This PDF file contains the front matter associated with SPIE Proceedings Volume 12847, including the Title Page, Copyright information, Table of Contents, and Conference Committee information.

So far, medical confocal reflectance microscopy is based on continuous wave (cw) laser diodes operating at 830 nm, and medical multiphoton tomography (MPT) is conducted with tunable water-cooled 80 MHz femtosecond titanium:sapphire lasers with mean powers less than 50 mW. The femtosecond laser beam is guided through an optical arm. The major application is high-resolution label-free skin imaging (“optical virtual biopsy”) to detect skin cancer. MPT has also been used to test anti-aging and pharmaceutical components in situ.

Here, we report on the use of an ultracompact femtosecond fiber laser for high-resolution tissue imaging. In particular, the development of the PRISM AWARD 2024 winning multimodal multiphoton tomograph based on an ultracompact air-cooled 50/80 MHz fiber laser operating at 780 nm is presented. The 18x9x3.5 cm³ laser head, consisting of a pulse compression unit and a SHG crystal, is positioned inside the 360° imaging head. An optical arm or a fiber delivery for transmitting the ultrashort near-infrared laser beam is no longer required. Interestingly, the femtosecond laser pulses, used for two-photon autofluorescence and SHG imaging, are also employed to realize simultaneous high-resolution (submicron) one-photon confocal microscopy. In addition, optical metabolic imaging (OMI) by time-correlated single photon counting and fluorescence lifetime imaging (FLIM) of autofluorescent coenzymes can be performed. The fifth imaging modality of this multimodal device is white LED far-field imaging for dermoscopy and to define regions of interest for confocal and multiphoton analysis.

The novel “green” 230 W femtosecond fiber laser tomograph can be operated by batteries and charged by sunlight due to the reduced power consumption by 75 % when using the fiber laser system compared to the tunable titanium:sapphire laser. High-resolution confocal and multiphoton imaging with compact fiber lasers in remote areas and on the bedside of the patient becomes a reality.

We present a femtosecond laser system at 920 nm delivering ultrashort pulses via a hollow-core photonic bandgap fiber (HC-PBGF). The laser system is designed to simplify two-photon microscopy applications and can be used for miniaturized two-photon microscopes. While previously presented solutions have been tailored to a specific length and dispersion coefficient of the HC-PBGF, we now show a compact and flexible scheme for dispersion compensation which is compatible with a wide range of fiber types and lengths.

In addition, this new approach fully maintains the capability of software-controlled dispersion compensation in the range from 0 to -40,000 fs² after the pulse delivery fiber. Hence, the dispersion of common two-photon microscopes can be pre-compensated in order to obtain compressed pulses at the sample plane. Our newly developed system displays excellent long-term fiber coupling stability under varying environmental conditions. It is capable of polarization-preserving femtosecond pulse delivery at 920 nm and reaches Watt-level power after the delivery fiber, making it suitable for in-vivo brain imaging of GCaMP in mouse models.

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of sub-cellular structures with spatial resolution well below the diffraction limit. The higher the STED beam intensity, the higher the resolution, but at the cost of increased photo-damage, which significantly limits the application of STED microscopy in live specimens. The separation by lifetime tuning (SPLIT) technique uses a time-resolved acquisition and a phasor-based analysis to efficiently distinguish photons emitted from the center and from the periphery of the effective fluorescent region, thus improves the resolution of STED microscopy without increasing the STED beam intensity. Furthermore, the SPLIT method is combined with a deep learning-based phasor analysis algorithm termed flimGANE (fluorescence lifetime imaging based on a generative adversarial network), to improve the robustness of SPLIT-STED allowing improving the resolution up to 1.45 folds at the half of the depletion laser beam intensity.

Understanding the complexity of life requires that it be studied with as many dimensions as possible in each experiment. STELLARIS 8 DIVE multiphoton system enables flexible spectral detection and the combination of lifetime-based information with deep imaging beyond 1 mm.

