xin Cheng, Xiaoliang Sunney Xie, Coherent Raman scattering microscopy, CRC press, 2016.

The extraction of fluorophore lifetimes in a biological sample provides useful information about the probe environment that is not readily available from fluorescence intensity alone. Cell membrane potential, pH, concentration of oxygen ([O₂]), calcium ([Ca²⁺]), NADH and other ions and metabolites are all regularly measured by lifetime-based techniques. These measurements provide invaluable knowledge about cell homeostasis, metabolism and communication with the cell environment. Fluorescence lifetime imaging microscopy (FLIM) produces spatial maps with time-correlated singlephoton counting (TCSPC) histograms collected and analyzed at each pixel, but traditional TCSPC analysis is often hampered by the low number of photons that can reasonably be collected while maintaining high spatial resolution. More important, traditional analysis fails to employ the spatial linkages within the image. Here, we present a different approach, where we work under the assumption that mixtures of a global set of lifetimes (often only 2 or 3) can describe the entire image. We determine these lifetime components by globally fitting precise decays aggregated over large spatial regions of interest, and then we perform a pixel-by-pixel calculation of decay amplitudes (via simple linear algebra applied to coarser time-windows). This yields accurate amplitude images (Decay Associate Images, DAI) that contain stoichiometric information about the underlying mixtures while retaining single pixel resolution. We collected FLIM data of dye mixtures and bacteria expressing fluorescent proteins with a two-photon microscope system equipped with a commercial single-photon counting card, and we used these data to benchmark the gDAI program.

Spectroscopic stimulated Raman scattering (SRS) is a label-free chemical imaging modality enabling visualization of molecules in living systems with high specificity. Among various spectroscopic SRS imaging methods, a convenient way is through linearly chirping two femtosecond lasers and tuning their temporal delay, which in turn corresponds to different Raman shifts. Currently, the acquisition speed using a resonant mirror is 3 seconds (80 microseconds per spectrum), which is insufficient for imaging samples with high motility. In this work, we aim to push the imaging speed using a 50-kHz polygon scanner as a delay line tuner, achieving a speed of 20 microseconds per spectrum. At such high speeds, to overcome the signal level decrease due to reduced signal integration time, we apply a U-Net deep learning framework, which first takes pairs of spectroscopic SRS images at different speeds as training samples, with high-speed, low-signal images as input and low speed, high-signal ones as output. After training, the network is capable of rapidly transforming a low-signal spectroscopic image to a high-signal version. Consequently, our design can generate ultrafast spectroscopic SRS image while maintaining the signal level comparable to the output with longer signal integration time.

Broadband coherent anti-Stokes Raman scattering (B-CARS) in a hybrid 2-color/3-color excitation regime has been shown to be a photon-efficient method of generating CARS across the entire vibrational region of interest (fingerprint and C-H stretch). We extend here our spectral interferometric polarized CARS (SIP-CARS) approach to a hybrid 2-color/3-color excitation regime, demonstrate that the power/polarization dependence for both 2- color and 3-color excitation agrees with theory, explore the application of SIP-CARS for the special case of anisotropic, birefringent materials (using collagen) and present in vivo lipid imaging of different mutations of the nematode Caenorhabditis elegans with modified lipid distributions. The extension of SIP-CARS to a hybrid excitation scheme makes possible a fast, all-optical and quantitative method for Raman imaging.

Voltage imaging has become an emerging technique to record membrane potential change in living cells. Yet, compared to the conventional electrophysiology, imaging approaches are still limited to relative membrane potential changes, losing important information conveyed by absolute value of membrane voltage. This challenge comes from several factors affecting the signal intensity, such as concentration, illumination intensity, and photobleaching. Spectroscopy is a quantitative method that shows potential to report the state of molecules in situ. Here, we apply electronic pre-resonance stimulated Raman scattering (SRS) imaging to detect near-infrared absorbing microbial rhodopsin voltage sensors in E. coli. The use of newly developed near-infrared microbial rhodopsins (Ganapathy et. al. 2017. JACS, 2017, 139(6):2338- 44) enables electronic pre-resonance SRS imaging with single cell sensitivity. By spectral profile analysis, we identified voltage-sensitive SRS peaks. The spectral signature can be used as part of a quantitative approach to measure membrane potential and enable mapping of absolute voltage in a neural network.

