Journal articles: 'Multi-photon excitation microscopy'

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Piston, David W. "Multi-Photon Excitation Microscopy: An Old Idea in Quantum Theory Applied to Modern Scientific Problems." Microscopy and Microanalysis 6, S2 (August 2000): 1180–81. http://dx.doi.org/10.1017/s1431927600038393.

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Multi-photon excitation microscopy provides attractive advantages over confocal microscopy for three-dimensionalry resolved fluorescence imaging and photochemistry. The most commonly used type of multi-photon excitation is two-photon excitation where simultaneous absorption of two photons leads to a single quantitized event. The powerful advantages of using two-photon excitation microscopy arise from the basic physical principle that the absorption depends on the square of the excitation intensity. In practice, two-photon excitation is generated by focusing a single pulsed laser through the microscope. As the laser beam is focused, the photons become more crowded, but the only place at which they are crowded enough to generate an appreciable amount of two-photon excitation is at the focus. Above and below the focus, the photon density is not high enough for two of them to interact with a single fluorophore at the same time. This dramatic difference between confocal and two-photon excitation microscopy is shown in Fig. 1.

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ROTHSTEIN, EMILY C., MICHAEL NAUMAN, SCOTT CHESNICK, and ROBERT S. BALABAN. "Multi-photon excitation microscopy in intact animals." Journal of Microscopy 222, no. 1 (April 2006): 58–64. http://dx.doi.org/10.1111/j.1365-2818.2006.01570.x.

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Masters, Barry R., and Peter T. C. So. "Multi-photon Excitation Microscopy and Confocal Microscopy Imaging of In Vivo Human Skin: A Comparison." Microscopy and Microanalysis 5, no. 4 (July 1999): 282–89. http://dx.doi.org/10.1017/s1431927699990311.

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Abstract: We compare here multi-photon excitation microscopy and tandem scanning reflected light confocal microscopy for the microscopic observation of human skin in vivo. Multi-photon excitation is induced by a 80-MHz pulse train of femtosecond laser pulses at 780 nm wavelength. This nonlinear microscopic technique is inherently suitable for tissue fluorescence imaging because of its deeper penetration depth and lower specimen photodamage. This technique has noninvasively obtained tissue structural information in human epidermis and dermis. Alternatively, tandem scanning confocal light microscopy based on a white light source can provide video-rate image acquisition with high resolution and high contrast. Reflected light confocal methods have been used to obtain images from the skin surface to the epidermal–dermal junction. The relative merits of these two techniques can be identified by comparing three-dimensionally resolved images obtained from the forearm skin of the same volunteer.

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Cheng, Ping-chin, Chi-Kuang Sun, Fu-Jen Kao, and Bai-Ling Lin. "Non-linear Spectral Microscopy-Multi-Photon Fl, SHG and THG." Microscopy and Microanalysis 7, S2 (August 2001): 1026–27. http://dx.doi.org/10.1017/s1431927600031202.

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The non-linear nature of multi-photon fluorescence (FL) excitation, SHG and THG restricts the signal detecting volume to the vicinity of the focal point. As a result, the technology has intrinsic optical sectioning capability. The use of multi-photon fluorescence excitation also allows micro-fluorometry at high spatial resolution. Figure 1 shows a conventional optical micrograph of maize protoplasts, the time lapse fluorescence spectral change from a single chloroplast is shown in FIG 2. Under high intensity illumination, biological specimen not only emits fluorescence, but also generates harmonic emissions. in addition to the Ti-sapphire laser commonly used in multiphoton microscopy, the use of ultra-fast Cr-fosterite laser made simultaneous detecting two- and three-photon fluorescence, SHG and THG possible. in addition to the fluorescence signals generated by multi-photon excitation process, non-linear phenomena such as harmonic generation can also provide useful information about the structure and optical properties of a specimen (Kao et al., 2000). Simultaneous recording the spectral response in an image (x-y-λ) can provide insight about the nature of the signal.

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Kao, F. J., B. L. Lin, and P. C. Cheng. "Multi-photon Fluorescence Micro-spectroscopy of Plant Tissues." Microscopy and Microanalysis 6, S2 (August 2000): 808–9. http://dx.doi.org/10.1017/s1431927600036539.

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Considering its non-linear nature, two-photon excitation may generate very different spectral response in samples when compared with single photon excitation. It is thus necessary to measure the two-photon spectra of samples, so that the two-photon fluorescence microscopic images can be properly interpreted. Fluorescence spectra obtained from bulk samples may not provide useful information for microscopy. For instance, due to the relatively small contribution to the total fluorescence intensity, a small number of fluorescent particles in a generally fluorescing specimen may escape detection when the spectrum of the specimen as a whole is obtained. Under two-photon excitation, the background noise can be greatly reduced due to the naturally limited excitation volume of focused laser beam. In addition, signals resulted from second harmonic generation (SHG) may be mixed with low level broad-band background autofluorescence which is commonly found in biological specimen. Therefore, measuring fluorescence spectrum from a micro-focused volume is essential for the proper interpretation of multi-photon fluorescence images.

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Guo, Yong, Hongyi Han, Luwei Wang, Yinru Zhu, Xinwei Gao, Zhigang Yang, Xiaoyu Weng, Wei Yan, and Junle Qu. "Label free deep penetration single photon microscopic imaging with ultralong anti-diffracting beam." Applied Physics Letters 121, no. 2 (July 11, 2022): 023701. http://dx.doi.org/10.1063/5.0097959.

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Label free single photon microscopic imaging has natural advantages in noninvasive in vivo tissue imaging such as high resolution and rapid imaging speed. Although label free multi-photon microscopy can be used for imaging thick tissue samples, it requires high excitation light power and is phototoxic to the samples. Conventional label free single photon microscopy requires lower excitation light power, but it has limited imaging depth. Observing some highly scattering thick tissue samples with single photon microscopy is a great challenge. To solve the problem, we developed a label free deep penetration single photon microscopic imaging technique with an ultralong anti-diffracting (UAD) beam. The penetrating ability of the UAD beam was verified by passing through turbid media and performed with autofluorescence of chloroplasts in fresh Epipremnum aureum leaves. Benefiting from the anti-diffracting properties and the elongated focal depth of the UAD beam, single photon UAD microscopy has deeper penetration depth and better anti-scattering ability and is one of the ideal methods to observe the deep structure of biological samples.

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Cheng, P. C., B. L. Lin, F. J. Kao, C. K. Sun, and I. Johnson. "Multi-Photon Fluorescence Spectroum of Common Nucleic Acid Probes." Microscopy and Microanalysis 6, S2 (August 2000): 820–21. http://dx.doi.org/10.1017/s143192760003659x.

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Fluorescent probes are commonly used in biological fluorescence microscopy for tracking specific structures and sub-cellular compartments, and for indicating cellular ionic conditions. Recent development in multi-photon fluorescence microscopy has greatly expanded the usage of fluorescent probes in biomedical research. Considering its non-linear nature, two-photon excitation may generate very different fluorescence spectral response in the sample when compared with single photon excitation. It is thus necessary to measure the two-photon spectra of various fluorescent probes, so that two-photon fluorescence microscopy may be operated effectively and the images properly interpreted. This report represents the first installment of a continued effort in characterizing the multi-photon fluorescence spectra of commonly used bio-probes.Two-photon fluorescence spectra excited with near infrared at 780nm were obtained with a SpectraPro-500 spectrophotometer (Acton Research) equipped with a TE-cooled PMT and coupled to a Spectra-Physics Tsunami Ti-sapphire laser pumped by a Coherent Verdi solid-state laser operated at 85MHz, l00fs pulse.