We report on a multimodal 230 W multiphoton tomograph, MPTcompact, featuring “green photonics”. This includes an air-cooled femtosecond fiber laser of low energy consumption instead of the 1 kW femtosecond laser tomographs with water-cooled tunable titanium:sapphire laser. The fiber laser tomograph can be operated with two 12V, 33Ah VRLA batteries.

Flexible solar panels connected via an Anderson power plug have been employed to recharge the system without removing batteries from the medical cart. Applications of this autonomous operating tomograph are high-resolution skin imaging to obtain optical biopsies directly at the patient’s location, including remote areas and on battlefields. Furthermore, on-site in vivo deep tissue multimodal imaging (autofluorescence, SHG, FLIM, confocal reflectance), e.g., on trees, algae, plants, and animals, is possible.

Fluorescent indicators allow the monitoring of physiological parameters in biological tissues by measuring changes in the brightness of the indicator upon binding of its ligand. Quantitative measurement of these parameters in vivo with high spatio-temporal resolution using multiphoton microscopy often requires the use of fluorescence ratiometric measurements. However, ratiometric measurements can be biased at depth in biological tissues due to absorption and scattering of the photons involved in the process.

Here we have developed a mathematical model that takes into account the chemical properties of the sensors, the spectral optical properties of the biological tissue and the optical system to provide quantitative measurements of the concentration of the sensor ligand. We have also developed a software implementing this model. Both can be used to obtain unbiased measurements of different physiological biomarkers from multiphoton images.

At the present, the two clinical biomarkers used to monitor diabetic progression are blood glucose and HbA1c. However, advanced glycation end products (AGEs) have been shown to contribute to diabetic pathogenesis, and there is interest in the use of AGEs in tissues as long-term glycemic markers. In this study, we investigate the in vitro rate of fluorescent AGEs (fAGEs) formation with multiphoton microscopy in different porcine tissues (aorta, cornea, kidney, dermis, and tendon) from glucose, galactose, and fructose, three primary monosaccharides found in human diets. These results may be of value in developing long-term glycemic biomarkers for diabetes.

Microplastic studies are crucial due to their impact on human health and the environment. However, the lack of a standardized method for microplastic identification and quantification hinders comprehensive analysis. Our research addresses these challenges by employing the advanced imaging technique Multimodal Multiphoton Tomography (MPT) including two-photon autofluorescence (AF), fluorescence lifetime imaging (FLIM) with phasor analysis, second harmonics generation (SHG), and reflectance confocal microscopy (RCM) with 50/80 MHz femtosecond laser pulses. The combined imaging approach allows also tracking of cosmetics with microplastics in-vivo in the human skin.

Fluorescent dyes exhibiting peak action cross-sections within the water absorption window are generally unsuitable for in vivo two-photon fluorescence imaging using photons of the same wavelength. We show that undertaking the two-photon excitation process with two photons of different wavelengths, namely non-degenerate two-photon excitation (ND-2PE), enables imaging in the water absorption window using two spatially and temporally overlapped excitation sources at 1300 nm and 1600 nm. We explore the relative brightness spectra of indocyanine green (ICG) and assess its suitability for imaging at wavelengths susceptible to water absorption. Further, we demonstrate damage-free in vivo imaging of the rodent cortex vascular structure up to 1.2 mm using ND-2PE.