Hyperspectral stimulated Raman scattering (SRS) microscopy allows imaging of complex chemical mixtures and analysis cellular metabolites with high specificity. However, current SRS imaging is not implemented to address the cell heterogeneity issue, which can only be resolved by statistical analysis of a large amount of cells through cytometry. We developed a high-speed hyperspectral SRS image cytometry platform based on multiplex excitation, acquiring a Raman spectrum of 200 wavenumbers in 5 microseconds. This platform enables measurement of <100 cells per second. Multiple chemical signatures, featuring different cellular organelles such as lipids, endoplasmic reticulum, nucleus, and cytoplasm can be segmented. Statistical analysis over a large amount of cells reveals unprecedented details about cell metabolic changes after drug treatment.

Two-Photon Microscopy (TPM) can provide three-dimensional morphological and functional contrast in vivo. Through proper staining, TPM can be utilized to create virtual, HE equivalent images and thus can improve throughput in histology-based applications. We previously reported on a new light source for TPM that employs a compact and robust fiber-amplified, directly modulated laser. This laser is pulse-to-pulse wavelength switchable between 1064 nm, 1122 nm, and 1186 nm with an adjustable pulse duration from 50ps to 5ns and arbitrary repetition rates up to 1MHz at kW-peak powers. Despite the longer pulse duration, it can achieve similar average signal levels compared to fs-setups by lowering the repetition rate to achieve similar cw and peak power levels. The longer pulses lead to a larger number of photons per pulse, which yields single shot fluorescence lifetime measurements (FLIM) by applying a fast 4 GSamples/s digitizer. In the previous setup, the wavelengths were limited to 1064 nm and longer. Here, we use four wave mixing in a non-linear photonic crystal fiber to expand the wavelength range down to 940 nm. This wavelength is highly suitable for imaging green fluorescent proteins in neurosciences and stains such as acridine orange (AO), eosin yellow (EY) and sulforhodamine 101 (SR101) used for histology applications. In a more compact setup, we also show virtual HE histological imaging using a direct 1030 nm fiber MOPA.

A multimodal multiphoton microscopy (MPM) is developed to acquire two-photon and three-photon signals simultaneously, including two-photon excitation fluorescence (TPEF), second harmonic generation (SHG), and third harmonic generation (THG). We have developed a miniature multimodal MPM system based on a dual-wavelength Erdoped fiber laser, which includes the fundamental pulse at 1580 nm to excite THG and the frequency-doubled pulse at 790 nm to excite TPEF and SHG. The laser is coupled by a single mode fiber into a miniature MPM imaging probe. Label-free imaging by TPEF, SHG, and THG are demonstrated on biological samples, obtained from intrinsic fluorophores, collagen, lipid and interface.

We propose and demonstrate a novel multiphoton frequency-domain fluorescence lifetime imaging microscopy (MPM-FD-FLIM) system that is able to generate 3D lifetime images in deep scattering tissues. The imaging speed of FD-FLIM is improved using phase multiplexing, where the fluorescence signal is split and mixed with the reference signal from the laser in a multiplexing manner. The system allows for easy generation of phasor plots, which not only address multi-exponential decay problems but also clearly represent the dynamics of the fluorophores being investigated. Lastly, a sensorless adaptive optics setup is used for FLIM imaging in deep scattering tissues. The capability of the system is demonstrated in fixed and living animal models, including mice and zebrafish.

The light attenuation in biological tissues, caused by scattering and absorption, is a main issue in deep imaging. While the signal from the focal volume in multiphoton imaging is mostly generated by ballistic photons, both ballistic and scattered fluorescence photons contribute to the detected signal. The impact of emission wavelengths on deep imaging has not been investigated experimentally before. Here we perform a systematic comparison of the fluorescence attenuation in tissue at the emission wavelengths of 520 nm, 615 nm and 711 nm in three-photon imaging of mouse brain vasculature in vivo. Our results show that the impact of the emission wavelengths on multiphoton imaging depth is small compared to the excitation wavelengths.

Our group has recently developed a method for characterizing distribution of a topical drug within skin using two-photon fluorescence lifetime imaging (FLIM) and phasor analysis. Here, we expand on this work by describing a multimodal approach for rapidly visualizing multiple components in tissue using FLIM and coherent Raman imaging (CRI). By employing a non-Euclidian FLIM phasor analysis for a three-component system informed with the vibrational signature of one of the components retrieved with CRI, we were able to semi-quantitatively describe the spatial distribution of drugs in tissue with molecular specificity and cellular resolution.

Cirrhosis is defined as the histological development of regenerative nodules surrounded by fibrous bands in response to chronic liver injury that leads to portal hypertension and end stage liver disease. Conventional techniques are insufficient to precisely describe the internal structure, heterogeneous cell populations and the dynamics of biological processes of the diseased liver. Currently, multiphoton microscopy with fluorescence lifetime imaging is actively introduced to biomedical research. This technic is 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. In this study we investigated metabolic changes and collagen fibers formation in the rat liver with induced cirrhosis based on the fluorescence of the metabolic co-factors (NAD(P)H, FAD) and a second harmonic signal by multiphoton microscopy with FLIM and SHG mode. Moreover we studied ex vivo liver samples of patients with cirrhosis. We presented a separate analysis of NADH and NADPH to estimate the contribution of energy metabolism and lipogenesis in the metabolic changes. 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.