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Mertz, J. "Molecular photodynamics involved in multi-photon excitation fluorescence microscopy." European Physical Journal D - Atomic, Molecular and Optical Physics 3, no. 1 (August 1, 1998): 53–66. http://dx.doi.org/10.1007/s100530050148.

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Schweitzer, Andreas, Heinz Eipel, and Christoph Cremer. "Rapid image acquisition in multi-photon excitation fluorescence microscopy." Optik 115, no. 3 (2004): 115–20. http://dx.doi.org/10.1078/0030-4026-00339.

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Goswami, Debabrata, Dhiman Das, and Soumendra Nath Bandyopadhyay. "Resolution enhancement through microscopic spatiotemporal control." Faraday Discussions 177 (2015): 203–12. http://dx.doi.org/10.1039/c4fd00177j.

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Operating at biologically benign conditions, multi-photon fluorescence imaging microscopy has benefitted immensely from recent developments in microscopic resolution enhancement. Fluorescence microscopy continues to be the best choice for experiments on live specimens, however, multi-photon fluorescence imaging often suffers from overlapping fluorescence of typical dyes used in microscopy, limiting its scope. This limitation has been the focus of our research where we show that by making simple modifications to the laser pulse structure, it is possible to resolve these overlapping fluorescence complications. Specifically, by using pairs of femtosecond pulses with variable delay in place of single pulse excitation, we show controlled fluorescence excitation or suppression of one of the fluorophores over the other through wave-packet interferometry. Such an effect prevails even after the fluorophore coherence timescale, which effectively results in a higher spatial resolution. Here we extend the effect of our pulse-pair technique to microscopic axial resolution experiments and show that such pairs of pulses can also ‘enhance’ axial resolution.

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Masters, Barry R., and Peter T. C. So. "Confocal microscopy and multi-photon excitation microscopy of human skin in vivo." Optics Express 8, no. 1 (January 1, 2001): 2. http://dx.doi.org/10.1364/oe.8.000002.

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Denk, Winfried. "Multi-Photon Microscopy, High Resolution Imaging Deep in Strongly Scattering Specimens." Microscopy and Microanalysis 3, S2 (August 1997): 301–2. http://dx.doi.org/10.1017/s1431927600008394.

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Imaging small structures substantially below the tissue surface in living specimens poses special challenges mainly because light is scattered by ever present refractive index inhomogeneities. Confocal microscoy removes the blurring caused by scattered and out-of-focus light but does so only at the expense of photodynamic damage that is often unacceptable when observing live specimens.Multi-photon absorption microscopy[l] solves these problems because excitation is virtually limited to the focal plane. Out-of-focus photobleaching and photodamage are therefore eliminated. In scattering samples substantial improvements accrue even for the focal plane because, different from confocal microscopy, where only ballistic fluorescenc photons can be used, in the multi-photon microscope scattered photons can be utilized in addition [2-4], provided whole-field detection is used[5].Many questions in the study of the nervous system require the investigation of intact portions of neural tissue in order to preserve the multiply branched processes of neurons, often extending over hundreds of microns, together with the local nervous circuitry.

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Yan, Wei, Yangrui Huang, Luwei Wang, Jin Li, Yong Guo, Zhigang Yang, and Junle Qu. "Multi-Color Two-Photon Microscopic Imaging Based on A Single-Wavelength Excitation." Biosensors 12, no. 5 (May 6, 2022): 307. http://dx.doi.org/10.3390/bios12050307.

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Two-photon probes with broad absorption spectra are beneficial for multi-color two-photon microscopy imaging, which is one of the most powerful tools to study the dynamic processes of living cells. To achieve multi-color two-photon imaging, multiple lasers and detectors are usually required for excitation and signal collection, respectively. However, one makes the imaging system more complicated and costly. Here, we demonstrate a multi-color two-photon imaging method with a single-wavelength excitation by using a signal separation strategy. The method can effectively solve the problem of spectral crosstalk by selecting a suitable filter combination and applying image subtraction. The experimental results show that the two-color and three-color two-photon imaging are achieved with a single femtosecond laser. Furthermore, this method can also be combined with multi-photon imaging technology to reveal more information and interaction in thick biological tissues.

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Malak, Henryk. "Up-Conversion and Two-Photon Excitation Fluorescence Properties of Phloxine B." Microscopy and Microanalysis 5, S2 (August 1999): 500–501. http://dx.doi.org/10.1017/s1431927600015828.

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A dye, Phloxine B, a common food coloring and one of the active components of a photoreactive insecticide was recently approved by Food and Drug Administration as D&C Red #28 for use in drugs and cosmetics. Phloxine B is also one of the most widely use stain in fluorescence microscopy. However, in spite of the widespread interest in multi-photon spectroscopy and imaging, no information is available on the electronic transitions properties of Phloxine B with red edge fluorescence excitation and with multi-photon excitation.In the present report we described the steady state and time-resolved fluorescent properties of Phloxine B with up-convert photon excitation and with two-photon excitation. We examined the electronic transitions properties of Phloxine B when was excited by femtosecond pulses from a mode-locked titanium sapphire laser or a mode-locked optical parametric oscillator laser. Phloxine B under excitation wavelengths above 775 nm was found to display two-photon excitation fluorescence with a spectrum maximum at 580 nm, which emission is consistent with one-photon excitation fluorescence spectrum. At the red edge excitation of Phloxine B, from 590 nm to 650 nm, we observed one-photon excitation fluorescence indicating that Phloxine B behaves like upconverting dye with one-photon excitation process.

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Choe, Kibaek, Yusaku Hontani, Tianyu Wang, Dimitre Ouzounov, Kristine Lai, Ankur Signh, Wendy Béguelin, Ari M. Melnick, and Chris Xu. "Intravital three-photon microscopy of entire mouse lymph node." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 86.4. http://dx.doi.org/10.4049/jimmunol.204.supp.86.4.

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Abstract For the last 20 years, intravital confocal and two-photon microscopy have been powerful tools to explore dynamic immune cell behavior in mouse lymph node. However, they can only image ~100 and ~300 μm depth respectively while a peripheral lymph node of adult mouse has 600 – 1000 μm thickness. Here, we visualized whole thickness of adult mouse popliteal lymph node with intravital three-photon microscopy. Three-photon excitation significantly improved a signal-to-background ratio compared to two-photon excitation even at the same excitation wavelength. The capability to image the full depth enabled to achieve the 3D volume of entire lymph node vasculature in vivo by tiling multiple z-stack images. We observed DsRed-expressing lymphocyte migration in LYVE1+ lymphatic sinus at 600 μm depth of lymph node where conventional two-photon microscopy was normally inaccessible. In addition, we demonstrated the capability multi-color imaging by using Cγ1-Confetti mice of which each germinal center B cells stochastically expresses one of 4 different fluorescent proteins (CFP, GFP, YFP and RFP). Intravital three-photon microscopy has the potential to shed light on unknown immune cell behavior in deeper regions of the lymph node in vivo just as two-photon microscopy did during the last 20 years at the shallower depth.

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Buist, Muller, Squier, and Brakenhoff. "Real time two-photon absorption microscopy using multi point excitation." Journal of Microscopy 192, no. 2 (November 1998): 217–26. http://dx.doi.org/10.1046/j.1365-2818.1998.00431.x.

