Journal articles: 'Multi-Photon microscope'

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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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van Hook, Lee. "Entangled Microscopy." Microscopy Today 7, no. 3 (April 1999): 6–7. http://dx.doi.org/10.1017/s1551929500064038.

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The major factor in light microscopy limiting resolution in practice is not so much the wavelength of light and the resolving power of microscope optics, but the scattering of iight by the specimen. This extraneous scattered light interferes with the light used to image the specimen, effectively reducing the contrast of the imaging light and causing other annoying problems, There have been several inventions dedicated to solving this problem: various forms of interference-based microscopies, confocal microscopy, and most recently, multi-photon confocal microscopy.

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Jason Kirk. "Beyond the Hype - Is 2-Photon Microscopy Right for You?" Microscopy Today 11, no. 2 (April 2003): 26–29. http://dx.doi.org/10.1017/s1551929500052469.

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Confocal microscopes have come a long way in the past decade. Not only are they more stable and easier to use than ever before, but their cost has dropped enough that multi-user facilities are finding competition from individual labs using the new breed of "personal" confocals. In fact it has, in some cases, become the de facto standard for fluorescence imaging regardless of whether the user actually has requirements for it or not.But, researchers always have an ear out for something better. Enter 2-photon microscopy (2PLSM). The “bigger & badder” cousin of the confocal microscope has become a new weapon in the arsenal of a microscopy industry that caters to researchers who can't wait to fill their labs with the latest and greatest imaging systems. Advertised by the industry and researchers alike as a technique that seems to solve most of the problems that plague confocal, “2-photon” has become the new buzzword in the vocabulary of researchers eager to enhance their fluorescence applications.

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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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So, P. T. C., C. Y. Dong, C. Buhler, and E. Gratton. "Time-Resolved Stimulated-Emission Fluorescence Microscope." Proceedings, annual meeting, Electron Microscopy Society of America 54 (August 11, 1996): 278–79. http://dx.doi.org/10.1017/s042482010016385x.

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Time-resolved stimulated-emission fluorescence microscopy is a novel technique for obtaining super-diffraction limited spatial resolution and sub-nanosecond time resolution using a multi-photon process. This technique is inspired by traditional asynchronous stimulated-emission pump-probe spectroscopy. Fluorescence sample is first excited by a pump laser pulse, tuned to the molecular absorption band of the molecule. Within the chromophore lifetime, a second probe pulse, tuned to the emission band, stimulates fluorescence emission.The spatial resolution enhancement originates from the bilinear dependence of the stimulated emission efficiency on both the pump and probe beam intensities. At the objective focal point, the stimulated emission point spread function is the product of the point spread functions of the pump and probe beams. This situation is mathematically equivalent to both the confocal and the two-photon methods. 3-D depth discrimination and superior spatial resolution is expected.

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Chen, Dihan, Mindan Ren, Dapeng Zhang, Jialong Chen, Songyun Gu, and Shih-Chi Chen. "Design of a multi-modality DMD-based two-photon microscope system." Optics Express 28, no. 20 (September 24, 2020): 30187. http://dx.doi.org/10.1364/oe.404652.

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Chuah, J., and D. Holburn. "Scalable and Configurable Multi-pixel CMOS Photon Detector for the Scanning Electron Microscope." Microscopy and Microanalysis 18, S2 (July 2012): 1222–23. http://dx.doi.org/10.1017/s1431927612007969.

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Pegoraro, A., A. Ridsdale, RK Lyn, JP Pezacki, and A. Stolow. "Simple High Performance Multi-modal Coherent Anti-Stokes Raman Scattering (CARS) Microscopy Based on a Two-Photon Microscope." Microscopy and Microanalysis 14, S2 (August 2008): 758–59. http://dx.doi.org/10.1017/s1431927608086911.

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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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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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Gaustad, Jon-Vidar, Trude G. Simonsen, Lise Mari K. Hansem, and Einar K. Rofstad. "Intravital microscopy of tumor vessel morphology and function using a standard fluorescence microscope." European Journal of Nuclear Medicine and Molecular Imaging 48, no. 10 (February 19, 2021): 3089–100. http://dx.doi.org/10.1007/s00259-021-05243-0.

