Multi Photon Research Articles

Retraction Note: Multi photon micro material analysis based on Raman spectroscopy biosensor for cancer detection using biomarker with deep learning techniques

Retraction Note: Multi photon micro material analysis based on Raman spectroscopy biosensor for cancer detection using biomarker with deep learning techniques

Correlative multiphoton-STED microscopy of podocyte calcium levels and slit diaphragm ultrastructure in the renal glomerulus

In recent years functional multiphoton (MP) imaging of vital mouse tissues and stimulation emission depletion (STED) imaging of optically cleared tissues allowed new insights into kidney biology. Here, we present a novel workflow where MP imaging of calcium signals can be combined with super-resolved STED imaging for morphological analysis of the slit diaphragm (SD) within the same glomerulus. Mice expressing the calcium indicator GCaMP3 in podocytes served as healthy controls or were challenged with two different doses of nephrotoxic serum (NTS). NTS induced glomerular damage in a dose dependent manner measured by shortening of SD length. In acute kidney slices (AKS) intracellular calcium levels increased upon disease but showed a high variation between glomeruli. We could not find a clear correlation between intracellular calcium levels and SD length in the same glomerulus. Remarkably, analysis of the SD morphology of glomeruli selected during MP calcium imaging revealed a higher percentage of completely disrupted SD architecture than estimated by STED imaging alone. Our novel co-imaging protocol is applicable to a broad range of research questions. It can be used with different tissues and is compatible with diverse reporters and target proteins.

Analysis of age-related changes in the left ventricular myocardium with multiphoton microscopy

Aging induces cardiac remodeling, resulting in an increase in the risk of suffering heart diseases, including heart failure. Collagen deposition increases with age and, together with sarcomeric changes in cardiomyocytes, may lead to ventricular stiffness. Multiphoton (MP) microscopy is a useful technique to visualize and detect variations in cardiac structures in a label free fashion. Here, we propose a method based on MP imaging (both two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) modalities) to explore and objectively quantify age-related structural differences in various components of cardiac tissues. Results in transmural porcine left ventricle (LV) sections reveal significant differences when comparing samples from young and old animals. Collagen and myosin SHG signals in old specimens are respectively 3.8x and >6-fold larger than in young ones. Differences in TPEF signals from cardiomyocyte were ∼3x. Moreover, the increased amount of collagen in old specimens results in a more organized pattern when compared to young LV tissues. Since changes in collagen and myosin are associated with cardiac dysfunction, the technique used herein might be a useful tool to accurately predict and measure changes associated with age-related myocardium fibrosis, tissue remodeling and sarcomeric alterations, with potential implications in preventing heart disease.

Dynamic Mode Decomposition of Multiphoton and Stimulated Emission Depletion Microscopy Data for Analysis of Fluorescent Probes in Cellular Membranes.

An analysis of the membrane organization and intracellular trafficking of lipids often relies on multiphoton (MP) and super-resolution microscopy of fluorescent lipid probes. A disadvantage of particularly intrinsically fluorescent lipid probes, such as the cholesterol and ergosterol analogue, dehydroergosterol (DHE), is their low MP absorption cross-section, resulting in a low signal-to-noise ratio (SNR) in live-cell imaging. Stimulated emission depletion (STED) microscopy of membrane probes like Nile Red enables one to resolve membrane features beyond the diffraction limit but exposes the sample to a lot of excitation light and suffers from a low SNR and photobleaching. Here, dynamic mode decomposition (DMD) and its variant, higher-order DMD (HoDMD), are applied to efficiently reconstruct and denoise the MP and STED microscopy data of lipid probes, allowing for an improved visualization of the membranes in cells. HoDMD also allows us to decompose and reconstruct two-photon polarimetry images of TopFluor-cholesterol in model and cellular membranes. Finally, DMD is shown to not only reconstruct and denoise 3D-STED image stacks of Nile Red-labeled cells but also to predict unseen image frames, thereby allowing for interpolation images along the optical axis. This important feature of DMD can be used to reduce the number of image acquisitions, thereby minimizing the light exposure of biological samples without compromising image quality. Thus, DMD as a computational tool enables gentler live-cell imaging of fluorescent probes in cellular membranes by MP and STED microscopy.