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Fisher, Wait G., and Eric A. Wachter. "Improved Signal Processing in Multi-Photon Imaging." Microscopy and Microanalysis 6, S2 (August 2000): 800–801. http://dx.doi.org/10.1017/s1431927600036497.

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Multi-photon excitation has been used in microscopy for nearly a decade, providing a number of demonstrated advantages over other methods for fluorescence imaging. Because excitation is achieved using longer, less energetic light, photodamage and photobleaching of the sample are reduced. Furthermore, since excitation occurs only at the focal point, this approach allows the practical collection of three-dimensionally resolved fluorescence images of live cells. However, due to the small two-photon cross-section of most fluorophores, pulsed lasers are required to generate detectable signal levels. This is due to the quadratic dependence of twophoton absorption on the instantaneous power of the laser. Typically, these lasers are pulsed at very high repetition frequencies, on the order of 106 pulses per second with pulse durations of a few hundreds of femtoseconds. For example, a titanium:sapphire (Ti:S) laser mode-locked at 76 Mhz can provide up to 100,000 watts of instantaneous power and is ideal for exciting two-photon events.

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Chang, Ching-Wei, and Mary-Ann Mycek. "Precise fluorophore lifetime mapping in live-cell, multi-photon excitation microscopy." Optics Express 18, no. 8 (April 9, 2010): 8688. http://dx.doi.org/10.1364/oe.18.008688.

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Masters, B. R., P. T. C. So, and E. Gratton. "Optical Biopsy of In Vivo Human Skin: Multi-photon Excitation Microscopy." Lasers in Medical Science 13, no. 3 (October 1, 1998): 196–203. http://dx.doi.org/10.1007/s101030050074.

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Matsumoto, Naoya, Alu Konno, Takashi Inoue, Koyo Watanabe, and Shigetoshi Okazaki. "Amplitude-modulation-type multi-ring mask for two-photon excitation scanning microscopy." OSA Continuum 4, no. 6 (May 18, 2021): 1696. http://dx.doi.org/10.1364/osac.419804.

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Vlieg, Redmar C., and John van Noort. "Multiplexed two-photon excitation spectroscopy of single gold nanorods." Journal of Chemical Physics 156, no. 9 (March 7, 2022): 094201. http://dx.doi.org/10.1063/5.0073208.

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Plasmonic metallic nanoparticles are commonly used in (bio-)sensing applications because their localized surface plasmon resonance is highly sensitive to changes in the environment. Although optical detection of scattered light from single particles provides a straightforward means of detection, the two-photon luminescence (TPL) of single gold nanorods (GNRs) has the potential to increase the sensitivity due to the large anti-Stokes shift and the non-linear excitation mechanism. However, two-photon microscopy and spectroscopy are restricted in bandwidth and have been limited by the thermal stability of GNRs. Here, we used a scanning multi-focal microscope to simultaneously measure the two-photon excitation spectra of hundreds of individual GNRs with sub-nanometer accuracy. By keeping the excitation power under the melting threshold, we show that GNRs were stable in intensity and spectrum for more than 30 min, demonstrating the absence of thermal reshaping. Spectra featured a signal-to-noise ratio of >10 and a plasmon peak width of typically 30 nm. Changes in the refractive index of the medium of less than 0.04, corresponding to a change in surface plasmon resonance of 8 nm, could be readily measured and over longer periods. We used this enhanced spectral sensitivity to measure the presence of neutravidin, exploring the potential of TPL spectroscopy of single GNRs for enhanced plasmonic sensing.

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NEMOTO, Tomomi, Ryosuke KAWAKAMI, and Terumasa HIBI. "Improvement in Tissue Penetration Depth and Spatial Resolution of Multi-Photon Laser Excitation Microscopy." Review of Laser Engineering 41, no. 2 (2013): 107. http://dx.doi.org/10.2184/lsj.41.2_107.

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Kantelhardt, Sven Rainer, Jan Leppert, Jan Werner Kantelhardt, Erich Reusche, Gereon Hüttmann, and Alf Giese. "Multi-photon excitation fluorescence microscopy of brain-tumour tissue and analysis of cell density." Acta Neurochirurgica 151, no. 3 (February 24, 2009): 253–62. http://dx.doi.org/10.1007/s00701-009-0188-6.

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Dal Fovo, Alice, Mikel Sanz, Mohamed Oujja, Raffaella Fontana, Sara Mattana, Riccardo Cicchi, Piotr Targowski, et al. "In-Depth Analysis of Egg-Tempera Paint Layers by Multiphoton Excitation Fluorescence Microscopy." Sustainability 12, no. 9 (May 8, 2020): 3831. http://dx.doi.org/10.3390/su12093831.

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The non-invasive depth-resolved imaging of pictorial layers in paintings by means of linear optical techniques represents a challenge in the field of Cultural Heritage (CH). The presence of opaque and/or highly-scattering materials may obstruct the penetration of the radiation probe, thus impeding the visualization of the stratigraphy of paintings. Nonlinear Optical Microscopy (NLOM), which makes use of tightly-focused femtosecond pulsed lasers as illumination sources, is an emerging technique for the analysis of painted objects enabling micrometric three-dimensional (3D) resolution with good penetration capability in semi-transparent materials. In this work, we evaluated the potential of NLOM, specifically in the modality of Multi-Photon Excitation Fluorescence (MPEF), to probe the stratigraphy of egg-tempera mock-up paintings. A multi-analytical non-invasive approach, involving ultraviolet-visible-near infrared (UV-Vis-NIR) Fiber Optics Reflectance Spectroscopy, Vis-NIR photoluminescence, and Laser Induced Fluorescence, yielded key-information for the characterization of the constituting materials and for the interpretation of the nonlinear results. Furthermore, the use of three nonlinear optical systems allowed evaluation of the response of the analyzed paints to different excitation wavelengths and photon doses, which proved useful for the definition of the most suitable measurement conditions. The micrometric thickness of the paint layers, which was not measurable by means of Optical Coherence Tomography (OCT), was instead assessed by MPEF, thus demonstrating the effectiveness of this nonlinear modality in probing highly-scattering media, while ensuring the minimal photochemical disturbance to the examined materials.

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George, Nicholas M., Arianna G. Polese, Greg Futia, Baris Ozbay, Wendy Macklin, Emily Gibson, Aviva Abosch, Diego Restrepo, and Brian E. Moore. "2507 A novel multi-photon microscopy method for neuronavigation in deep brain stimulation surgery." Journal of Clinical and Translational Science 2, S1 (June 2018): 2–3. http://dx.doi.org/10.1017/cts.2018.40.