We present a completely label-free technology that captures eight complementary contrasts describing the metabolic, chemical, structural, and dynamic profiles of the tissue microenvironment with a single laser source, called VAMPIRE (Versatile Autofluorescence lifetime imaging, Multiharmonic generation, Polarization-sensitive Interferometry, and Raman scattering in Epi-detection) microscopy. VAMPIRE microscopy maximizes the spectral utility of light-matter interactions by creating four nonlinear (second harmonic generation, two-channel two-photon fluorescence, coherent Raman scattering) and two linear interactions (backscattering, birefringence) simultaneously with a single laser.
Innovations in each modality improved the overall speed and sensitivity. Fast fluorescence lifetime imaging microscopy (FLIM) was accelerated with our computational photon counting algorithm called single-and-multi photon peak event detection (SPEED), capable of counting up to 250% photon rates. Dual-channel fast two-photon FLIM was achieved by compressed sensing of analog photocurrents on a field-programmable gate array on board the digitizer. We developed a new and faster method for hyperspectral coherent Raman microscopy using supercontinuum generation and custom pulse shapers, which facilitated rapid tuning to desired vibrational states. Polarization multiplexing in optical coherence imaging enabled compressed sensing of birefringence. Finally, computational approaches to maximize the information from these complementary contrasts yielded new insights into the processes within the tissue microenvironment. VAMPIRE microscopy is the nexus of label-free microscopy research, advances in optoelectronic technologies, and our innovations in computational and multimodal imaging for diverse applications.

The thymus plays a critical role in the adaptive immune system as the site of T cells maturation. Unfortunately, the thymus is susceptible to damage by acute insults which can negatively impact patient health outcomes. Despite significant medical interest, research into the thymus has been limited due to a lack of intravital imaging methods to study the thymus in situ. Our lab previously developed a method of intravital adhesive-stabilized two-photon microscopy of the thymus in mice to overcome the limitations of previous imaging methods. However, this method was limited by the permanent attachment of a support structure to the thymus, which prevented repeat imaging of the same thymus across multiple timepoints.

To enable longitudinal study of the thymus, we developed a vacuum-stabilized video-rate two-photon endoscope capable of imaging the native thymus in mice. The optical performance and ability of the endoscope to visualize the thymus was described. The stability of the endoscope to visualize the thymus was also described. Future longitudinal examination of the thymus is likely to expand the scientific communities’ understanding of thymus biology and its relationship to overall human health.

Collagen fibrils are nanoscale biological ropes which are the primary load bearing structures within the human body. Due to the non-centrosymmetric structure and high nonlinearity of the triple helical collagen molecule, collagen fibrils are able to produce significant second harmonic generation (SHG) when placed at the focus of an ultrafast laser. There have been many publications which have utilized polarization resolved SHG (PSHG) for investigating collagen organization in tissues, mainly for applications in disease diagnosis. However so far there has been little work on investigating individual isolated collagen fibrils using PSHG. This presents an exciting opportunity for research since PSHG imaging of individual fibrils can help us to gain a better understanding of PSHG images of collections of fibrils in tissues, and therefore improve the utility of PSHG as a potential diagnostic tool. Additionally, the high sensitivity of PSHG to changes in molecular structure can provide us with a better understanding of the nanoscale origins of injuries to tissues such as skin, tendons, and ligaments. Here we will review our recent work which has applied PSHG to study individual collagen fibrils, including investigation of how out of plane orientation affects PSHG images of collagen, and how differences in hydration alter collagen ultrastructure. We also present new data on the effect of applied tension on individual fibrils.

Lipid and protein metabolism play important roles in the aging process of ovary. High resolution in situ optical imaging provides a powerful approach to study the metabolic dynamics of macro-molecules such as lipids and proteins. Here, we applied a multimodal imaging platform integrated with D2O probing stimulated Raman scattering (DO-SRS), multiphoton fluorescence (MPF), and second harmonic generation (SHG) to explore metabolic changes of biomolecules in Drosophila ovaries during aging process. In this study, the sub-cellular spatial distribution of de novo protein synthesis, lipogenesis and redox ratio in ovaries are quantitatively imaged and examined in different ages. The regulation of diets on aging-dependant changes of ovary metabolism are investigated. Our results provide valuable insights for the underlying mechanism of ovary aging and how to intervene the aging process to achieve a better health

An advantage of two-photon microscopy is its sectioning capabilities, however, in practice it can often suffer from out of focus contamination. Here, we report the new application of strong acousto-optic modulation of two-photon excited fluorescence. We demonstrate how this effect enhances image detail and contrast and improves optical sectioning in vivo. Requiring no changes to the optical path or image acquisition parameters, our method is simple to implement and compatible with standard multiphoton microscopes.

