Two-Photon Excitation Spectra of Various Fluorescent Proteins within a Broad Excitation Range.

Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720.

Improvement of the Similarity Spectral Unmixing Approach for Multiplexed Two-Photon Imaging by Linear Dimension Reduction of the Mixing Matrix.

Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into pathophysiologic processes in vivo. Simultaneous multiplexed imaging is required to understand the dynamic orchestration of the multiple cellular and non-cellular tissue compartments defining immune responses. Here, we present an improvement of our previously developed method, which allowed us to achieve multiplexed dynamic intravital two-photon imaging, by using a synergistic strategy. This strategy combines a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an unmixing algorithm based on the calculation of spectral similarities with previously measured fluorophore fingerprints. The improvement of the similarity spectral unmixing algorithm here described is based on dimensionality reduction of the mixing matrix. We demonstrate its superior performance in the correct pixel-based assignment of probes to tissue compartments labeled by single fluorophores with similar spectral fingerprints, as compared to the full-dimensional similarity spectral unmixing approach.

Coregistered Spectral Optical Coherence Tomography and Two-Photon Microscopy for Multimodal Near-Instantaneous Deep-Tissue Imaging.

Two-photon microscopy (2PM) has brought unique insight into the mechanisms underlying immune system dynamics and function since it enables monitoring of cellular motility and communication in complex systems within their genuine environment-the living organism. However, use of 2PM in clinical settings is limited. In contrast, optical coherence tomography (OCT), a noninvasive label-free diagnostic imaging method, which allows monitoring morphologic changes of large tissue regions in vivo, has found broad application in the clinic. Here we developed a combined multimodal technology to achieve near-instantaneous coregistered OCT, 2PM, and second harmonic generation (SHG) imaging over large volumes (up to 1,000 × 1,000 × 300 µm3 ) of tendons and other tissue compartments in mouse paws, as well as in mouse lymph nodes, spleens, and femurs. Using our multimodal imaging approach, we found differences in macrophage cell shape and motility behavior depending on whether they are located in tendons or in the surrounding tissue compartments of the mouse paw. The cellular shape of tissue-resident macrophages, indicative for their role in tissue, correlated with the supramolecular organization of collagen as revealed by SHG and OCT. Hence, the here-presented approach of coregistered OCT and 2PM has the potential to link specific cellular phenotypes and functions (as revealed by 2PM) to tissue morphology (as highlighted by OCT) and thus, to build a bridge between basic research knowledge and clinical observations. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Phasor-Based Endogenous NAD(P)H Fluorescence Lifetime Imaging Unravels Specific Enzymatic Activity of Neutrophil Granulocytes Preceding NETosis.

Time-correlated single-photon counting combined with multi-photon laser scanning microscopy has proven to be a versatile tool to perform fluorescence lifetime imaging in biological samples and, thus, shed light on cellular functions, both in vitro and in vivo. Here, by means of phasor-analyzed endogenous NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence lifetime imaging, we visualize the shift in the cellular metabolism of healthy human neutrophil granulocytes during phagocytosis of Staphylococcus aureus pHrodo™ beads. We correlate this with the process of NETosis, i.e., trapping of pathogens by DNA networks. Hence, we are able to directly show the dynamics of NADPH oxidase activation and its requirement in triggering NETosis in contrast to other pathways of cell death and to decipher the dedicated spatio-temporal sequence between NADPH oxidase activation, nuclear membrane disintegration and DNA network formation. The endogenous FLIM approach presented here uniquely meets the increasing need in the field of immunology to monitor cellular metabolism as a basic mechanism of cellular and tissue functions.

MarShie: a clearing protocol for 3D analysis of single cells throughout the bone marrow at subcellular resolution.

Analyzing immune cell interactions in the bone marrow is vital for understanding hematopoiesis and bone homeostasis. Three-dimensional analysis of the complete, intact bone marrow within the cortex of whole long bones remains a challenge, especially at subcellular resolution. We present a method that stabilizes the marrow and provides subcellular resolution of fluorescent signals throughout the murine femur, enabling identification and spatial characterization of hematopoietic and stromal cell subsets. By combining a pre-processing algorithm for stripe artifact removal with a machine-learning approach, we demonstrate reliable cell segmentation down to the deepest bone marrow regions. This reveals age-related changes in the marrow. It highlights the interaction between CX3CR1+ cells and the vascular system in homeostasis, in contrast to other myeloid cell types, and reveals their spatial characteristics after injury. The broad applicability of this method will contribute to a better understanding of bone marrow biology.