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OBJECTIVES/SPECIFIC AIMS: The goal for this project is to determine the feasibility of using a novel multi-photon fiber-coupled microscope to aid surgeons in localizing STN during surgeries. In order to accomplish this goal, we needed to identify the source of a strong autofluorescent signal in the STN and determine whether we could use image classification methods to automatically distinguish STN from surrounding brain regions. METHODS/STUDY POPULATION: We acquired 3 cadaveric brains from the University of Colorado Anschutz Medical Campus, Department of Pathology. Two of these brains were non-PD controls whereas 1 was diagnosed with PD. We dissected a 10 square centimeter region of midbrain surrounding STN, then prepared this tissue for slicing on a vibratome or cryostat. Samples were immuno-labeled for various cellular markers for identification, or left unlabeled in order to observe the autofluorescence for image classification. RESULTS/ANTICIPATED RESULTS: The border of STN is clearly visible based on the density of a strong autofluorescent signal. The autofluorescent signal is visible using 2-photon (850–1040 nm excitation) and conventional confocal microscopy (488–647 nm excitation). We were also able to visualize blood vessels with second harmonic generation. The autofluorescent signal is quenched by high concentrations of Sudan-black B (0.5%–5%), and is primarily localized in microtubule-associated protein-2 (MAP2)+ cells, indicating that it is likely lipofuscin accumulation in neurons. Smaller lipofuscin particles also accumulate in microglia, identified based on ionized calcium binding adopter 1 (Iba1)+ labeling. We anticipate that colocalization analysis will confirm these qualitative observations. Using 2-photon images of the endogenous autofluorescent signal in these samples, we trained a logistic regression-based image classifier using features derived from gray-level co-occurrence matrices. Preliminary testing indicates that our classifier performed well, with a mean accuracy of 0.89 (standard deviation of 0.11) and a Cohen’s Kappa value of 0.76 (standard deviation of 0.24). We are currently using coherent anti-Stokes Raman scattering and third harmonic imaging to identify different features of myelin that can be used to distinguish between these regions and expect similar results. DISCUSSION/SIGNIFICANCE OF IMPACT: Traditional methods for localizing STN during DBS surgery include the use of stereotactic coordinates and multi-electrode recording (MER) during implantation. MERs are incredibly useful in DBS surgeries, but require penetration of brain structures in order to infer location. Using multi-photon microscopy techniques to aid identification of STN during DBS surgeries offers a number of advantages over traditional methods. For example, blood vessels can be clearly identified with second harmonic generation, something that is not possible with MER. Multi-photon microscopy also allows visualization deep into tissue without actually penetrating it. This ability to look within a depth of field is useful for detection of STN borders based on autofluorescent cell density. When combined with traditional stereotactic information, our preliminary image classification methods are a fast, reliable way to provide surgeons with extra information concerning their location in the midbrain. We anticipate that future advancements and refinements to our image classifier will only increase accuracy and the potential applications and value. In summary, these preliminary data support the feasibility of multi-photon microscopy to aid in the identification of target brain regions during DBS surgeries. The techniques described here complement and enhance current stereotactic and electrophysiological methods for DBS surgeries.

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Neu, Thomas R., Stefan Woelfl, and John R. Lawrence. "Three-dimensional differentiation of photo-autotrophic biofilm constituents by multi-channel laser scanning microscopy (single-photon and two-photon excitation)." Journal of Microbiological Methods 56, no. 2 (February 2004): 161–72. http://dx.doi.org/10.1016/j.mimet.2003.10.012.

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Robbins, Emma, Stéphanie Leroy-Lhez, Nicolas Villandier, Marek Samoć, and Katarzyna Matczyszyn. "Prospects for More Efficient Multi-Photon Absorption Photosensitizers Exhibiting Both Reactive Oxygen Species Generation and Luminescence." Molecules 26, no. 20 (October 19, 2021): 6323. http://dx.doi.org/10.3390/molecules26206323.

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The use of two-photon absorption (TPA) for such applications as microscopy, imaging, and photodynamic therapy (PDT) offers several advantages over the usual one-photon excitation. This creates a need for photosensitizers that exhibit both strong two-photon absorption and the highly efficient generation of reactive oxygen species (ROS), as well as, ideally, bright luminescence. This review focuses on different strategies utilized to improve the TPA properties of various multi-photon absorbing species that have the required photophysical properties. Along with well-known families of photosensitizers, including porphyrins, we also describe other promising organic and organometallic structures and more complex systems involving organic and inorganic nanoparticles. We concentrate on the published studies that provide two-photon absorption cross-section values and the singlet oxygen (or other ROS) and luminescence quantum yields, which are crucial for potential use within PDT and diagnostics. We hope that this review will aid in the design and modification of novel TPA photosensitizers, which can help in exploiting the features of nonlinear absorption processes.

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OTOMO, Kohei, Terumasa HIBI, Takashi MURATA, Hirotaka WATANABE, Ryosuke KAWAKAMI, Hiroshi NAKAYAMA, Mitsuyasu HASEBE, and Tomomi NEMOTO. "Multi-point Scanning Two-photon Excitation Microscopy by Utilizing a High-peak-power 1042-nm Laser." Analytical Sciences 31, no. 4 (2015): 307–13. http://dx.doi.org/10.2116/analsci.31.307.

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Gualda, Emilio J., George Filippidis, Kristalia Melessanaki, and Costas Fotakis. "Third-Harmonic Generation and Multi-Photon Excitation Fluorescence Imaging Microscopy Techniques for Online Art Conservation Diagnosis." Applied Spectroscopy 63, no. 3 (March 2009): 280–85. http://dx.doi.org/10.1366/000370209787598898.

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Faraldi, F., G. J. Tserevelakis, G. Filippidis, G. M. Ingo, C. Riccucci, and C. Fotakis. "Multi photon excitation fluorescence imaging microscopy for the precise characterization of corrosion layers in silver-based artifacts." Applied Physics A 111, no. 1 (January 16, 2013): 177–81. http://dx.doi.org/10.1007/s00339-013-7548-z.

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Bickford, Lissett, Jiantang Sun, Kun Fu, Nastassja Lewinski, Vengadesan Nammalvar, Joseph Chang, and Rebekah Drezek. "Enhanced multi-spectral imaging of live breast cancer cells using immunotargeted gold nanoshells and two-photon excitation microscopy." Nanotechnology 19, no. 31 (June 24, 2008): 315102. http://dx.doi.org/10.1088/0957-4484/19/31/315102.

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Liu, I.-Ting, Chia-Sheng Yen, Wen-Lung Wang, Hung-Wen Tsai, Chang-Yao Chu, Ming-Yu Chang, Ya-Fu Hou, and Chia-Jui Yen. "Predict Early Recurrence of Resectable Hepatocellular Carcinoma Using Multi-Dimensional Artificial Intelligence Analysis of Liver Fibrosis." Cancers 13, no. 21 (October 23, 2021): 5323. http://dx.doi.org/10.3390/cancers13215323.

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Background: Liver fibrosis is thought to be associated with early recurrence of hepatocellular carcinoma (HCC) after resection. To recognize HCC patients with higher risk of early recurrence, we used a second harmonic generation and two-photon excitation fluorescence (SHG/TPEF) microscopy to create a fully quantitative fibrosis score which is able to predict early recurrence. Methods: The study included 81 HCC patients receiving curative intent hepatectomy. Detailed fibrotic features of resected hepatic tissues were obtained by SHG/TPEF microscopy, and we used multi-dimensional artificial intelligence analysis to create a recurrence prediction model “combined index” according to the morphological collagen features of each patient’s non-tumor hepatic tissues. Results: Our results showed that the “combined index” can better predict early recurrence (area under the curve = 0.917, sensitivity = 81.8%, specificity = 90.5%), compared to alpha fetoprotein level (area under the curve = 0.595, sensitivity = 68.2%, specificity = 47.6%). Using a Cox proportional hazards analysis, a higher “combined index” is also a poor prognostic factor of disease-free survival and overall survival. Conclusions: By integrating multi-dimensional artificial intelligence and SHG/TPEF microscopy, we may locate patients with a higher risk of recurrence, follow these patients more carefully, and conduct further management if needed.

