This PDF file contains the front matter associated with SPIE Proceedings Volume 11965, including the Title Page, Copyright information, Table of Contents, and Conference Committee listings.

Localization microscopy circumvents the diffraction limit by identifying and measuring the positions of numerous subsets of individual fluorescent molecules, ultimately producing an image whose resolution depends on the uncertainty and density of localization, and whose capabilities are compatible with imaging living specimens. Spectral resolution can be improved by incorporating a dichroic or dispersive element in the detection path of a localization microscope, which can be useful for separation of multiple probes imaged simultaneously and for detection of changes in emission spectra of fluorophores resulting from changes in their environment. These methodological advances enable new biological applications, which in turn motivate new questions and technical innovations. As examples, we present fixed-cell imaging of the spike protein SARS-CoV2 (S) and its interactions with host cell components. Results show a relationship between S and the lipid phosphatidylinositol (4,5)-bisphosphate (PIP2). These findings have ramifications for several existing models of plasma membrane organization.

We report on the use of a modified multimodal multiphoton CARS tomograph for time-resolved auto-fluorescence (FLIM) and auto-phosphorescence imaging (PLIM) of biological tissues. Time-correlated single photon counting (TCSPC) was applied to measure fluorescence and phosphorescence decay curves per pixel. For PLIM, the repetition rate of the 80 MHz tunable titanium:sapphire femtosecond laser was reduced by an acousto-optic modulator. We present FLIM and PLIM data of non-labelled bones after heating. The phosphorescence behavior shows a strong dependence on the heat treatment. This is of special interest for forensic and archeological research.

The optical properties of the skin including reflectance are affected by aging, dehydration, and diseases and, therefore, can be used for non-invasive diagnosis. Multiphoton tomography (MPT) based on near-infrared (NIR) femtosecond (fs) lasers provide 3D optical biopsies with high-resolution (~300 nm). MPT has been used as a rapid diagnostic tool e.g., for the detection of malignant melanoma, the optimization of treatments as well as the investigation of the live 3D architecture of skin under various clinical conditions. So far, imaging is based on endogenous fluorophores e.g., elastin and metabolic coenzymes NAD(P)H and FAD as well as second harmonic generation (SHG) of the extracellular matrix protein collagen. The new tomograph MPTcompact is a multimodal high-resolution optical imaging tomograph with a reflectance confocal microscopy (RCM) module. The system employs 80 MHz NIR femtosecond laser pulses at 780 nm. In contrast to conventional RCM with a continuous wave NIR laser, the new tomograph provides the possibility of RCM with femtosecond laser pulses. The use of these ultrashort laser pulses has the advantage of confocal one-photon imaging (RCM) as well as two-photon autofluorescence and SHG imaging. This paper demonstrates clinical high-resolution reflectance confocal imaging of in vivo skin of volunteers and patients with dermatological disorders, in particular, malignant pigmented lesions.

Optical-fiber delivery increases the flexibility of experimental setups. Fiber delivery of fs pulses, however, is impractical with conventional optical fibers due their low-intensity damage threshold and nonlinearities which distort the temporal and spectral pulse shapes. Recently developed kagome hollow-core photonics crystal fibers can transmit ultra-short highpeak power pulses without disturbing pulse characteristics even in a broad wavelength range. The application outside of controlled laboratory settings, nevertheless, remains impeded by the sensitivity of the power transmission to movement and bending. In order to overcome this limitation, we have developed a continuous feedback-loop to stabilize the fiber output power by a fast online-control of the input power such that motion-caused transmission changes of the fiber are rapidly and continuously compensated. This is illustrated with a table-top setup. Furthermore, in order to demonstrate the improved flexibility, which is in particular beneficial for applications that require frequent positioning changes, the powerstabilized ultrashort pulse-fiber delivery is employed in a multiphoton tomograph.