Imaging the brain in its natural state with high spatial resolution is a challenging task for visualization techniques. Two-photon microscopy offers a better approach to image deep brain tissue due to its inherent sectioning ability compared to single-photon confocal microscopy. Despite this, the optical heterogeneity of the skull severely compromises imaging contrast and spatial resolution, limiting imaging depth to the superficial layer. Recently, a new adaptive optics method called alpha-FSS has been applied to three-photon microscopy, enabling high-resolution imaging in deep brain tissue through the intact skull and demonstrating strong ability to correct large aberrations and scattering. In this study, we combined alpha-FSS adaptive optics with two-photon microscopy and demonstrated that this method works well in two-photon imaging system as well, which is much more widely used than three-photon microscopy. Using this system, we achieved subcellular resolution transcranial imaging of layer 5 pyramidal neurons up to 650um below pia in living mice. We also demonstrated the ability to perform functional calcium imaging with high sensitivity, as well as high-precision laser-mediated microsurgery through thinned skull. In summary, we applied alpha-FSS to two photon system and proved its ability to achieve near non-invasive high-resolution imaging in deep brain tissue.

Metabolic Fluorescence Lifetime Imaging Microscopy (FLIM) algorithms have emerged as powerful tools for unraveling the intricate dynamics of cellular and tissue metabolism. FLIM algorithms offer unique insights into cellular metabolic processes that transcend traditional imaging methods. By combining Metabolic FLIM with Phosphorescence Lifetime Imaging Microscopy (PLIM), it becomes possible to assess cellular metabolic states, such as oxygen consumption, redox states, pH levels, and energy production pathways.

In our investigation, we employed a combination of 2-photon (2P) excited FLIM and PLIM techniques, along with timecorrelated single-photon counting (TCSPC) detection. Through this, we made a significant discovery of bromine indirubin derivatives that exhibit a PLIM/dFLIM signal in two living cell lines. Notably, indirubin is a natural dye, and though renowned for its anti-tumor properties, its mechanism of action remains to be fully investigated.

Under normoxic conditions, the PLIM signal of indirubin exhibited a value of 62 ns living cells, while under hypoxic conditions, it increased significantly to 107ns. This observation demonstrates the potential of these indirubins as highly reliable oxygen consumption sensors. Moreover, our investigation revealed that bromine indirubin had a profound impact on cellular metabolism, prompting a shift from oxidative phosphorylation to glycolysis.

Through our research, we aim to demonstrate that these techniques offer valuable insights into cellular metabolism, covering the way for deeper understanding and potential breakthroughs in various fields of biology and medicine.

Protein oligomerization is involved in a broad range of signal transduction pathways and regulatory cellular mechanisms, and therefore plays a role in several human diseases ranging from cancer to neurodegenerative diseases. It is a dynamic and often transient state change, showing spatial heterogeneity in a living cell. Despite the physiological importance of protein oligomerization and aggregation, its quantification in live cells with high spatial and temporal resolution remains a challenge. Number and Brightness (N&B) is a fluorescence fluctuation spectroscopy technique based on fast laser scanning microscopy and sensitive photon counting detection, that enables quantification of absolute protein counts and oligomeric state in each pixel of a light microscopy image. In this work, we implement a new N&B analysis tool in the VistaVision software and demonstrate its power in quantitatively measuring the protein oligomerization and aggregation states in live cells. We use this tool to demonstrate that the yeast G1/S transcription factor protein Mbp1 forms small oligomers in nitrogen-limited environments.