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Abstract Purpose The purpose of the study was to demonstrate the performance and possible applications of an intravital microscopy assay using a standard fluorescence microscope. Methods Melanoma and pancreatic ductal adenocarcinoma xenografts were initiated in dorsal window chambers and subjected to repeated intravital microscopy. The entire tumor vasculature as well as the normal tissue surrounding the tumor was imaged simultaneously with high spatial and temporal resolution. Vascular morphology images were recorded by using transillumination, and vascular masks were produced to quantify vessel density, vessel diameter, vessel segment length, and vessel tortuosity. First-pass imaging movies were recorded after an intervenous injection of a fluorescent marker and were used to investigate vascular function. Lymphatics were visualized by intradermal injections of a fluorescent marker. Results The intravital microscopy assay was used to study tumor growth and vascularization, tumor vessel morphology and function, tumor-associated lymphatics, and vascular effects of acute cyclic hypoxia and antiangiogenic treatment. The assay was sensitive to tumor-line differences in vascular morphology and function and detected tumor-induced lymphatic dilation. Acute cyclic hypoxia induced angiogenesis and increased the density of small diameter vessels and blood supply times, whereas antiangiogenic treatment selectively removed small-diameter vessels, reduced blood supply times, and induced hypoxia. Moreover, the window chamber was compatible with magnetic resonance imaging (MRI), and parametric images derived by dynamic contrast-enhanced MRI were shown to reflect vascular morphology and function. Conclusions The presented assay represents a useful and affordable alternative to intravital microscopy assays using confocal and multi-photon microscopes.

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Kim, Youngsik, Phat Lu, Tom D. Milster, and Khanh Kieu. "Hyper-numerical aperture (NA = 28) microscope using λ = 156 µm femtosecond source for multi-photon imaging." Biomedical Optics Express 4, no. 10 (August 29, 2013): 1786. http://dx.doi.org/10.1364/boe.4.001786.

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Schönhense, G., K. Medjanik, O. Fedchenko, A. Zymaková, S. Chernov, D. Kutnyakhov, D. Vasilyev, et al. "Time-of-flight photoelectron momentum microscopy with 80–500 MHz photon sources: electron-optical pulse picker or bandpass pre-filter." Journal of Synchrotron Radiation 28, no. 6 (November 1, 2021): 1891–908. http://dx.doi.org/10.1107/s1600577521010511.

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The small time gaps of synchrotron radiation in conventional multi-bunch mode (100–500 MHz) or laser-based sources with high pulse rate (∼80 MHz) are prohibitive for time-of-flight (ToF) based photoelectron spectroscopy. Detectors with time resolution in the 100 ps range yield only 20–100 resolved time slices within the small time gap. Here we present two techniques of implementing efficient ToF recording at sources with high repetition rate. A fast electron-optical beam blanking unit with GHz bandwidth, integrated in a photoelectron momentum microscope, allows electron-optical `pulse-picking' with any desired repetition period. Aberration-free momentum distributions have been recorded at reduced pulse periods of 5 MHz (at MAX II) and 1.25 MHz (at BESSY II). The approach is compared with two alternative solutions: a bandpass pre-filter (here a hemispherical analyzer) or a parasitic four-bunch island-orbit pulse train, coexisting with the multi-bunch pattern on the main orbit. Chopping in the time domain or bandpass pre-selection in the energy domain can both enable efficient ToF spectroscopy and photoelectron momentum microscopy at 100–500 MHz synchrotrons, highly repetitive lasers or cavity-enhanced high-harmonic sources. The high photon flux of a UV-laser (80 MHz, <1 meV bandwidth) facilitates momentum microscopy with an energy resolution of 4.2 meV and an analyzed region-of-interest (ROI) down to <800 nm. In this novel approach to `sub-µm-ARPES' the ROI is defined by a small field aperture in an intermediate Gaussian image, regardless of the size of the photon spot.