The performance of SwissSPAD2 (SS2), a large scale, widefield time-gated CMOS SPAD imager developed for fluorescence lifetime imaging, has recently been described in the context of visible range and fluorescence lifetime imaging microscopy (FLIM) of dyes with lifetimes in the 2.5 – 4 ns range. Here, we explore its capabilities in the NIR regime relevant for small animal imaging, where its sensitivity is lower and typical NIR fluorescent dye lifetimes are much shorter (1 ns or less). We carry out this study in a simple macroscopic imaging setup based on a compact NIR picosecond pulsed laser, an engineered diffuser-based illumination optics, and NIR optimized imaging lens suitable for well-plate or small animal imaging. Because laser repetition rates can vary between models, but the synchronization signal frequency accepted by SS2 is fixed to 20 MHz, we first checked that a simple frequency-division scheme enables data recording for different laser repetition rates. Next, we acquired data using different time gate widths, including gates with duration longer than the laser period, and analyzed the resulting data using both standard nonlinear least-square fit (NLSF) and phasor analysis. We show that the fixed synchronization rate and large gate widths characterizing SS2 (10 ns and over) are not an obstacle to accurately extracting lifetime in the 1 ns range and to distinguishing between close lifetimes. In summary, SS2 and similar very large gated SPAD imagers appear as a versatile alternative to other widefield time-resolved detectors for NIR fluorescence lifetime imaging, including preclinical molecular applications.

Fluorescence lifetime imaging microscopy (FLIM) is an important technique to understand the chemical microenvironment in cells and tissues since it provides additional contrast compared to conventional fluorescence imaging. When two fluorophores within a diffraction limit are excited, the resulting emission leads to nonlinear spatial distortion and localization effects in intensity (magnitude) and lifetime (phase) components. To address this issue, in this work, we provide a theoretical model for convolution in FLIM to describe how the resulting behavior differs from conventional fluorescence microscopy. We then present a Richardson-Lucy (RL) based deconvolution including total variation (TV) regularization method to correct for the distortions in FLIM measurements due to optical convolution, and experimentally demonstrate this FLIM deconvolution method on a multi-photon microscopy (MPM)-FLIM images of fluorescent-labeled fixed bovine pulmonary arterial endothelial (BPAE) cells.

The cornea is the primary refractive component of the eye with a central connective tissue (stroma) which is bounded by stratified epithelium (forms the anterior surface) and endothelium (forms the posterior monolayer). It is transparent to visible light and hence amenable to fluorescence spectroscopy. Here we report on developing a dedicated ophthalmic time-resolved confocal scanning microfluorometer (OTR-CSMF) for depth-resolved transcorneal spectroscopy. The instrument combines a confocal microfluorometer, nanostage, digital frequency domain unit for rapid lifetime acquisition, and a corneal perfusion system. Using a 40x objective (0.8 NA; wd = 3.3 mm; water), the instrument offers depth scanning with an axial resolution of ~1.3 μm and single-molecule detection sensitivity, validated by correlation spectroscopy with fluorescein (100 pM). In addition, the device can resolve fluorescence lifetimes from 100 ps to 100 ms. With porcine corneas ex vivo, topical administration of Rhodamine B showed fluorescence peaks in the epithelium and endothelium consistent with its lipophilicity and consequent accumulation in the cellular layers. Moreover, the high axial resolution of the instrument revealed (a) fluorescence discontinuities at the interface between epithelium and stroma and between stroma and endothelium, and (b) fluorescence spikes in the stroma corresponding to dye accumulation in the keratocytes. These data indicate transcorneal transport of Rhodamine B by sequential diffusion and partitioning. The lifetime of Rhodamine B revealed characteristic variations across the depth, potentially due to variable dye accumulation and/or local variations in the refractive index. Similar experiments with the relatively hydrophilic fluorescein showed high fluorescence in the stroma compared to the cellular layers. Overall, OTR-CSMF is optimized for transcorneal fluorescence spectroscopy. Accordingly, we envision applying the device to register transcorneal dynamics of pO2, temperature, pH, fluorescent drug surrogates, and electrolytes, among other parameters in the future. These measurements can be expected to revitalize investigations related to drug discovery and the pathophysiology of corneal disorders.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique that provides a totally new dimension to quantitate the fluorescent probe, in addition to its steady-state intensity and spectral profiles. As the fluorescence lifetime is a parameter specific of each fluorophore, its measurement is instrumental in the identification of the fluorophore in a mixture: this opens a new opportunity for multiplex imaging and improves imaging sensitivity (signal-to-noise ratio) and resolution in many applications. More importantly, with peculiar selectivity of probes, FLIM can provide quantitative information of the probe microenvironment (ions, pH, oxygen content, local electrical fields, index of refractions, etc.); FLIM is one of the most robust ways of quantifying FRET for studying protein-protein interactions in live specimens. Today, turn-key FLIM instruments are a mature technology available from several companies, making the technique easily accessible. However, a major challenge for the user is still the analysis and interpretation of FLIM data. In combination with the digital frequency domain FLIM (known as FastFLIM), the phasor plots approach has been demonstrated to be an effective solution to tackle many challenges in FLIM data analysis. In this report, we describe several tools using the phasor plots method available in the ISS VistaVision software multiimage phasor analysis module, and demonstrate their utilities with examples for various applications including time-resolved multiplexing, NADH imaging, STED and FRET.

