Epifluorescence Imaging Research Articles

The inotropic and arrhythmogenic effects of acutely increased late INa are associated with elevated ROS but not oxidation of PKARIα.

Acute stimulation of the late sodium current (INaL) as pharmacologically induced by Anemonia toxin II (ATX-II) results in Na+-dependent Ca2+ overload and enhanced formation of reactive oxygen species (ROS). This is accompanied by an acute increase in the amplitude of the systolic Ca2+ transient. Ca2+ transient amplitude is determined by L-type Ca2+-mediated transsarcolemmal Ca2+ influx (ICa) into the cytosol and by systolic Ca2+ release from the sarcoplasmic reticulum (SR). Type-1 protein kinase A (PKARIα) becomes activated upon increased ROS and is capable of stimulating ICa, thereby sustaining the amplitude of the systolic Ca2+ transient upon oxidative stress. We aimed to investigate whether the increase of the systolic Ca2+ transient as acutely induced by INaL (by ATX-II) may involve stimulation of ICa through oxidized PKARIα. We used a transgenic mouse model in which PKARIα was made resistant to oxidative activation by homozygous knock-in replacement of redox-sensitive Cysteine 17 with Serine within the regulatory subunits of PKARIα (KI). ATX-II (at 1 nmol/L) was used to acutely enhance INaL in freshly isolated ventricular myocytes from KI and wild-type (WT) control mice. Epifluorescence and confocal imaging were used to assess intracellular Ca2+ handling and ROS formation. A ruptured-patch whole-cell voltage-clamp was used to measure INaL and ICa. The impact of acutely enhanced INaL on RIα dimer formation and PKA target structures was studied using Western blot analysis. ATX-II increased INaL to a similar extent in KI and WT cells, which was associated with significant cytosolic and mitochondrial ROS formation in both genotypes. Acutely activated Ca2+ handling in terms of increased Ca2+ transient amplitudes and elevated SR Ca2+ load was equally present in KI and WT cells. Likewise, cellular arrhythmias as approximated by non-triggered Ca2+ elevations during Ca2+ transient decay and by diastolic SR Ca2+-spark frequency occurred in a comparable manner in both genotypes. Most importantly and in contrast to our initial hypothesis, ATX-II did not alter the magnitude or inactivation kinetics of ICa in neither WT nor KI cells and did not result in PKARIα dimerization (i.e., oxidation) despite a clear prooxidant intracellular environment. The inotropic and arrhythmogenic effects of acutely increased INaL are associated with elevated ROS, but do not involve oxidation of PKARIα.

Visualizing alpha-synuclein and iron deposition in M83 mouse model of Parkinson's disease in vivo.

Abnormal alpha-synuclein (αSyn) and iron accumulation in the brain play an important role in Parkinson's disease (PD). Herein, we aim to visualize αSyn inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. The fluorescent pyrimidoindole derivative THK-565 probe was characterized by means of recombinant fibrils and brains from 10- to 11-month-old M83 mice. Concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging were subsequently performed in vivo. Structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 T as well as scanning transmission x-ray microscopy (STXM) were performed to characterize the iron deposits in the perfused brains. Immunofluorescence and Prussian blue staining were further performed on brain slices to validate the detection of αSyn inclusions and iron deposition. THK-565 showed increased fluorescence upon binding to recombinant αSyn fibrils and αSyn inclusions in post-mortem brain slices from patients with PD and M83 mice. Administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 min post-intravenous injection by wide-field fluorescence compared to nontransgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe3+ form, as evinced by the STXM results. In conclusion, we demonstrated in vivo mapping of αSyn by means of noninvasive epifluorescence and vMSOT imaging and validated the results by targeting the THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo.

MDB-108. MRI-GUIDED FOCUSED ULTRASOUND BLOOD BRAIN/TUMOR BARRIER DISRUPTION FOR AUGMENTATION OF ANTIBODY DELIVERY TO HIGH-RISK MEDULLOBLASTOMAS