Experimental and computational study of the gas-phase reaction of O(1D) Atoms with VF5.

The reactions of O((1)D) atoms with VF(5) at room temperature have been studied by time-resolved laser magnetic resonance at the buffer gas (SF(6)) pressure of 6 Torr. The O((1)D) atoms were produced by the photodissociation of ozone using an excimer laser (KrF, 248 nm). By monitoring the kinetics of FO radical formation, the bimolecular rate constant of O((1)D) consumption in collisions with VF(5) has been determined to be k(VF(5)) = (7.5 ± 2.2) × 10(-11) cm(3) s(-1). The branching ratio for the channel producing FO radicals (k(8a)) has been found to be k(8a)/k(VF(5)) = 0.11 ± 0.02. Quantum chemical calculations at the CCSD(T)/CBS level of theory give evidence that the reactions of O((1)D) with VF(5) proceed via the VF(4)OF intermediate. The enthalpy of the reaction leading to this intermediate formation was calculated to be -245.8 kJ/mol. In qualitative agreement with the experimental results, the reaction channel O((1)D) + VF(5) → FO + VF(4) (8a) turned out to be 72.9 kJ/mol energetically more favorable than the channel O((1)D) + VF(5) → F + OVF(4) (8b). The dissociation enthalpy of the OVF(4) radical was calculated to be very low (18.1 kJ/mol); hence, the decay of OVF(4) to F + OVF(3) should proceed very fast. The molecular channel O((1)D) + VF(5) → F(2) + VF(3)O, though being most favorable thermodynamically, is kinetically unimportant.

Method for Multiplexed Dynamic Intravital Multiphoton Imaging.

Intravital two-photon microscopy enables monitoring of cellular dynamics and communication of complex systems, in genuine environment-the living organism. Particularly, its application in understanding the immune system brought unique insights into pathophysiologic processes in vivo. Here we present a method to achieve multiplexed dynamic intravital two-photon imaging by using a synergistic strategy combining a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an effective unmixing algorithm based on the calculation of spectral similarities with previously acquired fluorophore fingerprints. Our unmixing algorithm allows us to distinguish 7 fluorophore signals corresponding to various cellular and tissue compartments by using only four detector channels.

Intravital quantification reveals dynamic calcium concentration changes across B cell differentiation stages.

Calcium is a universal second messenger present in all eukaryotic cells. The mobilization and storage of Ca2+ ions drives a number of signaling-related processes, stress-responses, or metabolic changes, all of which are relevant for the development of immune cells and their adaption to pathogens. Here, we introduce the Förster resonance energy transfer (FRET)-reporter mouse YellowCaB expressing the genetically encoded calcium indicator TN-XXL in B lymphocytes. Calcium-induced conformation change of TN-XXL results in FRET-donor quenching measurable by two-photon fluorescence lifetime imaging. For the first time, using our novel numerical analysis, we extract absolute cytoplasmic calcium concentrations in activated B cells during affinity maturation in vivo. We show that calcium in activated B cells is highly dynamic and that activation introduces a persistent calcium heterogeneity to the lineage. A characterization of absolute calcium concentrations present at any time within the cytosol is therefore of great value for the understanding of long-lived beneficial immune responses and detrimental autoimmunity.

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells.

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.

Excited Cl((2)P(1/2)) atoms: yield from the photodissociation of SOCl(2) and collisional deactivation by NO(2), CCl(3)H, C(2)H(4), C(3)H(6), and SOCl(2).

Rate constants for the collisional deactivation of spin-orbitally excited Cl* ( identical withCl((2)P(1/2))) atoms by some selected gases at T = 298 K have been determined using time-resolved laser magnetic resonance (LMR) techniques. Cl* atoms were produced by photodissociation of SOCl(2) at 248 nm, and the relative quantum yield of Cl* atoms is determined to be 0.52 +/- 0.03. This yield is much larger than the yield at 235 nm (0.35 +/- 0.06). The rate constants for the relaxation of Cl* (x10(-11) cm(3)/s, +/- 2sigma) by NO(2)(1.5 +/- 0.4), C(2)H(4) (18 +/- 5), CCl(3)H (1.8 +/- 0.4), CH(3)-CH=CH(2) (16 +/- 4), and SOCl(2) (0.62 +/- 0.2) are reported for the first time. All of them are pressure-independent, and in all cases the dominant channel is physical quenching. It was established that the spin-orbital excitation of chlorine atoms decreases the probability of chemical reactions in collisions with propylene molecules. The rate constants of the reactions of ground state Cl((2)P(3/2)) atoms with C(2)H(4), CH(3)-CH=CH(2), and SOCl(2) at T = 298 K were found to be (5.0 +/- 1) x 10(-30) cm(6)/s (P(Ar) = 8-15 Torr), (5.2 +/- 1) x 10(-11) cm(3)/s (P(Ar) = 9-12 Torr), and (6 +/- 4) x 10(-14) cm(3)/s, respectively; the first one is termolecular, the last two are bimolecular, and the buffer gas is Ar.