RETRACTED ARTICLE: Multi photon micro material analysis based on Raman spectroscopy biosensor for cancer detection using biomarker with deep learning techniques

RETRACTED ARTICLE: Multi photon micro material analysis based on Raman spectroscopy biosensor for cancer detection using biomarker with deep learning techniques

A novel multi-anode MCP-PMT with Cherenkov radiator window

With the proposal of the road map of Time-of-Flight positron emission tomography (TOF-PET) with a Coincidence Time Resolution (CTR) of 10 ps by Paul Lecoq et al. in 2020, the technology based on TOF puts forward higher requirements on the performance of its core optical devices. In this manuscript, we developed a kind of Multi-Anode micro channel plate photomultiplier tube (MCP-PMT) based on Cherenkov radiator window. By using the Cherenkov radiator as the optical window of Multi-Anode MCP-PMT directly, the optical interface between the scintillator and the PMT window in conventional TOF-PET is eliminated, reducing light loss and further improve the time resolution of TOF-PET. The anode of this MCP-PMT can be split into a finely spaced array of collecting electrodes to achieve good positional sensitivity. In this paper, the single photon detection capability and time resolution of this kind of MCP-PMT are evaluated. The transit time spread (TTS) at single photon mode can reach 42.9 ps, and the TTS at multi photon mode is less than 10 ps. The CTR based on this kind of MCP-PMT is 156.7 ± 10.6 ps FWHM, which is equivalent to a position resolution of 23.5 ± 1.6 mm along a line of response (LOR). The performance uniformity of this kind MCP-PMT has also been calibrated.

Biomimetic Remineralization of an Extracellular Matrix Collagen Membrane for Bone Regeneration.

Natural extracellular matrix (ECM) collagen membranes are frequently used for bone regeneration procedures. Some disadvantages, such as rapid degradation and questionable mechanical properties, limit their clinical use. These membranes have a heterologous origin and may proceed from different tissues. Biomineralization is a process in which hydroxyapatite deposits mainly in collagen fibrils of the matrices. However, when this deposition occurs on the ECM, its mechanical properties are increased, facilitating bone regeneration. The objective of the present research is to ascertain if different membranes from distinct origins may undergo biomineralization. Nanomechanical properties, scanning electron (SEM) and multiphoton (MP) microscopy imaging were performed in three commercially available ECMs before and after immersion in simulated body fluid solution for 7 and 21 d. The matrices coming from porcine dermis increased their nanomechanical properties and they showed considerable mineralization after 21 d, as observed in structural changes detected through SEM and MP microscopy. It is hypothesized that the more abundant crosslinking and the presence of elastin fibers within this membrane explains the encountered favorable behavior.

Dental Age Estimation Using Multiphoton Microscopy: A Potential Tool for Forensic Science.

Normal aging affects the different structures of teeth, in particular, the dentine. These changes are useful in forensic disciplines as a tool for age estimation. Although multiphoton (MP) microscopy has been used to explore dental pieces, a relationship between age and MP response of the human dentine has not been proposed yet. The relationship between MP signals and natural dentine aging is investigated herein. An index of age (INAG) combining two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) images has been used to quantify these changes. The results show that the INAG significantly decreases with age. Moreover, peritubular dentine size and collagen internal properties are also modified with age. This information confirms the usefulness of this technique in forensic age estimation after disasters (natural or manmade) with a lack of comprehensive fingerprint database. Courts and other government authorities might also benefit from this tool when the official age of individuals under special circumstances is required for legal or medical reasons.

Studies of the First Electronically Excited State of 3-Fluoropyridine and Its Ionic Structure by Means of REMPI, Two-Photon MATI, One-Photon VUV-MATI Spectroscopy and Franck–Condon Analysis

3-Fluoropyridine has been investigated by means of resonance-enhanced multi photon ionization, mass-analyzed threshold ionization (MATI) and one-photon VUV-MATI spectroscopy to study the effect of m-fluorine substitution on the involved states.