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Lavin, C. A., W. A. Mohler, H. H. Keating, and J. G. White. "Capturing Developmental Events of the C. Elegans Embryo by High Pressure Freezing After Monitoring by a Multi-Photon Imaging System." Microscopy and Microanalysis 3, S2 (August 1997): 291–92. http://dx.doi.org/10.1017/s1431927600008345.

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High pressure freezing enables the rapid arrest of developmental events without prefixation. Standard chemical fixation is a time dependent event and may cause artifacts in sensitive cytoskeletal components. We are studying two developmental events in embryonic Caenorhabditis elegans: that involve changes in the cytoskeleton: spindle alignment and membrane fusion. The mitotic spindle undergoes rapid rotational alignment prior to certain differentiative divisions. We are trying to capture these events by anticipation their timing and rapid freezing. Precursor hypodermal cells of embryonic C. elegans undergo a transition from individual cells to a syncytium at the onset of morphogenesis. In an effort to visualize the fusion events, embryos were stained with the vital probe FM4-64 to highlight cell membranes. Development was monitored by fluorescent microscopy using multiple-photon excitation imaging to minimize photobleaching while providing clear images of deep sections. Small cellulose capillary tubes, as described by Hohenberg for isolation and high pressure freezing of individual cells, were not of sufficient optical quality for monitoring by a laser-scanning microscope.

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Nevin, A., D. Comelli, I. Osticioli, G. Filippidis, K. Melessanaki, G. Valentini, R. Cubeddu, and C. Fotakis. "Multi-photon excitation fluorescence and third-harmonic generation microscopy measurements combined with confocal Raman microscopy for the analysis of layered samples of varnished oil films." Applied Physics A 100, no. 3 (April 8, 2010): 599–606. http://dx.doi.org/10.1007/s00339-010-5644-x.

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Zandt, Bas-Jan, Are Losnegård, Erlend Hodneland, Margaret Lin Veruki, Arvid Lundervold, and Espen Hartveit. "Semi-automatic 3D morphological reconstruction of neurons with densely branching morphology: Application to retinal AII amacrine cells imaged with multi-photon excitation microscopy." Journal of Neuroscience Methods 279 (March 2017): 101–18. http://dx.doi.org/10.1016/j.jneumeth.2017.01.008.

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Zandt, Bas-Jan, Jian Hao Liu, Margaret Lin Veruki, and Espen Hartveit. "AII amacrine cells: quantitative reconstruction and morphometric analysis of electrophysiologically identified cells in live rat retinal slices imaged with multi-photon excitation microscopy." Brain Structure and Function 222, no. 1 (March 7, 2016): 151–82. http://dx.doi.org/10.1007/s00429-016-1206-0.

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Brzoska, Tomasz, Tomasz W. Kaminski, Egemen Tutuncuoglu, Mark T. Gladwin, and Prithu Sundd. "Two Stage Intravital Imaging Mouse Model to Assess Venous Thromboembolism in Sickle Cell Disease." Blood 138, Supplement 1 (November 5, 2021): 3225. http://dx.doi.org/10.1182/blood-2021-154317.

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Abstract Sickle cell disease (SCD) patients have an increased risk of venous thromboembolism (VTE). Population-based studies demonstrated that VTE has a cumulative incidence rate of approximately 25% in adult SCD patients and is associated with higher risk of mortality. VTE in SCD is most commonly manifested as deep vein thrombosis (DVT) with associated pulmonary embolism (PE). Although autopsy studies have regularly discovered pulmonary thromboembolic lesions in SCD patients, the pathophysiology of VTE in SCD remains largely unknown mostly due to the lack of relevant animal VTE model. Understanding the mechanisms that promote VTE in SCD is imperative to identify its prevention and treatment measures. To elucidate the cellular, molecular and biophysical mechanisms of VTE in SCD we developed an innovative two stage intravital imaging analysis experimental model in SCD mice. DVT in SCD mice was induced by surgical ligation of femoral vein. Venous thrombus formation was visualized using intravital multi-photon-excitation (MPE) microscopy. Venous thrombus took the form of a large mass of elliptical shape which extended in the long-axis direction of the femoral vein. It was composed of fibrin, erythrocytes and sparse platelets. Over time, thrombus was infiltrated by migrating neutrophils. To trigger acute PE, femoral vein ligation was removed and the venous thrombus was observed to spontaneously detach and travel to the SCD mouse lung. This method allowed real time visualization of acute PE in vivo using MPE microscopy of intact lung in live breathing mice. Acute PE involved embolization of the pulmonary arterioles and the arteriolar bottle-necks located at the junction of pulmonary arterioles and capillaries. The embolization of arteriolar circulation led to loss of blood flow in the arterioles and the down-stream capillaries. Herein we introduce an intravital microscopy approach to probe VTE in SCD live mouse. Our model has potential application in investigating the molecular determinants of VTE associated with SCD as well as evaluating efficacy of new antithrombotic drugs. Disclosures Sundd: Bayer: Research Funding; CSL Behring Inc: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Song, Ying, Zongwei Xu, Tao Liu, Mathias Rommel, Hong Wang, Yufang Wang, and Fengzhou Fang. "Depth Profiling of Ion-Implanted 4H–SiC Using Confocal Raman Spectroscopy." Crystals 10, no. 2 (February 21, 2020): 131. http://dx.doi.org/10.3390/cryst10020131.

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For silicon carbide (SiC) processed by ion-implantation, dedicated test structure fabrication or destructive sample processing on test wafers are usually required to obtain depth profiles of electrical characteristics such as carrier concentration. In this study, a rapid and non-destructive approach for depth profiling is presented that uses confocal Raman microscopy. As an example, a 4H–SiC substrate with an epitaxial layer of several micrometers thick and top layer in nanoscale that was modified by ion-implantation was characterized. From the Raman depth profiling, longitudinal optical (LO) mode from the epitaxial layer and longitudinal optical phonon-plasmon coupled (LOPC) mode from the substrate layer can be sensitively distinguished at the interface. The position profile of the LOPC peak intensity in the depth direction was found to be effective in estimating the thickness of the epitaxial layer. For three kinds of epitaxial layer with thicknesses of 5.3 μm, 6 μm, and 7.5 μm, the average deviations of the Raman depth analysis were −1.7 μm, −1.2 μm, and −1.4 μm, respectively. Moreover, when moving the focal plane from the heavily doped sample (~1018 cm−3) to the epitaxial layer (~1016 cm−3), the LOPC peak showed a blue shift. The twice travel of the photon (excitation and collection) through the ion-implanted layer with doping concentrations higher than 1 × 1018 cm−3 led to a difference in the LOPC peak position for samples with the same epitaxial layer and substrate layer. Furthermore, the influences of the setup in terms of pinhole size and numerical aperture of objective lens on the depth profiling results were studied. Different from other research on Raman depth profiling, the 50× long working distance objective lens (50L× lens) was found more suitable than the 100× lens for the depth analysis 4H–SiC with a multi-layer structure.

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Sundd, Prithu, Maritza Ann Jimenez, Margaret F. Bennewitz, Tomasz Brzoska, Egemen Tutuncuoglu, Gregory J. Kato, and Mark T. Gladwin. "Hairy Platelet-Derived Extracellular Vesicles Promote Lung Vaso-Occlusion in Sickle Cell Disease." Blood 130, Suppl_1 (December 7, 2017): 958. http://dx.doi.org/10.1182/blood.v130.suppl_1.958.958.