The time-resolved fluorescence anisotropy of a biological sample can reveal rotational dynamics and structure of a fluorescent probe’s local environment. Here, methods are presented towards developing widefield time-resolved fluorescence anisotropy imaging (TR-FAIM) using a single photon avalanche diode (SPAD) array (QuantICAM). Such detector allows for simultaneous time-correlated single photon counting (TCSPC) measurements in each pixel with single-photon sensitivity and picosecond time resolution. Our method integrates the SPAD array with a widefield microscope, and automated rotating polarizers in the excitation and emission pathways to demonstrate TR-FAIM. We have shown the robustness of the method through spectroscopic measurements of the fluorophore PM546, and we have demonstrated the usefulness of simultaneous FLIM and TR-FAIM for studying properties of plasma membranes in live yeast cells.

Optical microscopy has been indispensable for visualizing biological structure and function, while it remains a challenge since the limited diffraction resolution and restricted imaging depth. Nonlinear multifocal structured illumination microscopy (MSIM) provides resolution-doubled images and good penetration. Furthermore, adaptive optics (AO) is an effective method to recover spatial resolution and signal-to-noise ratio (SNR) in deep tissues and complex environments. Thus, we present a non-inertial scanning nonlinear MSIM system combined with AO to realize super-resolution imaging with aberration correction in vivo. Our strategy is designed to correct both laser and fluorescence paths simultaneously using a spatial light modulator and a deformable mirror respectively, providing better results than the individual path corrections. Furthermore, traditional approaches for MSIM image reconstruction at the expense of speed. Many raw images and iteration times are required for the reconstruction; besides, four steps in MSIM are separately accomplished in the reconstruction procedures of these methods. This is complicated and time-consuming, limiting extensive adoption of MSIM for practical use. To address the issues, a deep convolutional neural network to learn a direct mapping from raw MSIM images to super-resolution image, which takes advantage of the computational advances of deep learning to accelerate the reconstruction. The successful implementation of AO MSIM and fast MSIM reconstruction have allowed for the dynamic morphological characteristics of zebrafish motoneurons in vivo.

In this study, we performed two-photon imaging of porcine skin and oral mucosa specimens under simulated tissue glycation. After 14 days of glycation treatment with fructose, AGEs were formed in both porcine skin and oral mucosa. The autofluorescence response of both skin and mucosa depended on the detection bandwidth with the wavelength region of 330-480 nm demonstrated the strongest detection of AGE autofluorescence.

With the aim of advancing modern neuroscience and sampling neurons at up to 100 kHz frame rates, our group is developing a novel Two-photon Line Excitation Array Detection (2p-LEAD) imaging modality. Performing high resolution two-photon imaging at such high sampling rates necessitates the deposition of a large number of photons within the focal volume, which in turn warrants high laser powers. Consequently, the risk of heating and thermal damage limits the imaging speed and depth. In contrast to point-scan two-photon imaging, where safe average laser power values of 200 mW with conventional objective cooling have been established, there are no thermal characterization studies in the case of line-scan imaging modalities that could enable us in determining maximum laser powers to prevent tissue heating damage. We recently demonstrated through numerical investigations that enhanced cooling strategies of imparting laminar flow to the objective immersion water layer while implementing laser duty cycles could potentially increase safe power levels up to 600 mW of average surface power in the case of point scanning. A clear understanding of the effects of laser dosimetry on optical parameters of line-scan systems is essential to determine safe power values that would prevent thermal damage. In this work, we perform 3D MC-FDM numerical simulations at 1035 nm wavelength with a novel beam focusing framework over a parameter space spanning average powers and imaging depth to predict optothermal interactions. With experimental validation studies on tissue phantoms, our work would establish a much-needed power threshold in two-photon line scanning, which is an emerging modality of choice for high-speed volumetric imaging systems.

We construct a universal image formation theory that covers almost all optical imaging systems with diverse (coherent, incoherent, linear, and nonlinear) light-matter interactions, by utilizing Feynman diagrams. To deal with all optical systems ranging from classical to cutting-edge instruments including OCT in one framework, we reformulate the theory in four dimensions. We incorporate the vacuum field to treat incoherent interactions by using the operator for electric field.