Improving Multiphoton Microscopy by Combining Spherical Aberration Patterns and Variable Axicons

Multiphoton (MP) microscopy is a well-established method for the non-invasive imaging of biological tissues. However, its optical sectioning capabilities are reduced due to specimen-induced aberrations. Both the manipulation of spherical aberration (SA) and the use of axicons have been reported to be useful techniques to bypass this limitation. We propose the combination of SA patterns and variable axicons to further improve the quality of MP microscopy images. This approach provides enhanced images at different depth locations whose quality is better than those corresponding to the use of SA or axicons separately. Thus, the procedure proposed herein facilitates the visualization of details and increases the depth observable at high resolution.

Helper T Cell Response to Factor VIII In Vivo Requires Several Anatomically Distinct Types of Antigen Presenting Cells

Inhibitor formation is the most serious complication of factor (F)VIII replacement therapy for hemophilia A. It has long been clear that FVIII inhibitors arise in a CD4 + T cell-dependent manner, but early events in the immune response leading to MHC-II presentation and CD4 + T cell activation remain obscure. Several types of antigen presenting cells (APCs) have been implicated in the uptake of FVIII, but their relevance in T cell activation has been unclear. This study aimed to pinpoint the roles of APCs in priming FVIII-specific CD4 + T cells in vivo.Several transgenic strains of mice on the C57BL/6J background were employed to perform in vivo antigen presentation assays involving one intravenous (IV) injection of 5 µg FVIII-OVA fusion protein (FOVA), which contained an MHC-II I-A b peptide epitope of chicken ovalbumin in place of the B domain of FVIII, and an adoptive transfer (via IV injection on the following day) of 5E6 transgenic OT-II CD4 + T cells, which express chicken ovalbumin-specific T cell receptor (TCR). Before adoptive transfers, OT-II T cells were stained with CellTrace Violet (CTV). Spleens were collected for flow cytometry analyses four days after the adoptive transfer. FOVA shows identical specific activity to that of B domain deleted FVIII.CD11c-DTR/GFP mice (which express diphtheria toxin receptor and green fluorescent protein under the CD11c promoter) (n=3) received two intraperitoneal (IP) injections of 100 ng diphtheria toxin (DT) or PBS only (n=3) one day before and on the day of FVIII-OVA injection to deplete CD11c high cells (i.e. dendritic cells, DCs, marginal zone, sinusoidal and metallophilic macrophages, MFs). DT treatment completely abrogated T cell proliferation in the animals (p=0.03), while on average 11.1% of CD4 +CTV + cells from the control animals were progenies of the transferred cells.XCR1-DTRvenus mice (which express DT receptor and the Venus variant of yellow fluorescent protein under the XCR1 promoter) were similarly treated with DT or PBS (n=4/group) one day before FOVA injection and one day after adoptive transfer to deplete type I conventional DCs (which make up ~80% of XCR1 + cells). XCR1 + cell depletion reduced T cell proliferation by ~50% (p=0.02). CD4 +CTV + cells from the DT-treated and control mice included 19.4% and 38.6% progenies, respectively.Hemophilia A (F8e16-/-) mice received 100 µg of marginal zone (MZ) B cell-depleting antibodies anti-CD11a and anti-CD49d (n=4) or isotype control antibodies (n=3) four and two days before FOVA injection. MZ B cell depletion completely abrogated T cell proliferation (p=0.02), while on average 31% of CD4 +CTV + cells from the control animals were progenies of the transferred cells. In another experiment, hemophilia A mice were injected with GdCl 3 (n=4), which inactivates MFs, or PBS (n=3) one day before and on the day of FOVA administration. MF inactivation completely abrogated T cell proliferation in all but 1 animal (p=0.03), while on average 15.9% of CD4 +CTV + cells from the control animals were progenies of the transferred cells.To visualize APC-T cell interactions in vivo, we performed multiphoton (MP) intravital microscopy (IVM) of inguinal lymph nodes (LNs) in CD11c-DTR/GFP mice. The animals received adoptive transfers of 1E7 CTV-stained OT-II CD4 + T cells IV ~24 hours and 5 µg FOVA 20, 5 or 1 hour before IVM delivered intradermally (ID) to target the skin-draining LNs. Control animals received adoptive cell transfer only. For IVM, the LNs were surgically exposed in live, anesthetized animals. Five hours after FOVA injection, multiple CTV + OT-II T cells formed clusters around GFP + cells throughout the T cell zone with several motile T cells surveying the B cell follicle. Twenty hours after FOVA injection, CTV + T cells densely populated and demarcated the T-B border in the LNs. CTV + T cells were nearly absent from the inguinal LNs in the control animals.We propose that activation of CD4 + T cells in response to FVIII requires a complex interaction of multiple types of APCs, which occupy distinct compartments in the secondary lymphoid organs that FVIII antigen traverses en route to cognate CD4 + T cells. In the spleen, where response to FVIII primarily takes place, antigens larger than 60 kDa (such as FVIII at 280 kDa) do not freely flow into the white pulp and instead need to be ferried by APCs. While DCs ultimately activate CD4 + T helper cells, MFs shuttle the antigen to DCs and may also provide innate activation immune signals. DisclosuresNo relevant conflicts of interest to declare.