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Abstract Background: Acute chest syndrome (ACS) is a type of acute lung injury and the leading cause of mortality in Sickle Cell Disease (SCD). Current treatments for ACS are primarily supportive, and there is a critical need for rescue therapies. ACS is often a sequela of acute systemic vaso-occlusive crisis and preceded by thrombocytopenia. However, the role of platelets in the pathogenesis of ACS remains largely unknown. Methods: We used our validated model of vaso-occlusive crisis in transgenic, humanized SCD mice, which is triggered by intravenous challenge with nanogram levels of the TLR4 ligand, lipopolysaccharide (LPS). Platelet-neutrophil aggregates and blood flow in the lung microcirculation was visualized in real time in vivo, using multi-photon-excitation microscopy of intact lung in live SCD mice. SCD or control human blood was perfused through microfluidic channels in vitro and neutrophil-platelet aggregation was visualized using fluorescence microscopy. Platelet derived extracellular vesicles were characterized using nanoparticle tracking and biochemical approaches. Results: We have made a novel finding that the arrest of blood flow and injury in the lung is secondary to blockade of pulmonary arterioles by platelet-neutrophil aggregates. Using in vitro microfluidic studies, we confirmed that platelet-neutrophil aggregation is higher in LPS-treated SCD patient blood compared with healthy controls, and this correlates with increased numbers of platelet-derived extracellular vesicles (EVs) that express IL-1β. Our studies also reveal that platelet-neutrophil aggregation in pulmonary arterioles of SCD mice is associated with an increase in peripheral blood levels of platelet-derived EVs containing IL-1β. Remarkably, inhibition of TLR4 or TLR4/NLRP3-inflammasome activated caspase-1, or inhibition of IL-1β signaling, attenuated release of platelet EVs and platelet-neutrophil aggregation in the lung arterioles of SCD mice in vivo and SCD human blood in vitro . Conclusions: TLR4 and NLRP3-inflammasome-mediated caspase-1 activation in platelets during vaso-occlusive crisis leads to release of IL-1β-containing EVs into the circulation. These circulating platelet EVs promote platelet-neutrophil aggregation in pulmonary arterioles, which results in arrest of blood flow in the lung, leading to ACS. Therapeutic inhibition of TLR4/NLRP3-caspase-1 signaling in platelets or IL-1β signaling is a potential therapy for ACS in SCD patients. Acknowledgments: This study was supported by 1R01HL128297-01 (P.S.) and VMI startup funds (P.S.). Disclosures No relevant conflicts of interest to declare.

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Vats, Ravi, Egemen Tutuncuoglu, Jesus Tejero, Gray Shaw, and Prithu Sundd. "Tandem P-Selectin Glycoprotein Ligand Immunoglobulin Prevents Lung Vaso-Occlusion in SCD Mice." Blood 132, Supplement 1 (November 29, 2018): 2364. http://dx.doi.org/10.1182/blood-2018-99-116742.

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Abstract Introduction: Sickle cell disease (SCD) is an autosomal-recessive-genetic disorder, which leads to red blood cell sickling, hemolysis and vaso-occlusion. Acute systemic painful vaso-occlusive crisis (VOC) is the predominant pathophysiology requiring emergency medical care by SCD patients. 10-20% of SCD patients hospitalized with VOC tend to develop acute chest syndrome (ACS), a type of lung injury within next few days, suggesting a role for pulmonary vaso-occlusion in ACS. This epidemiology also provides a window for therapeutic intervention provided treatments to prevent vaso-occlusion are available. Earlier, we have shown that VOC involves entrapment of large neutrophil-platelet aggregates in lung arterioles of SCD mice, which is inhibited following intravenous administration of P-selectin function blocking antibody. Recently, a tandem-P-selectin-glycoprotein-ligand-immunoglobulin (TSGL-Ig) with two P-selectin binding sites in tandem, has been shown to prevent P-selectin-dependent liver injury in mice. Here, we test the ability of TSGL-Ig in attenuating P-selectin dependent lung vaso-occlusion in SCD mice. Materials and Methods: Townes knock-in humanized SS (hα/hα:βS/βS) mice were used as SCD mice. SCD mice were intravenously (IV) challenged with 10 µmole/kg Oxy-hemoglobin (Oxy-Hb) without or with 100 µg of TSGL-Ig to trigger lung vaso-occlusion. Fluorescent anti-mouse mAbs against CD49b and Ly6G, and FITC dextran were IV administered for in vivo staining of platelets, neutrophils, and visualizing lung microvasculature, respectively. Presence or absence of lung vaso-occlusion was assessed using multi-photon excitation enabled quantitative fluorescence intravital lung microscopy (qFILM). Results and Discussion: IV administration of Oxy-Hb led to occlusion of pulmonary arterioles by large neutrophil-platelet aggregates. Remarkably, IV administration of TSGL-Ig significantly attenuated lung vaso-occlusion in SCD mice by reducing the number as well as size of neutrophil-platelet aggregates in the pulmonary arterioles of SCD mice. Conclusion: Systemic challenge with Oxy-Hb promotes lung vaso-occlusion in SCD mice. TSGL-Ig significantly reduced oxy-Hb induced lung vaso-occlusion in SCD mice, highlighting the potential of TSGL-Ig to prevent ACS. Disclosures No relevant conflicts of interest to declare.

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Pradhan, Tirthadipa, Prithu Sundd, Mark T. Gladwin, Satdarshan Pal Monga, and Gregory J. Kato. "Impaired Bile Secretion Promotes Chronic Liver Injury in Sickle Cell Disease." Blood 134, Supplement_1 (November 13, 2019): 3536. http://dx.doi.org/10.1182/blood-2019-131915.

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Hepatic crisis is an emergent complication affecting sickle cell disease (SCD) patients, however, the molecular mechanism of sickle cell hepatobiliary crisis remains poorly understood. We examined the liver pathophysiology of SCD using a humanized mouse model (townes SCD mice; homozygous for Hbatm1(HBA)Tow, homozygous for Hbbtm2(HBG1,HBB*)Tow). These mice have the major features (irreversibly sickled red cells, splenomegaly, anemia, multiorgan pathology) found in humans with SCD and, as such, represent a useful in vivo system to study hepatobiliary changes in SCD disease. SCD mice manifested progressive hepatomegaly, liver injury and hyperbilirubinemia. RNA-sequence analysis of total RNA from SCD mouse liver compared to control (AS) identified dysregulation of genes encoding proteins responsible for fibrosis, bile acid synthesis, bile transport and cholesterol metabolism. Immunohistochemical analysis confirmed inflammation, fibrosis and increased ductular reaction in SCD mice. To mechanistically address the cholestatic phenotype in SCD mice, we used our recently developed multi-photon-excitation (MPE) enabled in vivo real-time fluorescence microscopy of intact liver in live mice. We used Texas-Red (TXR) dextran to visualize the blood flow through liver sinusoids and carboxyfluorescein (used as a surrogate of bile flow) to visualize bile flow through biliary canaliculi. Real time imaging show that sinusoidal ischemia occurs in the liver of transgenic-humanized SCD mice under basal condition. Intravital imaging also revealed impaired bile secretion into the bile canaliculi, which was associated with loss of apical bile acid transporters and bile acid biosynthetic enzymes, hepatic bile accumulation, and activation of Farnesoid X receptor (FXR) and small heterodimer partner (Shp) in the liver of SCD mice. These findings are the first to identify that impaired bile acid synthesis and misexpression of bile transporters promote intrahepatic bile accumulation and impaired bile secretion, leading to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of new therapies to treat sickle cell hepatic crisis. Disclosures Gladwin: Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases . Kato:Novartis, Global Blood Therapeutics: Consultancy, Research Funding; Bayer: Research Funding.