B Cell Speed and B-FDC Contacts in Germinal Centers Determine Plasma Cell Output via Swiprosin-1/EFhd2.

Plasma cells secreting affinity-matured antibodies develop in germinal centers (GCs), where B cells migrate persistently and directionally over defined periods of time. How modes of GC B cell migration influence plasma cell development remained unclear. Through genetic deletion of the F-actin bundling protein Swiprosin-1/EF-hand domain family member 2 (EFhd2) and by two-photon microscopy, we show that EFhd2 restrains B cell speed in GCs and hapten-specific plasma cell output. Modeling the GC reaction reveals that increasing GC B cell speed promotes plasma cell generation. Lack of EFhd2 also reduces contacts of GC B cells with follicular dendritic cells in vivo. Computational modeling uncovers that both GC output and antibody affinity depend quantitatively on contacts of GC B cells with follicular dendritic cells when B cells migrate more persistently. Collectively, our data explain how GC B cells integrate speed and persistence of cell migration with B cell receptor affinity.

Infrared multiple photon dissociation of chloromethyltrifluorosilane.

Infrared multiphoton absorption and dissociation of chloromethyltrifluorosilane molecules under the action of pulsed transversely excited atmospheric pressure CO2 laser were experimentally studied. Dissociation products were analyzed. The dissociation proceeds via chlorine atom transfer from carbon to silicone. High degrees of silicon isotope separation were achieved. The presence of alpha-chlorine atom in a silicon organic compound brings about a significant improvement in multiple photon dissociation characteristics and an essential increase in isotopic selectivity.

Longitudinal intravital imaging of the femoral bone marrow reveals plasticity within marrow vasculature.

The bone marrow is a central organ of the immune system, which hosts complex interactions of bone and immune compartments critical for hematopoiesis, immunological memory, and bone regeneration. Although these processes take place over months, most existing imaging techniques allow us to follow snapshots of only a few hours, at subcellular resolution. Here, we develop a microendoscopic multi-photon imaging approach called LIMB (longitudinal intravital imaging of the bone marrow) to analyze cellular dynamics within the deep marrow. The approach consists of a biocompatible plate surgically fixated to the mouse femur containing a gradient refractive index lens. This microendoscope allows highly resolved imaging, repeatedly at the same regions within marrow tissue, over months. LIMB reveals extensive vascular plasticity during bone healing and steady-state homeostasis. To our knowledge, this vascular plasticity is unique among mammalian tissues, and we expect this insight will decisively change our understanding of essential phenomena occurring within the bone marrow.

Synergistic Strategy for Multicolor Two-photon Microscopy: Application to the Analysis of Germinal Center Reactions In Vivo.

Simultaneous detection of multiple cellular and molecular players in their native environment, one of the keys to a full understanding of immune processes, remains challenging for in vivo microscopy. Here, we present a synergistic strategy for spectrally multiplexed in vivo imaging composed of (i) triple two-photon excitation using spatiotemporal synchronization of two femtosecond lasers, (ii) a broad set of fluorophores with emission ranging from blue to near infrared, (iii) an effective spectral unmixing algorithm. Using our approach, we simultaneously excite and detect seven fluorophores expressed in distinct cellular and tissue compartments, plus second harmonics generation from collagen fibers in lymph nodes. This enables us to visualize the dynamic interplay of all the central cellular players during germinal center reactions. While current in vivo imaging typically enables recording the dynamics of 4 tissue components at a time, our strategy allows a more comprehensive analysis of cellular dynamics involving 8 single-labeled compartments. It enables to investigate the orchestration of multiple cellular subsets determining tissue function, thus, opening the way for a mechanistic understanding of complex pathophysiologic processes in vivo. In the future, the design of transgenic mice combining a larger spectrum of fluorescent proteins will reveal the full potential of our method.