Abstract BACKGROUND Medulloblastoma is the most common pediatric brain cancer, and among its four molecular subgroups, group 3 (G3MB) harbors the worst prognosis (5-year survival rate <50%). Effective treatment of G3MB remains difficult due to disease complexity, its challenging niche within the immunologically- and pharmacologically-privileged central nervous system, and significant risk of long-term developmental deficits due to treatment toxicities. MRI-guided focused ultrasound (MRgFUS) holds the potential to revolutionize care as a non-invasive, non-ionizing approach for spatially precise intervention in G3MBs. Given that the utility of FUS in MBs remains unknown, we implemented FUS-mediated blood brain/tumor barrier disruption (BBB/BTB-D) in a high-fidelity model of G3MB and evaluated its impact on localized antibody delivery – with an eye toward future applications in immuno-oncology. METHODS An orthotopic syngeneic model was established via stereotactic implantation of G3MB cells (Gfi1/c-MYC driven) into the cerebellum. G3MB growth kinetics were validated via serial MRI. Multiple protocols for naive cerebellum or G3MB BBB/BTB-D were established with a 1.15 MHz FUS system under 9.4T MRI guidance. Across both settings, mice were sonicated with concomitant intravenous administration of microbubbles and fluorescently-labeled IgG. Safety was confirmed by acoustic emissions monitoring, susceptibility-weighted imaging, histology, and acute activity monitoring. Antibody delivery was evaluated via ex vivo epifluorescence imaging. RESULTS Post-contrast T1w-MRI depicted clear evidence of localized BBB/BTB-D in naive and G3MB mice. No damage was noted by imaging or histopathologic analysis, and mice displayed normal cardiorespiratory and motor functions following FUS. FUS conferred significantly increased epifluorescence signal in targeted cerebellum (1.3x) and G3MBs (1.5x) relative to controls, indicating improved antibody access. CONCLUSIONS Our findings support the safety and feasibility of FUS BBB/BTB-D in naive and G3MB-bearing cerebellum. We demonstrate that FUS markedly improves antibody penetrance within G3MBs. Ongoing studies are evaluating FUS in combination with novel immunotherapeutic antibodies for G3MB therapy.

Mechanisms of synapse-to-nucleus calcium signalling in striatal neurons and impairments in Huntington's disease.

Huntington's disease (HD) is a monogenic disorder with autosomal dominant inheritance. In HD patients, neurons in the striatum and cortex degenerate, leading to motor, psychiatric and cognitive disorders. Dysregulated synaptic function and calcium handling are common in many neurodegenerative diseases, including HD. N-methyl-D-aspartate (NMDA) receptor function is enhanced in HD at extrasynaptic sites, altering the balance of calcium-dependent neuronal survival versus death signalling pathways. Endoplasmic reticulum (ER) calcium handling is also abnormal in HD. The ER, which is continuous with the nuclear envelope, is purportedly involved in nuclear calcium signalling; based on this, we hypothesised that nuclear calcium signalling is altered in HD. We explored this hypothesis with calcium imaging techniques, including simultaneous epifluorescent imaging of cytosolic and nuclear calcium using jRCaMP1b and GCaMP3 sensors, respectively, in striatal spiny projection neurons in cortical-striatal co-cultures from the YAC128 mouse model of HD. Our data show contributions from a variety of calcium channels to nuclear calcium signalling. NMDA receptors (NMDARs) play an essential role in initiating action potential-dependent calcium signalling to the nucleus, and ryanodine receptors (RyR) contribute to both cytosolic and nuclear calcium signals. Unlike previous reports in glutamatergic hippocampal and cortical neurons, we found that in GABAergic striatal neurons, L-type voltage-gated calcium channels (CaV) contribute to cytosolic, but not nuclear calcium signalling. Calcium imaging also suggests impairments in nuclear calcium signalling in HD striatal neurons, where spontaneous action potential-dependent calcium transients in the nucleus were smaller in YAC128 striatal neurons compared to those of wild-type (WT). Our results elucidate mechanisms involved in action potential-dependent nuclear calcium signalling in GABAergic striatal neurons, and have revealed a clear deficit in this signalling in HD.

Focal Recording of Synaptic Currents from Single Boutons at the Drosophila Larval Neuromuscular Junction.

Focal recording is an extracellular method for studying synaptic transmission at the Drosophila larval neuromuscular junction (NMJ) designed for the study of synaptic activity of one or a few synaptic boutons rather than the ensemble activity of all the boutons as occurs with intracellular recording or two-electrode voltage-clamp. This is a useful technique for investigating the properties of different motor neurons that innervate the same muscle, applying statistical analysis to discrete synaptic events, and investigating the heterogeneity of synaptic release properties among boutons. A compound microscope with epifluorescent imaging capability is very helpful but not essential; any GFP Drosophila strain that labels the nerve terminal or synaptic boutons can be used to locate the boutons. A particularly useful strain is Mhc-CD8-Sh-GFP, containing a GFP molecule that is expressed in muscle, localizes to the postsynaptic apparatus, and outlines boutons. Vital fluorescent dyes (such as 4-Di-2-Asp) may also be applied to the dissected preparation to help locate boutons. The microscope should be equipped for differential interference contrast (DIC or Nomarski) optics if fluorescence is not used.

Low-cost 3D printed lenses for brightfield and fluorescence microscopy.