In microscopy, quantitative information such as fluorescence lifetime allows researchers to provide detailed information about the microenvironment and the physicochemical state of the molecule under study. However, the high number of influencing factors might be an explanation for the strongly deviating values of fluorescent lifetimes for the same fluorophore as reported in literature. This could be the reason for the impression that inconsistent results are obtained depending on which FLIM technique is used. To clarify this controversy, the two most common as well as two newly developed techniques for measuring fluorescence lifetimes in the time-domain (TD) and in the frequency-domain (FD) were implemented in one single microscopy setup. Furthermore, we applied each of them to a variety of fluorophores under different environmental conditions such as pH-value, temperature, solvent polarity, etc., along with distinct state forms that depend, for example, on the concentration. From a vast amount of measurement results, both setup- and sample-dependent parameters were extracted and represented using a single display form, the phasor-plot. The measurements showed consistent results between the techniques and revealed which of the tested parameters has the strongest influence on the fluorescence lifetime. In addition, together with the instant FLIM setup we present a new technique that we coined Speed-Up PhasE-Resolved (SUPER)-FLIM, which we adapted from elsewhere such that it combines the advantages from TD- with FD-FLIM. For the first time, we demonstrate that this method opens the door for very fast (124ns/px) FLIM measurements required for imaging with a resonant galvo scanner.

In the heart, action potentials travel through the tissue to orchestrate muscle contraction and make the organ an efficient pump. Scar tissues caused by myocardial infarction impairs electrical conduction. In the rabbit heart, action potentials propagate from the endocardium to the epicardium with a conduction velocity of 30 cm/s. Therefore, a rapid vertical scan is necessary to observe this transmural cardiac conduction at cellular resolution. Here we present an implementation of a versatile remote focusing module, compatible with retrofitting to commercial two-photon microscopes and capable of 0.3 kHz rate axial scanning over the range of 100 μm in cardiac tissue without disturbing the sample or the sample objective. We discuss the necessary optimization to compensate for pulse broadening, power losses and optical aberrations. We demonstrate fast imaging of cardiac cell structure in functionally viable rabbit ventricular slice model. We will apply this system to resolve cardiac electrical signal propagation transmurally in healthy and infarcted hearts.

The advances in the field of optics and laser technologies are helpful to visualize and investigate the metabolism in live animals. Fluorescence lifetime imaging (FLIM) is a non-invasive optical technique to measure the fluorescence lifetime of the fluorophore towards its applications where fluorescence intensity-based techniques do not provide sufficient and accurate information to discriminate auto-fluorescent species. In contrast to conventional single photon excitation, two-photon excitation improves the penetration depth with low scattering of the longer wavelength to examine thick biological samples. In this work, we explain our 2photon-FLIM methodology for imaging mouse brains via a cranial window to investigate the changes in the NAD(P)H bound coenzyme, in both wild type and Alzheimer disease (AD) animal models before and after stimulation with nutrients. Moreover, phasor plot analysis has been used for removing lower a₂(%) fraction from the metabolic trajectory.

Stimulated Raman scattering (SRS) technique has been widely used in biomedical imaging, in which the CH band is usually used for detecting biomolecules. However, Raman peaks have the nonlinear identity limitation in which a Raman peak can be comprised of multiple molecules and a molecule can manifest itself in multiple Raman peaks. In this study, we applied a circularly polarized light for SRS (CP-SRS) imaging to examine the effect of polarization on differentiating CH vibrational modes. We observed that CP exerted a significant suppression on CH₂ bonds compared to CH₃ bonds, allowing for visualizing pure protein instead of protein-lipid complexes. This distinctive physics-informed unmixing method has the potential to enhance the chemical sensitivity of SRS imaging, displaying important implications in label-free biomedical imaging field.