Mitochondrial dysfunction is increasingly being recognized in many pathologies. Mitochondria, the power houses of cells have central roles to play in energy metabolism and apoptosis. Structure-Function studies designed towards characterizing and understanding defects in mitochondrial metabolism, dynamics and biogenesis in pathologies and response to treatments would provide insight into mitochondrial dysfunction. A 2-step imaging approach was used; (a) Zeiss 880/980 Airyscan Super Resolution microscopy to understand mitochondrial morphological response to treatment and (b) Fluorescence Lifetime Imaging (FLIM) -B&H TCSPC lifetime board coupled to a Zeiss 780 to track metabolic changes in HeLa cells by following the auto-fluorescent metabolic co-enzyme NAD(P)H. FLIM signatures, the lifetimes and the relative fractions of bound and free states of NAD(P)H and FAD are generated with multiphoton excitation by a pulsed femto-second infra-red laser. Publications suggest that FLIM multiphoton laser power requirements for NAD(P)H and FAD may not be well optimized, which could result in injurious effects to cells. We have characterized two photon (2p)- laser induced changes at the cellular level, particularly in mitochondria. Live-cell FLIM measurements were conducted on stage in HeLa cells by gradually increasing the laser average power, followed by the assessment of phototoxic effects. Our results show that NAD(P)H-a2%, the enzyme-bound fraction increases with rising laser average power, inducing cytotoxic damaging effects. As elevated NAD(P)H-a2% is also shown after drug treatment, sub-optimal laser power can be falsely interpreted as drug treatment response. Our study demonstrates how the laser power optimization at the specimen plane is critical in FLIM.

To develop new methods for the diagnosis and treatment of such a widespread disease as steatosis, there is still a lack of fundamental biological knowledge about various aspects of the functioning of the liver tissue at the cellular level. In our work, we assessed the metabolic state of hepatocytes, as well as the collagen content in the liver tissue with induced steatosis using the modern label-free minimally invasive methods of multiphoton laser scanning microscopy with TPEF mode (Two-Photon Excited Fluorescence) and SHG mode (Second Harmonic Generation), equipped with FLIM (Fluorescence-Lifetime Imaging Microscopy). Using multiphoton microscopy, it was shown that during the development of steatosis, it is possible to identify areas with a reduced NAD(P)H autofluorescence signal in damaged hepatocytes. Using SHG we showed a gradual accumulation of collagen in the liver tissue with induced steatosis, however, extensive areas of fibrosis were not detected even at the advanced stages of the pathology. Using FLIM, we studied the specific features of the energy metabolism of hepatocytes based on data on the lifetimes of various forms of NAD(P)H and their relative contributions. It has been revealed that there is a gradual decrease in the intensity of oxidative phosphorylation, accompanied by the rise in the intensity of lipogenesis in the liver tissue with induced steatosis. Such results are consistent with the data of histological analysis. The results obtained in this work can be useful for developing new criteria for express intraoperative assessment of liver pathology at the cellular level in the clinic.