Covering the ends of long bones, articular cartilage provides a smooth, lubricated surface to absorb impact and distribute loads during movement so that underlying bone is protected. This function is facilitated by a complex and well-organized extracellular matrix (ECM). Being the only cell type in articular cartilage, chondrocytes are responsible for maintaining the homeostasis of the cartilage ECM; as such, the viability of chondrocytes is a critical parameter to reflect the quality of the cartilage. Most prevalent cell viability assays rely on dye staining and thereby cannot be performed for longitudinal monitoring or in-vivo assessment. Here we demonstrate that two-photon autofluorescence (TPAF) microscopy distinguishes live cells from dead cells in intact ex-vivo cartilage tissues, which provides a non-invasive method to assess cell viability. In our study, the endogenous fluorophores such as nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were used to image chondrocytes in cartilages on rat tibia condyles immediately after harvesting. Second harmonic generation (SHG) imaging was also performed to examine the integrity of the extracellular and pericellular matrix. On the same tissue, the cell viability assay with Calcein-AM and Ethidium homodimer-1 (EthD-1) labeling was used as a gold standard to identify live or dead cells. We found that live cells presented stronger NAD(P)H fluorescence than dead cells in general and were readily identified if pseudo colors were used for showing two-channel images. Owing to its non-destructive nature, this method holds the potential value in assessing cell viability of engineered or living tissues without dye labeling.

To study cancer progression and drug response, researchers have developed methods to derive tumor organoids from primary tissues, which not only have the same proteomic and genetic abnormalities as the malignant disease but also better replicate tumor behaviors than 2-dimensional culture models. It has been shown that tumor organoids can be used to predict treatment response, understand drug resistance, and study tumor heterogeneity at the individual-patient level. Whereas large-scale production of patient-derived organoids in standard flat-bottom 1,536-well plates has recently been demonstrated for cytotoxicity screening of 3,300 approved drugs, no suitable functional imaging tool can provide rapid 3-dimensional (3D) evaluation over a wide range of cellular states in these mass-produced organoids. The traditional two-photon scanning microscopy is too slow, while the conventional light-sheet microscopy is not compatible with microwell plates. Here we propose a microplate-compatible single-objective multiphoton light-sheet microscopy (SOMP- LSM) that can provide high imaging speed for 3D imaging analysis of organoids and deeper imaging depth with sub-cellular resolution. Our simulation of 3D point-spread function simulation for the SO-MP-LSM shows that our imaging system can achieve 270 nm lateral resolution and 800 nm axial resolution deep into organoids.

We present numerical simulations and experiments employing two-photon excited fluorescence in a sum-frequency mixing scheme which could be used for colocalization experiments in biophysics. By means of numerical calculations using the Debye approximation, the point spread functions (PSF) of each focused laser beam as well as the resulting PSF of the two-color two-photon (2C2P) excitation are calculated and discussed. Experiments are performed on Streptococcus pneumoniae and on surface filaments (“pili”) of the bacteria. Two different fluorescent labels were used for staining the bacteria themselves as well as the surface filaments as structure of interest. Since one fluorophore is excited by one single laser and the other label only in the combination of both lasers, intrinsic colocalization of the signals on the nanometer scale is ensured. The two-color two-photon excitation is performed by an ultrashort fiber laser system with synchronized emission wavelengths at λ₁ = 780 nm and λ₂ = 1030 nm and pulse durations around 100 fs. The multiphoton microscopy approach provides high resolution and allows for three-dimensional imaging of bacteria in a volume of (5 x 5 x 5) μm³.

We describe the novel method of pbICS that enables us to determine the complex aggregation distributions of molecules. The method is applied to determine the clustering status of the EGF receptor on the surface of a CHO cell.

Two-photon microscopy (TPM) has shown its great impact in studying neuronal activity of live animals. Commercial TPM systems require the animals under study to be restrained under the microscope and the 3D images are obtained typically by using bulky galvo scanning mirrors, which limits the flexibility of animal brain imaging to a large extent. To study freely moving animals, miniaturization of the optical scanning system is the key. In this work, a miniature TPM probe has been developed based on an electrothermal MEMS mirror that can be driven under low voltage. The MEMS mirror has an initial tilt angle after fabrication, and its footprint is 3 mm x 4 mm and reflective mirror plate is aluminumcoated with an equivalent diameter of 2.5 mm. This MEMS probe can be directly adapted to a commercial TPM system. In addition to the MEMS mirror for laser beam scanning, inside the MEMS probe head there are a fixed mirror to fold the laser beam and a high NA polymer lens for focusing. This miniature probe can realize an FOV of 3.5°, or a scanning range of 150 μm. This MEMS probe head is compact with a size of 8 mm x 16 mm, which can be further scaled down.