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Ko, Cheng-Hao, Janos Kirz, Harald Ade, Erik Johnson, Steven Hulbert, Erik Anderson, and Dieter Kern. "Second-generation scanning photoemission microscope at the National Synchrotron Light Source." Proceedings, annual meeting, Electron Microscopy Society of America 51 (August 1, 1993): 650–51. http://dx.doi.org/10.1017/s0424820100149088.

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We are commissioning a new generation scanning photoemission microscope (X1-SPEM II) at beamline X1A of the National Synchrotron Light Source (NSLS). Our first generation scanning photoemission microscope (X1-SPEM I) was the first to achieve submicron resolution. One of the major improvements is the replacement of the home-made single pass cylindrical mirror analyzer with a high energy resolution, multi-channel Hemispherical Sector Analyzer (HSA). The alignment scheme for the optical elements has also been redesigned. The advantages of these two major improvements will be discussed.A photoemission microscope requires a high brightness source and a good focusing scheme. In most cases, a monochromator is placed between the photon source and the focusing optical elements. Our X1-SPEM uses the soft x-ray undulator at the NSLS as a high brightness source. A Fresnel zone plate is coherently illuminated by the monochromatic beam selected by the spherical grating monochromator (250-800 eV range) to form a microprobe, less then 0.2 μm in size.

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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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Walter, Peter, Micheal Holmes, Razib Obaid, Lope Amores, Xianchao Cheng, James P. Cryan, James M. Glownia, et al. "The DREAM Endstation at the Linac Coherent Light Source." Applied Sciences 12, no. 20 (October 19, 2022): 10534. http://dx.doi.org/10.3390/app122010534.

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Free-electron lasers (FEL), with their ultrashort pulses, ultrahigh intensities, and high repetition rates at short wavelength, have provided new approaches to Atomic and Molecular Optical Science. One such approach is following the birth of a photo electron to observe ion dynamics on an ultrafast timescale. Such an approach presents the opportunity to decipher the photon-initiated structural dynamics of an isolated atomic and molecular species. It is a fundamental step towards understanding single- and non-linear multi-photon processes and coherent electron dynamics in atoms and molecules, ultimately leading to coherent control following FEL research breakthroughs in pulse shaping and polarization control. A key aspect for exploring photoinduced quantum phenomena is visualizing the collective motion of electrons and nuclei in a single reaction process, as dynamics in atoms/ions proceed at femtosecond (10−15 s) timescales while electronic dynamics take place in the attosecond timescale (10−18 s). Here, we report on the design of a Dynamic Reaction Microscope (DREAM) endstation located at the second interaction point of the Time-Resolved Molecular and Optical (TMO) instrument at the Linac Coherent Light Source (LCLS) capable of following the photon–matter interactions by detecting ions and electrons in coincidence. The DREAM endstation takes advantage of the pulse properties and high repetition rate of LCLS-II to perform gas-phase soft X-ray experiments in a wide spectrum of scientific domains. With its design ability to detect multi-ions and electrons in coincidence while operating in step with the high repetition rate of LCLS-II, the DREAM endstation takes advantage of the inherent momentum conservation of reaction product ions with participating electrons to reconstruct the original X-ray photon–matter interactions. In this report, we outline in detail the design of the DREAM endstation and its functionality, with scientific opportunities enabled by this state-of-the-art instrument.

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Burns, Kory, Benjamin Bischoff, Christopher M. Barr, Khalid Hattar, and Assel Aitkaliyeva. "Photo-exfoliation of MoS₂ quantum dots from nanosheets: an in situ transmission electron microscopy study." Nanotechnology 33, no. 8 (November 29, 2021): 085601. http://dx.doi.org/10.1088/1361-6528/ac357c.