P10.02 Combined methods of a micropump system and a chronic cranial window allows tumor observation with multi photon laser scanning microscopy under continuous treatment

Abstract BACKGROUND Glioblastomas are notoriously therapy resistant tumors. As opposed to other tumor entities, no major advances in therapeutic success have been made in the past decades. This has been calling for a deeper biological understanding of the tumor, its growth and resistance patterns. We have been using a xenograft glioma model, where human glioblastoma cells are implanted under chronic cranial windows and studied longitudinally over many weeks and months using multi photon laser scanning microscopy (MPLSM). To test the effect of (new) drugs, a stable and direct delivery system avoiding the blood-brain-barrier has come into our interest. MATERIAL AND METHODS We implanted cranial windows and fluorescently labeled human glioblastoma stem-like cells into NMRI nude mice to follow up on the tumor development in our MPLSM model. After tumor establishment, an Alzet® micropump was implanted to directly deliver agents via a catheter system continuously over 28 days directly under the cranial window onto the brain surface. Using the MPLSM technique, the continuous delivery and infusion of drugs onto the brain and into the tumor was measured over many weeks in detail using MPLSM. RESULTS The establishment of the combined methods allowed reliable concurrent drug delivery over 28 days bypassing the blood-brain-barrier. Individual regions and tumor cells could be measured and followed up before, and after the beginning of the treatment, as well as after the end of the pump activity. Fluorescently labelled drugs were detectable in the MPLSM and its distribution into the brain parenchyma could be quantified. After the end of the micropump activity, further MPLSM measurements offer the possibility to observe long term effects of the applied drug on the tumor. CONCLUSION The combination of tumor observation in the MPSLM and concurrent continuous drug delivery is a feasible and reliable method for the investigation of (novel) anti-tumor agents, especially drugs that are not blood-brain-barrier penetrant. Morphological or even functional changes of individual tumor cells can be measured under and after treatment. These techniques can be used to test new drugs targeting the tumor, its tumor microtubes and tumor cells networks, and measure the effects longitudinally.

Insight into diffusive and convective processes affecting gold nanoparticles microclustering by multiphoton photoreduction

Multi-photon direct laser writing (MP-DLW) in polymeric matrices doped with a tetrachloroauric acid (HAuCl4) water solution allows creating clusters of gold nanoparticles (GNPs) inside the focus figure of a tightly focused ultrafast laser beam. The key physical phenomena involved in the process are analyzed, with the aim to assess the limits and potential of this promising technology. Multi Photon Absorption (MPA) triggers the photo-reduction of AuCl4– ions and the consequent creation of GNPs in the spotted volume. Thermal electronic decays lead to a local abrupt increase of the temperature, which influences the morphology of the created structures. At the same time, two different effects take place, related to the dehydration of the polymeric matrix, and the concentration gradient of the gold precursor upon localized photoreduction. Given their different timescales, these phenomena allow for controlling the GNPs density and size dispersity when a given energy dose is delivered in multiple exposures, tuning the delay between consecutive laser exposures. A simple yet effective experiment to estimate the temperature distribution at the micron-scale is also proposed.