We present the fabrication and implementation of low-cost optical quality 3D printed lenses, and their application as microscope objectives with different prescriptions. The imaging performance of the 3D printed lenses was benchmarked against commercially available optics including a 20 mm focal length 12.7 mm diameter NBK-7 plano-convex lens used as a low magnification objective, and a separate high magnification objective featuring three 6 mm diameter NBK-7 lenses with different positive and negative focal lengths. We describe the design and manufacturing processes to produce high-quality 3D printed lenses. We tested their surface quality using a stylus profilometer, showing that they conform to that of commercial glass counterpart lenses. The 3D printed lenses were used as microscope objectives in both brightfield and epi-fluorescence imaging of specimens including onion, cyanobacteria, and variegated Hosta leaves, demonstrating a sub-cellular resolution performance obtained with low-cost 3D printed optical elements within brightfield and fluorescence microscopy.

6]-Shogaol Induces Apoptosis of Murine Bladder Cancer Cells.

Bladder cancer is considered one of the most aggressive neoplasms due to its recurrence and progression profile, and even with the improvement in diagnosis and treatment methods, the mortality rate has not shown a declining trend in recent decades. From this perspective, the search and development of more effective and safer therapeutic alternatives are necessary. Phytochemicals are excellent sources of active principles with therapeutic potential. [6]-Shogaol is a phenolic compound extracted from the ginger rhizomes that has shown antitumor effects in a wide variety of cancer models. However, there is no record in the literature of studies reporting these effects in models of bladder cancer. Thus, this study aimed to investigate the in vitro cytotoxic and pro-apoptotic potential of [6]-Shogaol against murine bladder cancer urothelial cells (MB49). The cytotoxic effects of [6]-Shogaol on cell viability (MTT method), cell morphology (light microscopy), alteration of proliferative processes (clonogenic assay), oxidative stress pathway (levels of reactive oxygen species) and the induction of apoptotic events (flow cytometry and high-resolution epifluorescence imaging) were evaluated in murine urothelial bladder cancer cell lines (MB49), relative to non-tumor murine fibroblasts (L929). The results showed that [6]-Shogaol was able to induce concentration-dependent cytotoxic effects, which compromised cell viability, exhibiting an inhibitory concentration of 50% of cells (IC50) of 146.8 µM for MB49 tumor cells and 236.0 µM for L929 non-tumor fibroblasts. In addition to inhibiting and altering the proliferative processes if colony formation, it presented pro-apoptotic activity identified through a quantitative analysis and the observation of apoptotic phenotypes, events apparently mediated by the induction of nuclear fragmentation. The data presented suggest that [6]-Shogaol has a higher concentration-dependent cytotoxic and apoptosis-inducing potential in MB49 cells than in L929 fibroblasts. These results may contribute to the development of therapeutic alternatives for bladder cancer.

A novel modular opto-biomechatronics bioreactor for simultaneous isotropic mechanical stretch application and fluorescence microscopy under cell and tissue culture conditions

Mechanical stresses are an environmental challenge virtually all tissues in the body are exposed to and thus, are of fundamental interest to study cell reactions in mechanobiology. Yet, unlike acute short-term mechanical cell stimulations, long-term or cyclic mechano-stimulation as experienced in the body is difficult to reproduce. Bioreactors are designed to control cell culture conditions, but still, there are yet no technical solutions available to merge bioreactor and opto-biomechatronics technologies for cyclic stretch-applications and simultaneous live cell imaging. To close this gap, we have engineered an opto-biomechatronics module, consisting of our in-house developed IsoStretcher technology and customised epifluorescence optics, into an automated bioreactor platform. For this, redesigned polydimethylsiloxane (PDMS) chambers with closed geometry (∽700μL internal volume) to warrant sterile operation were developed. Those chambers could be flushed with cell solution for cell seeding in a sterile manner. The epifluorescence imaging module was engineered into the reactor underneath the IsoStretcher to allow for continuous image acquisition during long-term stretch cycles (hours to days). The system was validated on human fibroblast BJ foreskin cells, and Cal-520 Ca2＋ fluorescence was stably imaged using our in-built autofocus functionality. Cultures for 24h within the IsoStretcher-bioreactor preserved a normal cell morphology as compared to external incubator control cultures. Isotropic stretch was reliably transferred to the cell membranes. Our system with in-built bioreactor and opto-biomechatronics functionality provides a holistic technology platform for the growing field of mechanobiology to allow long-term observations of cultured single cells and confluent cell layers that are subjected to cyclic long-term isotropic stretch protocols.

Smart lattice light-sheet microscopy for imaging rare and complex cellular events.