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Abstract Fabrication of transition metal dichalcogenide quantum dots (QDs) is complex and requires submerging powders in binary solvents and constant tuning of wavelength and pulsed frequency of light to achieve a desired reaction. Instead of liquid state photoexfoliation, we utilize infrared laser irradiation of free-standing MoS2 flakes in transmission electron microscope (TEM) to achieve solid-state multi-level photoexfoliation of QDs. By investigating the steps involved in photochemical reaction between the surface of MoS2 and the laser beam, we gain insight into each step of the photoexfoliation mechanism and observe high yield production of QDs, led by an inhomogeneous crystalline size distribution. Additionally, by using a laser with a lower energy than the indirect optical transition of bulk MoS2, we conclude that the underlying phenomena behind the photoexfoliation is from multi-photon absorption achieved at high optical outputs from the laser source. These findings provide an environmentally friendly synthesis method to fabricate QDs for potential applications in biomedicine, optoelectronics, and fluorescence sensing.

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Yu, Xi, Fumihiro Itoigawa, and Shingo Ono. "Femtosecond Laser-Pulse-Induced Surface Cleavage of Zinc Oxide Substrate." Micromachines 12, no. 6 (May 21, 2021): 596. http://dx.doi.org/10.3390/mi12060596.

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The induction of surface cleavage along the crystalline structure of a zinc oxide substrate (plane orientation: 0001) by femtosecond laser pulses (wavelength: 1030 nm) has been reported; a scanning electron microscope image of the one-pulse (pulse energy: 6–60 μJ) irradiated surface shows very clear marks from broken hexagons. This cleavage process differs from the general laser-induced melt process observed on the surfaces of narrower-bandgap semiconductors and other metal materials. This phenomenon is discussed using a multi-photon absorption model, and the pulse-energy dependence of the cleavage depth (less than 3 μm) is quantitatively analyzed. Laser-induced cleavage is found not to occur under multi-pulse irradiation; when more than four pulses are irradiated upon the same spot, the general laser-induced melt process becomes dominant. This cleavage–melt shift is considered to be caused by the enhancement of absorption due to the initial pulses, which is supported by our measurement of cathodoluminescence.

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Reis, Marilia Alves dos, Jorge Mejia, Ilza Rosa Batista, Marycel Rosa Felisa Figols de Barboza, Solange Amorim Nogueira, Jairo Wagner, Francisco Romero Cabral, et al. "SPEM: a state-of-the-art instrument for high resolution molecular imaging of small animal organs." Einstein (São Paulo) 10, no. 2 (June 2012): 209–15. http://dx.doi.org/10.1590/s1679-45082012000200015.

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OBJECTIVE: To describe the Single Photon Emission Microscope (SPEM), a state-of-the-art instrument for small animal SPECT imaging, and characterize its performance presenting typical images of different animal organs. METHODS: SPEM consists of two independent imaging devices based on high resolution scintillators, high sensitivity and resolution Electron-Multiplying CCDs and multi-pinhole collimators. During image acquisition, the mouse is placed in a rotational vertical holder between the imaging devices. Subsequently, an appropriate software tool based on the Maximum Likelihood algorithm iteratively produces the volumetric image. Radiopharmaceuticals for imaging kidneys, heart, thyroid and brain were used. The mice were injected with 74 to 148 MBq/0,3mL and scanned for 40 to 80 minutes, 30 to 60 minutes afterwards. During this procedure, the animals remained under ketamine/xilazine anesthesia. RESULTS: SPEM images of different mouse organs are presented, attesting the imaging capabilities of the instrument. CONCLUSION: SPEM is an innovative technology for small animal SPECT imaging providing high resolution images with appropriate sensitivity for pre-clinical research. Its use with appropriate radiotracers will allow translational investigation of several animal models of human diseases, their pharmacological treatment and the development of potential new therapeutic agents.

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Hasegawa, Shigeru. "Multi-Photon Fluorescence Microscopy." Acta Histochemica et Cytochemica 31, no. 4 (1998): 293–96. http://dx.doi.org/10.1267/ahc.31.293.

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LEE, G. J., Y. H. JEONG, J. J. LEE, C. H. OH, E. K. KIM, Y. P. LEE, K. H. CHO, and D. W. SHIN. "PHOTONIC PATTERN FABRICATION IN FUSED SILICA, BK7, ANDGe-DOPED BOROPHOSPHOSILICA GLASS BY A FEMTOSECOND LASER." Journal of Nonlinear Optical Physics & Materials 14, no. 03 (September 2005): 305–9. http://dx.doi.org/10.1142/s0218863505002748.