In this work, we compare the performance of a quantitative scientific complementary metal-oxide semiconductor (qCMOS) camera to the sCMOS camera for multiphoton imaging of tissue specimens. We find that the qCMOS achieves a signal-to-background ratio that is ~2x and ~1.6x higher than that achieved by the sCMOS for twophoton fluorescence and second-harmonic generation (SHG) imaging, respectively. The field-of-view of the qCMOS camera is noticeably larger at ~1.3x that of the sCMOS. We also confirm that the qCMOS can spatially resolve features as fine as 12.5 μm in 200-μm thick tendon tissue, at a penetration depth of 140 μm, using SHG imaging. Our results highlight the applicability of the qCMOS for some multiphoton imaging applications.

Mesoscopic optical imaging of an ultra-large biological sample demands a large enough field of view (FOV) in order to reduce the requirement of extensive digital image stitching operations. For an objective lens with a specific magnification, when we continue extending the FOV, the adverse effects of optical aberrations become prominent. One of such effects is radial distortion towards the edges and corners of a square-shaped FOV. Consequently, when we attempt to mosaic/stitch multiple of such radially distorted adjacent tiles, it often becomes challenging to avoid artifacts or discontinuity of structures especially at the joining of such adjacent tiles. To address the same, we apply a preestimated opposite radial distortion to each acquired tile prior to applying an alignment algorithm. Employing a bruteforced approach, we one-time quantify a suitable compensating radial distortion. We apply this method in a custom-built large-FOV nonlinear optical microscope (NLOM) system and demonstrate an artifact-free mosaic-stitching operation.

This paper, originally published on 3 March 2022, was withdrawn on 23 May 2023 per authors' request.

Short irradiation of a cell with focused femtosecond laser pulses can make the membrane of the cell transiently permeable such that macro molecules from the surrounding microenvironment can enter. This laser-induced sub-micron optoporation is well tolerated and makes it possible to efficiently transfect a cell with desired nucleic acids (DNA/RNA) and, thereby, even to reprogram it. However, for high cell viability and high transfection efficiency individual cell targeting is required, which limits the number of addressable cells per time. We have investigated the laser-assisted cell transfection of adherent mammal and human cells by manually and, to increase the throughput, automatically targeting the laser focus onto the cells. A different strategy is to irradiate continuously flowing cells without individual targeting. This allows to increases the number of addressable cells per time, but at the cost of the efficiency, since not all cells are optimally hit. A precise control of the cell flow as well as the focusing conditions is crucial to maximize the laser-cell interaction in that case. This is possible with a microfluidic setup with flow control where the cells pass a light-sheet like focal region which consists of a scanned Bessel beam of femtosecond-laser pulses. We summarize and review our experimental approaches for laser transfection and non-viral optical cell reprogramming to generate induced pluripotent stem cells through the use of ultrashort near infrared laser pulses.