Production of PAH cations with narrow internal energy distribution using single nanosecond pulsed laser

The internal energy dynamics of three examples of PAHs are probed using a high resolution energy time of flight spectrometer. The measured H-loss rates are converted to internal energy values for a range of UV photon wavelengths. Multi step multi photon absorption scheme is proposed to estimate the possible internal energy values with reasonable accuracies. This was possible because of the ultrafast radiationless internal conversion process in PAHs and their structural rigidity. Thus, a method is proposed to produce PAH cations with narrow and known internal energy distribution using a single nanosecond pulsed laser, paving a way to study statistical unimolecular decay in PAHs very efficiently.

Glutathione As Biomarker for Neurodegenerative Diseases

Brain imaging is one of the main obstacles of modern disease diagnostics since it must allow accurate and quantitative measurements on a living organ that is placed in adults (in neonatal humans’ skull penetration is somewhat easier) inside a bone cage. Inside is the brain, the main function controller of our body that must be done in a non- invasive way on the living operation brain. Many spectroscopic methods are trying to overcome the difficult access to the brain: Photoacoustic microscopy, Confocal microscopy, Two-photon microscopy, Optical coherence tomography, Scanning Laser Acoustic Microscopy, Acoustic microscopy, Ultrasonography, positron tomography, fluorescence methods, photo caustic microscopy and multi (two) photon imaging spectroscopy. The situation is complex since the brain is wrapped in the blood brain barrier allowing only selected molecules to pass from the blood stream to and out of the brain. In this chapter, we will survey the current situation of brain diagnostics with the aid of the spectroscopic methods. Brain research is integrated in aging research as a major area of interest. Aging is in most cased coupled with the loss of brain function and dementia. The neurodegenerative diseases, although identified by Dr. Alzheimer and his collaborators more than a century ago, continue as the leading causes of mortality among the elderly. Brain research in trying to give hope to those people but unfortunately our understanding in this area is limited. Oxidative stress contributes to neurodegenerative diseases pathophysiology and progression. The target was to describe central and peripheral metabolites of redox metabolism and to describe correlations between glutathione status, age, and disease severity.

Single-phase and correlated-phase estimation with multiphoton annihilated squeezed vacuum states: An energy-balancing scenario

In the last years, several works have demonstrated the advantage of photon subtracted Gaussian states for various quantum optics and information protocols. In most of these works, it was not clearly investigated the relation between the advantages and the usual increasing energy of the quantum state related to photon subtraction. In this paper, we study the performance of an interferometer injected with multi photon annihilated squeezed vacuum states mixed with coherent states for both single and correlated phase estimation. For single phase estimation, albeit the use of multi-photon annihilated squeezed vacuum states at low mean photons per mode provide advantage compared to classical strategy, when the total input energies is held fixed, the advantage due to photon subtraction is completely lost. However, for the correlated case in analogous scenario, some advantage appears to come from both the energy rise and improvement in photon statistics. In particular quantum enhanced sensitivity with photon subtracted states appears more robust to losses, showing an advantage of about 30% with respect to squeezed vacuum state in case of realistic value of the detection efficiency.

STOCHASTIC PERTURBATION OF OPTICAL SOLITONS WITH QUADRATIC–CUBIC NONLINEAR REFRACTIVE INDEX

This work obtains mean free velocity of bright optical solitons in optical fibers that are studied with quadratic–cubic form of nonlinear refractive index. The perturbation terms are from multi–photon absorption, filters and stochastic effect. The Langevin equation is derived from soliton perturbation which lead to the mean free velocity of the soliton.

High-resolution resonance-enhanced multiphoton photoelectron circular dichroism.

Photoelectron circular dichroism (PECD) is a highly sensitive enantiospecific spectroscopy for studying chiral molecules in the gas phase using either single-photon ionization or multiphoton ionization. In the short pulse limit investigated with femtosecond lasers, resonance-enhanced multiphoton ionization (REMPI) is rather instantaneous and typically occurs simultaneously via more than one vibrational or electronic intermediate state due to limited frequency resolution. In contrast, vibrational resolution in the REMPI spectrum can be achieved using nanosecond lasers. In this work, we follow the high-resolution approach using a tunable narrow-band nanosecond laser to measure REMPI-PECD through distinct vibrational levels in the intermediate 3s and 3p Rydberg states of fenchone. We observe the PECD to be essentially independent of the vibrational level. This behaviour of the chiral sensitivity may pave the way for enantiomer specific molecular identification in multi-component mixtures: one can specifically excite a sharp, vibrationally resolved transition of a distinct molecule to distinguish different chiral species in mixtures.