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Photonic patterns were fabricated in fused silica, BK7, and Ge -doped borophosphosilica glass ( Ge -BPSG) using a focused femtosecond (fs)-laser beam. By focusing tens to hundreds of μJ fs-laser beam with a 10x microscope objective, we inscribed the semi-circular cavity patterns on the fused silica and the BK7. The inscribed hole diameters are 28 μm (fused silica) and 11 μm (BK7) at an input fluence of 71 J/cm2. This circular-cavity patterning is ascribed to the ablation via the multi-photon absorption process. For the application to functional devices, the surface relief gratings (SRGs) were made in fused silica and BK7 by focusing the fs-laser beam on the glass surface with a cylindrical lens and by translating the sample in the direction perpendicular to the focus line. The first-order diffraction efficiencies of the prepared SRGs are 34% (fused silica) and 14% (BK7). A refractive-index grating was also fabricated in the Ge -BPSG by using the two-beam interference method. The maximum index modulation of 2.5 × 10-3was obtained for 20,000 laser shots of 73 mJ/cm2per pulse. It is thought that the index modification occurs through the defect formation by the fs-laser irradiation.

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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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Zhang, Luwei, Xiaodong Jia, Yunzhe Wang, Yin Zhang, Anmin Chen, Junfeng Shao, and Changbin Zheng. "Effect of Femtosecond Laser Polarization on the Damage Threshold of Ta2O5/SiO2 Film." Applied Sciences 12, no. 3 (January 30, 2022): 1494. http://dx.doi.org/10.3390/app12031494.

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The study used linearly and circularly polarized femtosecond pulsed lasers to irradiate a Ta2O5/SiO2 film. Firstly, the damage thresholds of the film for linearly and circularly polarized femtosecond pulsed lasers were measured in 1-on-1 mode. The results showed that the damage threshold (1.70 J/cm2) under a circularly polarized laser was higher than that (1.68 J/cm2) under a linearly polarized laser. For femtosecond lasers, the multi-photon ionization cross-section under circular polarization was lower than that under linear polarization. The lower ionization rate under circular polarization led to a higher damage threshold compared to the case under linear polarization. Secondly, the damage morphology of the film irradiated by linearly and circularly polarized femtosecond lasers was observed by microscope. The damage caused by linearly polarized laser was more evident than that caused by the circularly polarized laser. Finally, the damage thresholds induced by linearly and circularly polarized femtosecond pulsed lasers were measured in S-on-1 (S = 2, 5, and 10) mode. For the same S value (2, 5, or 10), the damage threshold under the circularly polarized laser was higher than that under the linearly polarized laser. The damage thresholds under two polarized laser pulses decreased with an increase in the number of laser shots, indicating that repeated laser pulses had a cumulative effect on the damage of the film.

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Denk, Winfried. "Multi-photon microscopy in neuroscience." Microscopy and Microanalysis 9, S02 (August 2003): 1138–39. http://dx.doi.org/10.1017/s1431927603445698.

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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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KUMAZAKI, SHIGEICHI, MAKOTO HASEGAWA, MOHAMMAD GHONEIM, YUGO SHIMIZU, KENJI OKAMOTO, MASAYOSHI NISHIYAMA, HIROZO OH-OKA, and MASAHIDE TERAZIMA. "A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120." Journal of Microscopy 228, no. 2 (November 2007): 240–54. http://dx.doi.org/10.1111/j.1365-2818.2007.01835.x.

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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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Zamora, Ana, Michèle Moris, Rui Silva, Olivier Deschaume, Carmen Bartic, Tatjana N. Parac-Vogt, and Thierry Verbiest. "Visualization and characterization of metallo-aggregates using multi-photon microscopy." RSC Advances 11, no. 2 (2021): 657–61. http://dx.doi.org/10.1039/d0ra07263j.