Myoglobin is a protein that is expressed quite unevenly among different cell types. Nevertheless, it has been widely acknowledged that the Fe³⁺ state of myoglobin, metmyoglobin (metMb) has a broad functional role in metabolism, oxidative/nitrative regulation and gene networks. Accordingly, real-time monitoring of oxygenated, deoxygenated and metMb proportions- or, more broadly, of the mechanisms by which metMb is formed, presents a promising line of research. We had previously introduced a Förster resonance energy transfer (FRET) method to read out the deoxygenation/oxygenation states of myoglobin, by creating the targetable oxygen (O₂) sensor Myoglobin-mCherry. In this sensor, changes in myoglobin absorbance features that occur with lost O₂ occupancy -or upon metMb production control the FRET rate from the fluorescent protein to myoglobin. When O₂ is bound, mCherry fluorescence is only slightly quenched, but if either O₂ is released or met is produced, FRET will increase- and this rate competing with emission reduces both emission yield and lifetime. Nitric oxide (NO) is an important signal (but also a toxic molecule) that can oxidize myoglobin to metMb with absorbance increases in the red visible range. mCherry thus senses both met and deoxygenated myoglobin, which cannot be easily separated at hypoxia. In order to dissect this, we treat cells with NO and investigate how the Myoglobin-mCherry lifetime is affected by generating metMb. More discriminatory power is then achieved when the fluorescent protein EYFP is added to Myoglobin-mCherry, creating a sandwich probe whose lifetime can selectively respond to metMb while being indifferent to O₂ occupancy.

Lichen sclerosus (LS) is the most common vulvar dermatosis, which is characterized by damage to the connective tissue of the dermis. The affected area in lichen sclerosus is characterized by a sequential change in the main components of the connective tissue - collagen and elastin fibers. The affected area is polymorphic and remains poorly defined from a histological point of view. Among histopathologists, there are no unequivocal opinions on changes in the connective tissue of the dermis in LS. However, an assessment of the degree of dermis damage is important for the timely diagnosis of the condition and adequate treatment. Nonlinear microscopy includes second-harmonic generation (SHG) and twophoton autofluorescence (TPEF). SHG allows to selectively examine the signal from heterotypic collagen fibers of the dermis that contain type 1 collagen. TPEF allows to identify elastic fibers of the connective tissue matrix. It has been demonstrated that nonlinear microscopy allows visualizing the changes in the microstructure of collagen and elastin fibers. Three histological patterns were revealed as a result of the analysis of the nonlinear optical microscopy of the classical VLS. These histological patterns cannot be distinguished using histological stains and indicate a polymorphism of connective tissue changes. Nonlinear microscopy makes it possible to assess the changes in tissue structure, which is important for the histological interpretation of changes in the dermis and to clarify histological classification system in the future.

We report on a multimodal multiphoton microscopy (MPM) system with depth scanning. An Er-doped fiber laser with 1580 nm and 790 nm output provides the dual-wavelength multimodal capability and a shape-memory-alloy (SMA) based depth-scanning objective enables the depth scanning. Image stacks combining two-photon-excitation-fluorescence (TPEF), second-harmonic-generation (SHG), and third-harmonic-generation (THG) signals have been acquired on animal samples from the surface to over 200 μm underneath.

The biological relevance of nitric oxide (NO) in cells to processes of signaling, metabolic regulation, and disease treatment has become abundantly clear. NO or reactive oxygen species (ROS) can oxidize myoglobin to the met state (metMb; the Fe³⁺ state of myoglobin), a change accompanied with an altered absorbance profile in the visible region. Recent studies show that metMb has a broad functional role in metabolic pathways, oxidative/nitrative regulation and gene networks of many cells. Thus, real-time monitoring of the different charge states of myoglobin is a promising field of research. We previously introduced a Förster resonance energy transfer (FRET) sensor, EYFP-Myoglobin-mCherry, to measure the deoxygenation, oxygenation and met states of myoglobin, creating a simultaneous oxygen (O₂) and NO sensor. In this sandwich probe, the mCherry binary chimera lifetime responds to oxygenated vs. deoxygenated myoglobin, while the yellow fluorescent protein (YFP) lifetime selectively responds to metMb (while indifferent to O₂ concentration). We now use Citrine, a more robust YFP, in place of EYFP and append a mitochondrial targeting peptide sequence to specifically target mitochondria. We use fluorescence lifetime imaging (FLIM) of this mtCitrine-Myoglobin-mCherry sandwich probe while monitoring both oxygenation level and NO-induced met formation in mitochondria of mouse embryonic fibroblasts. We also test the NO response of Citrine alone to verify that the met sensitivity is specific to the Mb sandwich probe and not Citrine alone.