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Kaminski, Tomasz W., Tomasz Brzoska, Egemen Tutuncuoglu, Margaret V. Ragni, and Prithu Sundd. "Neutrophil Extracellular Traps Promote Joint Injury in Hemophilia." Blood 138, Supplement 1 (November 5, 2021): 990. http://dx.doi.org/10.1182/blood-2021-153645.

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Abstract Epidemiological evidence suggests that recurring episodes of joint-bleeding contribute to the development of hemophilic arthropathy (H) in 70-85% of hemophilia patients. Despite major advances in the treatment to prevent joint bleeding, HA continues to be a major morbidity affecting hemophilia patients and the etiological mechanism contributing to the progression of HA remains poorly understood. Recent evidence suggests that the accumulation of blood in the joints may lead to the release of erythrocyte-derived DAMPs (eDAMPs) such as heme and hemoglobin that can promote sterile inflammation, however, the innate immune pathways contributing to this pathophysiology remain unknown. In the study, we used a model of puncture-induced knee joint injury in FVIII-total knockout (F8TKO) mice and blood samples from hemophilia-A patients diagnosed with HA. Intravital multi-photon-excitation fluorescence intravital (in vivo) microscopy of injured synovium in live F8TKO or control mice was conducted to assess neutrophil-platelet aggregation and NETs generation in the knee-joint. Imaging-flow-cytometry and ELISA assays were used to estimate the number of circulating NETs in plasma of patients diagnosed with HA and mice after the knee-injury procedure. Scoring of the bleeding severity, histology, IHC and confocal imaging of joints were conducted to quantify the joint injury in mice. F8TKO but not control mice manifested knee-joint injury and severity of bleeding 5-days post knee-injury. Progression of knee-joint injury was associated with increased neutrophil accumulation and NETs shedding within the synovium of F8TKO mice. Circulating NETs were significantly abundant in the plasma of hemophilia patients diagnosed with HA and F8TKO mice following knee-injury but not plasma of control humans or mice. These findings are the first to suggest that NETs contribute to pathogenesis of HA in hemophilia. Currently, experiments are underway to identify the innate immune pathways that promote NETs shedding, leading to joint-damage in hemophilia. Disclosures Ragni: Takeda Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioverativ (Sanofi): Membership on an entity's Board of Directors or advisory committees; BioMarin Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Alnylam (Sanofi): Membership on an entity's Board of Directors or advisory committees; University of Pittsburgh: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees. Sundd: CSL Behring Inc: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

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Lu, Qiang, Shaoqun Zeng, Qingming Luo, and Yu Ruan. "Rapid modeling of two-dimensional multi-photon excitation microscopic imaging through turbid medium." Optics Communications 189, no. 4-6 (March 2001): 227–34. http://dx.doi.org/10.1016/s0030-4018(01)01033-1.

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Vats, Ravi, Egemen Tutuncuoglu, Jesus Tejero, Cheryl A. Hillery, Mark T. Gladwin, and Prithu Sundd. "Circulating Neutrophil Extracellular Traps in the Pathogenesis of Acute Chest Syndrome of Sickle Cell Disease." Blood 134, Supplement_1 (November 13, 2019): 3556. http://dx.doi.org/10.1182/blood-2019-130574.

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Introduction: Acute chest syndrome (ACS) is a type of acute lung injury and among the primary reasons for mortality and morbidity among Sickle Cell Disease (SCD) patients. Although epidemiologic evidence suggests that vaso-occlusion in the lung may serve as an antecedent to ACS, the cellular, molecular and biophysical mechanism of ACS is incompletely understood. Our recent findings revealed that the lung vaso-occlusion is enabled by the entrapment of embolic neutrophil-platelet aggregates in the pulmonary arterioles of transgenic humanized SCD mice. Recent evidence also suggests a role for neutrophil extracellular traps (NETs) in ACS. NETs are web-like structures of decondensed nuclear DNA decorated with citrullinated-histones (H3-cit) and neutrophil granule proteins. Interestingly, circulating nucleosomes and NETs fragments are elevated in SCD patient blood and the levels correlate with onset of ACS, however, the molecular mechanism that promotes generation of circulating NETs and the role of circulating NETs in promoting ACS remains poorly understood. Materials and Methods: Townes knock-in humanized SS (hα/hα:βS/βS) and AS (hα/hα:βA/βS) mice were used as SCD and control mice, respectively. SS and AS mice were intravenously (IV) administered 10 µmole/kg Oxy-Hb followed by Sytox orange, FITC-dextran or fluorescent anti-mouse mAbs against Ly6G, CD49b, H3cit, and neutrophil elastase for in vivo visualization of extracellular DNA, blood vessels, neutrophils, platelets and NETs, respectively. Pulmonary microcirculation was monitored using multi-photon-excitation enabled quantitative fluorescence intravital lung microscopy (qFILM). Results and Discussion: IV Oxy-Hb triggered the occlusion of pulmonary arterioles by neutrophil-platelet aggregates leading to loss of pulmonary blood flow in SCD but not control mice. Surprisingly, pulmonary vaso-occlusion in SCD mice was accompanied by the arrival of circulating cell free DNA (CFD) and NETs fragments into the pulmonary circulation. The cell free DNA (CFD) and NETs fragments entered the lung through the arterial circulation suggesting that they originated outside of lung. These cell free DNA (CFD) and NETs fragments contributed to lung vaso-occlusion and injury by promoting neutrophil-platelet aggregation in the lung arterioles. Conclusion: These findings reveal for the first time that circulating cell free DNA (CFD) and NETs fragments originating outside of lung contribute to pathogenesis of ACS. Currently, experiments are underway to identify the innate immune pathways that promote circulating NETs dependent lung injury in SCD. Disclosures Gladwin: Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Bayer Pharmaceuticals: Other: Co-investigator.

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Santos, Francisco A., Carlos E. R. Cardoso, José J. Rodrigues, Leonardo De Boni, and Luis M. G. Abegão. "Nonlinear Optical Materials: Predicting the First-Order Molecular Hyperpolarizability of Organic Molecular Structures." Photonics 10, no. 5 (May 8, 2023): 545. http://dx.doi.org/10.3390/photonics10050545.

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Experimental nonlinear optics (NLO) is usually expensive due to the high-end photonics and electronic devices needed to perform experiments such as incoherent second harmonic generation in liquid phase, multi-photon absorption, and excitation. Nevertheless, exploring NLO responses of organic and inorganic compounds has already opened a world of new possibilities. For example, NLO switches, NLO frequency converters, and a new way to obtain biological images through the incoherent second harmonic generation (SHG) originate from first-order molecular hyperpolarizability (β). The microscopic effect of the coherent or incoherent SHG is, in fact, the β. Therefore, estimating β without using expensive photonic facilities will optimize time- and cost-efficiency to predict if a specific molecular structure can generate light with double its incident frequency. In this work, we have simulated the β values of 27 organic compounds applying density functional theory (PBE0, TPSSh, wB97XD, B3LYP, CAM-B3LYP, and M06-2X) and Hartree–Fock methods using the Gaussian software package. The predicted β was compared with the experimental analogs obtained by the well-known Hyper–Rayleigh Scattering (HRS) technique. The most reliable functionals were CAM-B3LYP and M06-2X, with an unsigned average error of around 25%. Moreover, we have developed post-processing software—Hyper-QCC, providing an effortless, fast, and reliable way to analyze the Gaussian output files.