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Wu, Zhuojun, Timo Rademakers, Fabian Kiessling, Michael Vogt, Erik Westein, Christian Weber, Remco T. A. Megens, and Marc van Zandvoort. "Multi-photon microscopy in cardiovascular research." Methods 130 (November 2017): 79–89. http://dx.doi.org/10.1016/j.ymeth.2017.04.013.

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Ingram, Peter, Scott D. Davilla, and Ann LeFurgey. "Event Streamed Spectrum Imaging using Programmed Beam Acquisition in Biological Microprobe Analysis." Microscopy Today 17, no. 1 (January 2009): 44–47. http://dx.doi.org/10.1017/s1551929500055024.

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The general trend of microscopical investigation in biology from the 1950s to the early 1970s was towards obtaining structural information. This goal initially was met using heavy metal and/or aldehyde fixatives, room temperature dehydration with polar organic liquids, embedding with epoxy and acrylate resins, and thin sectioning at room temperature. By the mid 1970s, a perceptible change occurred in the direction of both light and analytical electron microscopy towards investigation of the chemical reactivity and composition of structures made visible with increasingly better spatial resolution for light and electron microscopes. During the past 25 years there have been considerable innovations in microanalytical techniques, including analytical electron and x-ray microscopy and microanalysis, secondary ion mass spectrometry, laser microprobe mass analysis, the scanning probe microscopies, and confocal/multi-photon microscopy. In addition, cryopreservation and the development of chromophores for visualization of molecular and ionic sites within individual living cells as well as membranes have redefined the goal of microscopical preservation: to stabilize cell structure and composition as they exist in the living state.

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Phillips, C. L., and V. H. Gattone. "Diagnostic Implications of Multi-Photon Fluorescent Microscopy." Microscopy and Microanalysis 19, S2 (August 2013): 238–39. http://dx.doi.org/10.1017/s1431927613003188.

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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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Lin, B. L., F. J. Kao, P. C. Cheng, and P. C. Cheng. "The Response of Maize Protoplasts to High Intensity Illumination in Multi-Photon Fluorescence Microscopy." Microscopy and Microanalysis 6, S2 (August 2000): 806–7. http://dx.doi.org/10.1017/s1431927600036527.

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Multi-photon fluorescence microscopy has been cited for its advantage in increased depth penetration due to low linear absorption coefficient of biological specimen in the near infrared (NIR) range. Using a pulsed laser, it is possible to efficiently excite two-photon fluorescence with a high peak power while keeping the average power low to avoid thermal and photochemical damages to the specimen. Currently, mode-locked Ti-sapphire and Cr-Forsterite lasers that generate sub-picosecond pulses are used as the light source for multi-photon fluorescence microscopy. Because of the need of high peak power for efficiently exciting two-photon fluorescence, the relationship between cell damage and peak power has become an interesting and much debated topic in the application of multi-photon fluorescence microscopy. It is conceivable that at high illumination intensity, non-linear photochemical processes may have impacts on cell physiology and viability in ways much different from low illumination in the linear domain.

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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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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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Zhang, Zongqi, Yizhu Chen, Tiantian Zhang, Lingyu Guo, Wenlong Yang, Junfeng Zhang, and Changqian Wang. "Role of Myoendothelial Gap Junctions in the Regulation of Human Coronary Artery Smooth Muscle Cell Differentiation by Laminar Shear Stress." Cellular Physiology and Biochemistry 39, no. 2 (2016): 423–37. http://dx.doi.org/10.1159/000445636.