We introduce the concept of broadband near-infrared molecular fieldoscopy. In this scheme, molecules are excited by femtosecond pulses in near-infrared spectral range and the complex electric field of their free induction decay is directly measured by means of electro-optic sampling. Few-cycle pulses centered at 2 µm and 1 µm are generated from a 5 kHz, Yb:YAG regenerative amplifier and employed for femtosecond excitation and electro-optic sampling, respectively. We chose water in an acetic acid solvent to demonstrate the first proof of principle measurement with the novel technique. The complex electric field of the combination bond of water molecules at 1930 nm at different molecular concentrations is detected and presented. We show the detection sensitivity of our time- domain technique is comparable to conventional specral-domain techniques. However, by employing a laser frontend with higher repetition rates, the detection sensitivity can be drastically enhanced. To the best of our knowledge, this is the first detection of the complex electric field of the molecular response in near-infrared spectral range. The new method holds promise for high-resolution overtone spectroscopy and microscopy with unparalleled sensitivity and specificity over the entire molecular fingerprint region.

In combination with laser scanning microscopes, optical imaging technologies based on time correlated single photon counting (TCSPC) are successfully used in fluorescence lifetime imaging microscopy (FLIM) providing monitoring of intracellular intrinsic metabolic coenzymes as NAD(P)H (nicotinamide adenine dinucleotide (phosphate)). Due to oxygen-dependent quenching of the phosphorescence of some compounds including transition metal complexes, the phosphorescence lifetime imaging microscopy (PLIM) can be used for evaluation of oxygen partial pressure (pO2). Using a multi-channel FLIM/PLIM system, we were able to monitor pO2 by PLIM simultaneously with NAD(P)H by FLIM providing complex metabolic and redox imaging of living cells and tissues.

Single molecule imaging techniques allow tracking dynamic behaviors of individual molecules, providing insight information of molecular processes that could be hidden by the ensemble average. Combining with the time-resolved imaging capability, the laser scanning confocal microscopy at the single molecule sensitivity allows quantitative multiparameter analyses of single molecule dynamics. Here, we describe the single molecule imaging tools in the ISS VistaVision software, including FCS, FCCS, PIE-FCCS, FLCS, PCH, FLIM, steady-state and time-resolved FRET, steady-state and time-resolved polarization anisotropy, burst analysis for single molecule FRET and stoichiometry, antibunching. We demonstrate with measurement examples how these techniques are used for studying photophysical properties and behaviors of single molecules, such as diffusion rates, molecular brightness, triplet time, rotational relaxation time, fluorescence lifetime. By using donor-acceptor FRET pair-labeled proteins, we detect changes in protein conformation and dynamics by quantitatively measuring FRET efficiency, stoichiometry and lifetime. This quantitative multi-parameter analysis approach gives researchers more opportunities for a better understanding of single molecule dynamics.

Second order susceptibility (SOS) microscopy is used as a contrast mechanism for distinguishing types I and III collagen in tissues. In addition, relative proportion of collagen type was determined by analyzing the histograms of SOS ratios. Our results show that second order susceptibility is an effective metric for differentiating types I and III collagen without extrinsic labeling.

Fluorescence lifetime imaging microscopy (FLIM) techniques are widely used in auto-fluorescence imaging to investigate dynamic metabolic states of the cells. Traditional FLIM imaging requires the cells to adhere to the coverslip to collect enough photons for FLIM data processing. Such conditions pose challenges to non-adherent cells, such as acute myeloid leukemia (AML) cells, because of cell motility. We developed a proto-type micro cell trapping array (MCTA) to immobilize cells in picoliter-size wells with location references. The array keeps non-adherent cells in referenced well locations, allows treatment on stage and re-imaging after time for ultimate cell segmentation analysis. Individual wells are analyzed by a pixel-based region-of-interest (ROI) to analyze cellular redox states. This single well trapping and analysis method allows to isolate treatment responses of a small number of cells, compare their range and predict early effect, which may have clinical applications in the context of cancer aggressiveness and treatment outcomes. We expanded the common intensity-based assay for cellular redox state by a Fluorescence Lifetime measurement, NAD(P)H-a2%, serving as an alternative metric. The new assay is flexible and can be applied to other non-adherent cell lines, expanding FLIM applications in both research and the clinic.