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Cheng, P. C., J. H. Chen, S. C. Hwang, C. K. Sun, D. B. Walden, and W. Y. Cheng. "3D Visualization of Maize Stem by MRI Technology." Microscopy and Microanalysis 7, S2 (August 2001): 100–101. http://dx.doi.org/10.1017/s143192760002657x.

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Recent development in confocal and multi-photon microscopy allows 3D imaging of plant tissue in high resolution. However, other than physical sectioning, macroscopical study of plant organs in 3D remains a difficult task. Among various available technologies for macroscopical imaging (e.g., Xray macro-tomography, optical coherent tomography and MRI), MRI is an ideal choice for its contrasting modality in volumetric imaging of soft tissues. A 3T Biospect MRI system (Brucker, Germany)(FIG 1) equipped with a 6cm inner diameter micro-quadrature coil (FIG 2) for RF transmission and reception of MRI signals was used in this study. Spin echo based RARE sequence was used to obtain T2 weighted images with TR/TE = 3160.5/58.5ms and field-of-view of 1.67cm × 1.67cm (256 × 256 pixels) at a slice thickness of 0.8mm. This corresponds to a voxel size of 65 × 65 × 800μm. Data was obtained within 1/2 hour with number-of-excitations (nex) set at 16. Figure 4 (a-x) shows a series of MRI sections through a stem node (the node below the main ear insertion) from field-grown maize (Zea mays, van Odyssey sweet corn). The stem was fixed in 1:3 EtOH/acetic acid, washed thoroughly in water prior to imaging. Air bubbles trapped in the tissue were removed by vacuuming, to avoid imaging artifact due to low magnetic susceptibility of air. Figure 5 (a-g) shows reconstructed longitudinal sections. Three-dimensional reconstruction (FIG. 3) was performed by using Vaytek VoxBlast™ and AutoQuant’s AutoVisulize 3D™ software. in combination with image segmentation and tracing tools, the MRI technology will greatly enhance our capability in the understanding of vascular architecture and its development in plants.

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Diaspro, Alberto, Paolo Bianchini, Giuseppe Vicidomini, Mario Faretta, Paola Ramoino, and Cesare Usai. "Multi-photon excitation microscopy." BioMedical Engineering OnLine 5, no. 1 (June 6, 2006). http://dx.doi.org/10.1186/1475-925x-5-36.

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Hoang, Van Thuy, Yassin Boussafa, Lynn Sader, Sébastien Février, Vincent Couderc, and Benjamin Wetzel. "Optimizing supercontinuum spectro-temporal properties by leveraging machine learning towards multi-photon microscopy." Frontiers in Photonics 3 (September 6, 2022). http://dx.doi.org/10.3389/fphot.2022.940902.

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Multi-photon microscopy has played a significant role in biological imaging since it allows to observe living tissues with improved penetration depth and excellent sectioning effect. Multi-photon microscopy relies on multi-photon absorption, enabling the use of different imaging modalities that strongly depends on the properties of the sample structure, the selected fluorophore and the excitation laser. However, versatile and tunable laser excitation for multi-photon absorption is still a challenge, limited by e.g. the narrow bandwidth of typical laser gain medium or by the tunability of wavelength conversion offered by optical parametric oscillators or amplifiers. As an alternative, supercontinuum generation can provide broadband excitations spanning from the ultra-violet to far infrared domains and integrating numerous fluorophore absorption peaks, in turn enabling different imaging modalities or potential multiplexed spectroscopy. Here, we report on the use of machine learning to optimize the spectro-temporal properties of supercontinuum generation in order to selectively enhance multi-photon excitation signals compatible with a variety of fluorophores (or modalities) for multi-photon microscopy. Specifically, we numerically explore how the use of reconfigurable (femtosecond) pulse patterns can be readily exploited to control the nonlinear propagation dynamics and associated spectral broadening occurring in a highly-nonlinear fiber. In this framework, we show that the use of multiple pulses to seed optical fiber propagation can trigger a variety of nonlinear interactions and complex propagation scenarios. This approach, exploiting the temporal dimension as an extended degree of freedom, is used to maximize typical multi-photon excitations at selected wavelengths, here obtained in a versatile and reconfigurable manner suitable for imaging applications. We expect these results to pave the way towards on-demand and real time supercontinuum shaping, with further multi-photon microscopy improvements in terms of spatial 3D resolution, optical toxicity, and wavelength selectivity.

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Kamada, Takafumi, Kohei Otomo, Takashi Murata, Kaito Nakata, Shota Hiruma, Ryota Uehara, Mitsuyasu Hasebe, and Tomomi Nemoto. "Low-invasive 5D visualization of mitotic progression by two-photon excitation spinning-disk confocal microscopy." Scientific Reports 12, no. 1 (January 17, 2022). http://dx.doi.org/10.1038/s41598-021-04543-7.

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AbstractNon-linear microscopy, such as multi-photon excitation microscopy, offers spatial localities of excitations, thereby achieving 3D cross-sectional imaging with low phototoxicity even in thick biological specimens. We had developed a multi-point scanning two-photon excitation microscopy system using a spinning-disk confocal scanning unit. However, its severe color cross-talk has precluded multi-color simultaneous imaging. Therefore, in this study, we introduced a mechanical switching system to select either of two NIR laser light pulses and an image-splitting detection system for 3- or 4-color imaging. As a proof of concept, we performed multi-color fluorescent imaging of actively dividing human HeLa cells and tobacco BY-2 cells. We found that the proposed microscopy system enabled time-lapse multi-color 3D imaging of cell divisions while avoiding photodamage. Moreover, the application of a linear unmixing method to the 5D dataset enabled the precise separation of individual intracellular components in multi-color images. We thus demonstrated the versatility of our new microscopy system in capturing the dynamic processes of cellular components that could have multitudes of application.

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Maity, Barun Kumar, and Sudipta Maiti. "Label-free imaging of neurotransmitters in live brain tissue by multi-photon ultraviolet microscopy." Neuronal Signaling 2, no. 4 (December 3, 2018). http://dx.doi.org/10.1042/ns20180132.

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Visualizing small biomolecules in living cells remains a difficult challenge. Neurotransmitters provide one of the most frustrating examples of this difficulty, as our understanding of signaling in the brain critically depends on our ability to follow the neurotransmitter traffic. Last two decades have seen considerable progress in probing some of the neurotransmitters, e.g. by using false neurotransmitter mimics, chemical labeling techniques, or direct fluorescence imaging. Direct imaging harnesses the weak UV fluorescence of monoamines, which are some of the most important neurotransmitters controlling mood, memory, appetite, and learning. Here we describe the progress in imaging of these molecules using the least toxic direct excitation route found so far, namely multi-photon (MP) imaging. MP imaging of serotonin, and more recently that of dopamine, has allowed researchers to determine the location of the vesicles, follow their intracellular dynamics, probe their content, and monitor their release. Recent developments have even allowed ratiometric quantitation of the vesicular content. This review shows that MP ultraviolet (MP-UV) microscopy is an effective but underutilized method for imaging monoamine neurotransmitters in neurones and brain tissue.

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Journal articles on the topic 'Multi-photon excitation microscopy'

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