Comparing the performance of a femto fiber-based laser and a Ti:sapphire used for multiphoton microscopy applications.

Ti:sapphire laser systems are the more extended excitation sources in multiphoton (MP) microscopy. Although tunable, the cost, size, and lack of portability often limit their use in some research fields. Femtosecond fiber-based lasers represent an attractive alternative since they are portable, compact, and affordable. Most MP applications using these devices employ wavelengths beyond 1000 nm. This work evaluates the performance of a mode-locked fiber-based laser emitting at 780 nm (within the spectral region often used with Ti:sapphire devices) for use in MP imaging microscopy. MP images acquired with this laser system have been compared with those obtained with a "regular" solid-state source. Results herein show that the images recorded with both laser sources were similar, independently of the depth location of the imaged plane. The structural information contained in the images hardly differed. Moreover, the images of deeper layers improved by means of adaptive optics were also similar. These ultrafast laser sources are expected to enhance the impact of MP microscopy in basic research, as well as in biomedical environments.

Monitoring Hydrogen Peroxide (H2O2) in live kidneys by Multiphoton Microscopy and genetically encoded probes

Hydrogen Peroxide (H2O2) is an important physiological factor in the control of renal function. In the kidney, H2O2 is enhanced in some forms of hypertension and in diabetes, where it may play a role in renal damage. We previously found that H2O2 potently stimulates renin release from mouse juxtaglomerular (JG) cells. While all cells have the capacity to produce H2O2, the relative production levels in the different cell types of the kidney cortex (glomeruli vs proximal tubules vs JG cells), is not clear. Most H2O2 measurements have been performed ex‐vivo or in isolated renal cells, but few measurements of H2O2 have been performed in vivo. To begin addressing this, we used a genetically encoded H2O2 sensor named HyPer and multi‐photon (MP) microscopy imaging of the live mouse kidney. Before using HyPer in vivo, experiments were conducted in cultured cells expressing HyPer to determine the best wavelength for MP excitation and emission, by monitoring the increase in fluorescence after addition of H2O2. Then, HyPer was transduced into the renal cortex by subcapsular infusion of a plasmid, for 7 days. After transduction, mice were anesthetized with isoflurane, the left kidney exteriorized and a carotid cannula for infusion was placed. The renal cortical vasculature and glomeruli were visualized by using Rhodmaine‐R18 simultaneously with HyPer Imaging. We observed strong HyPer fluorescence primarily in proximal tubules, and in a few cases inside glomeruli. We then tested the ability of PEG‐Catalase plus the H2O2 scavenger Ebselen to decrease the HyPer signal in proximal tubules. After finding a region to image, baseline measurements were obtained for 5 minutes at 15 sec intervals, and then Catalase/Ebselen was infused into the carotid artery. Fluorescence was monitored for another 25 minutes. We observed a steady decrease in fluorescence over time that reached 35±5% after 20 minutes (p<0.05, n=3), suggesting a decrease in H2O2. Monitoring H2O2 in specific cell types can be performed by using cell‐selective promoters to drive expression of HyPer. To monitor H2O2 in JG cells we generated adenoviruses encoding HyPer under control of the Ren1 (renin 1) promoter. Renal subcapsular catheters were used to infuse adenoviruses for 7 days. MP imaging was performed to locate glomeruli expressing Hyper in the afferent arteriole or glomerular pole. After finding this, baseline measurements were performed for five minutes and the Catalase/Ebselen was infused. We observed a steady decrease in fluorescence over time that reached 33±7% after 20 minutes (p<0.05, n=3), suggesting a decrease in H2O2 in JG cells expressing HyPer. We conclude that HyPer can be used to monitor and compare H2O2 levels in different cell types in the kidney cortex by using cell‐type specific promoters and MP microscopy. This method should allow the comparison of H2O2 levels in renal cortical cell types between different strains or during pathological conditions in live animals.Support or Funding InformationNIH 5R03DK105300‐03 to MMThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.