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Background/Aims: Smooth muscle cells may dedifferentiate into the synthetic phenotype and promote atherosclerosis. Here, we explored the role of myoendothelial gap junctions in phenotypic switching of human coronary artery smooth muscle cells (HCASMCs) co-cultured with human coronary artery endothelial cells (HCAECs) exposed to shear stress. Methods: HCASMCs and HCAECs were seeded on opposite sides of Transwell inserts, and HCAECs were exposed to laminar shear stress of 12 dyn/cm2 or 5 dyn/cm2. The myoendothelial gap junctions were evaluated by using a multi-photon microscope. Results: In co-culture with HCAECs, HCASMCs exhibited a contractile phenotype, and maintained the expression of differentiation markers MHC and H1-calponin. HCASMCs and HCAECs formed functional intercellular junctions, as evidenced by colocalization of connexin(Cx)40 and Cx43 on cellular projections inside the Transwell membrane and biocytin transfer from HCAECs to HCASMCs. Cx40 siRNA and 18-α-GA attenuated protein expression of MHC and H1-calponin in HCASMCs. Shear stress of 5 dyn/cm2 increased Cx43 and decreased Cx40 expression in HCAECs, and partly inhibited biocytin transfer from HCAECs to HCASMCs, which could be completely blocked by Cx43 siRNA or restored by Cx40 DNA transfected into HCAECs. The exposure of HCAECs to shear stress of 5 dyn/cm2 promoted HCASMC phenotypic switching, manifested by morphological changes, decrease in MHC and H1-calponin expression, and increase in platelet-derived growth factor (PDGF)-BB release, which was partly rescued by Cx43 siRNA or Cx40 DNA or PDGF receptor signaling inhibitor. Conclusions: The exposure of HCAECs to shear stress of 5 dyn/cm2 caused the dysfunction of Cx40/Cx43 heterotypic myoendothelial gap junctions, which may be replaced by homotypic Cx43/Cx43 channels, and induced HCASMC transition to the synthetic phenotype associated with the activation of PDGF receptor signaling, which may contribute to shear stress-associated arteriosclerosis.

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White, Nick, and Rachel Errington. "Multi-Photon Microscopy: Seeing More by Imaging Less." BioTechniques 33, no. 2 (August 2002): 298–303. http://dx.doi.org/10.2144/02332bi01.

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Niggli, Ernst. "Applications of multi-photon microscopy in cell physiology." Frontiers in Bioscience 9, no. 1-3 (2004): 1598. http://dx.doi.org/10.2741/1353.

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Wang, Ke, Nicholas G. Horton, and Chris Xu. "Going Deep: Brain Imaging with Multi-Photon Microscopy." Optics and Photonics News 24, no. 11 (November 1, 2013): 32. http://dx.doi.org/10.1364/opn.24.11.000032.

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Yates, Andrew. "Visualizing recall immune responses with multi-photon microscopy." Immunology 128, no. 4 (December 2009): 461–62. http://dx.doi.org/10.1111/j.1365-2567.2009.03195.x.

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Niesner, Raluca A., and Anja E. Hauser. "Recent advances in dynamic intravital multi-photon microscopy." Cytometry Part A 79A, no. 10 (September 8, 2011): 789–98. http://dx.doi.org/10.1002/cyto.a.21140.

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TANG ZHI-LIE, LIANG RUI-SHENG, and CHANG HONG-SEN. "THE IMAGING THEORY OF TWO-PHOTON AND MULTI-PHOTON CONFOCAL SCANNING MICROSCOPY." Acta Physica Sinica 49, no. 6 (2000): 1076. http://dx.doi.org/10.7498/aps.49.1076.

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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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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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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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Cheng, P. C., C. K. Sun, B. L. Lin, S. W. Chu, I. S. Chen, T. M. Liu, S. P. Lee, H. L. Liu, M. X. Kuo, and D. J. Lin. "Biological Photonic Crystals – Revealed by Multi-photon Nonlinear Microscopy." Microscopy and Microanalysis 8, S02 (August 2002): 268–69. http://dx.doi.org/10.1017/s1431927602100389.

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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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Extermann, Jerome, Luigi Bonacina, Enrique Cuña, Christelle Kasparian, Yannick Mugnier, Thomas Feurer, and Jean-Pierre Wolf. "Nanodoublers as deep imaging markers for multi-photon microscopy." Optics Express 17, no. 17 (August 14, 2009): 15342. http://dx.doi.org/10.1364/oe.17.015342.

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Journal articles on the topic 'Multi-Photon microscope'

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