Light-sheet microscopes enable rapid high-resolution imaging of biological specimens; however, biological processes span spatiotemporal scales. Moreover, long-term phenotypes are often instigated by rare or fleeting biological events that are difficult to capture with a single imaging modality. Here, to overcome this limitation, we present smartLLSM, a microscope that incorporates artificial intelligence-based instrument control to autonomously switch between epifluorescent inverted imaging and lattice light-sheet microscopy (LLSM). We apply this approach to two unique processes: cell division and immune synapse formation. In each context, smartLLSM provides population-level statistics across thousands of cells and autonomously captures multicolor three-dimensional datasets or four-dimensional time-lapse movies of rare events at rates that dramatically exceed human capabilities. From this, we quantify the effects of Taxol dose on spindle structure and kinetochore dynamics in dividing cells and of antigen strength on cytotoxic T lymphocyte engagement and lytic granule polarization at the immune synapse. Overall, smartLLSM efficiently detects rare events within heterogeneous cell populations and records these processes with high spatiotemporal four-dimensional imaging over statistically significant replicates.

Optimizing Lectin Staining Methodology to Assess Glycocalyx Composition of Legionella-Infected Cells

Introduction: Legionella is a gram-negative bacterium that replicates intracellularly within macrophages. Legionella utilizes effector proteins to hijack ER-Golgi vesicle trafficking to sustain proliferation in its intracellular niche. Legionella has a considerable influence on O-glycosylation but not N-glycosylation events in the Golgi of infected cells. This research aims to optimize the use of fluorescent lectins, which are proteins that bind carbohydrates, to effectively label host-cell glycocalyx during Legionella infection. Methods: Epifluorescence imaging or flow cytometry were used to optimize the lectin staining methodology. We noted that the most effective conditions for lectin-labeling were when live HeLa cells were incubated with lectins diluted in Hank’s balanced salt solution (HBSS) with 3% Bovine serum albumin (BSA) for 10-30 minutes at 4 °C. Results: Incubating suspended cells with lectins necessitated smaller lectin concentrations, whereas lectin labeling of adherent cells required considerably larger concentrations. Wheat germ agglutinin (WGA) lectin mean fluorescence intensity (MFI) was concentration-dependent, but Concanavalin A (ConA) and Maclura pomifera (MPA) MFIs did not alter substantially with increasing lectin concentrations. Discussion: The optimal lectin concentration required was lectin-specific and based on whether the lectin fluorescence was assessed using flow cytometry or epifluorescence. Furthermore, the use of phosphate-buffered saline (PBS) for lectin dilution, cell permeabilization for intracellular labelling, and incubation of lectins in fixed cells reduced productive labelling of lectins on cell surfaces because it inhibited the lectin's ability to effectively bind the associated carbohydrate structure. Conclusion: Further research using diverse lectins on U937 macrophages is necessary to reach a definitive conclusion on the effect of Legionella on the overall host-cell glycocalyx composition during infection of these relevant immune cells.

A turn-on fluorescence sensor based on water-soluble amino-pyridyl-benzothiazole-derivatives for measuring cellular Zn2+

Novel amino-pyridyl-benzothiazole-derivatives (PBT) were synthesized through cycloaddition/nucleophilic aromatic substitution reactions for the development of a turn-on fluorescence chemosensor that accurately determines Zn2+ in polar media. The selectivity, water-solubility, and overall analytical response were systematically optimized through incorporation of different aliphatic amines as substituents: pyrrolidine (PBT-Pyn), ethylenediamine (PBT-Et) or 1,3-propylenediamine (PBT-Pr). Those ligands presented intense dual radiative emissions in the blue/green region upon activation with a 395 nm LED, which were attributed to the local excitation and internal energy transfer states. Although no variations were observed upon the interaction of Zn2+ with PBT-Pyn, both turn-off and turn-on responses were observed upon its interaction with PBT-Et. These responses depended on the ligand concentration and the associated inner filter effects. Thereby, PBT-Pr was selected for a deeper evaluation towards photoluminescence determination of Zn2+ due to its exclusive turn-on response and major sensibility obtained at pH 7.0, making it very convenient for analysis in physiological media. Under those conditions, the detection limits were 1.9 µM and 4.7 µM for measurements in aqueous or ethanolic solutions, respectively, with linear ranges up to 40 and 80 μM. The selectivity and good biocompatibility of the chemosensor were demonstrated through cell-viability assays with MCF-7, 3T3-L1 and HT-29 cell lines. The probe was then applied for the real-time epifluorescence imaging of Zn2+, observing emission intensities and distribution profiles that correlated with the concentration and location of the target within living cells.

Opposing effects of β-2 and β-1 adrenergic receptor signaling on neuroinflammation and dopaminergic neuron survival in α-synuclein-mediated neurotoxicity

BackgroundNoradrenergic neurons in the locus coeruleus (LC) are the primary source of norepinephrine (NE) in the brain and degeneration of these neurons is reported in the early stages of Parkinson’s disease (PD), even prior to dopaminergic neuron degeneration in the substantia nigra (SN), which is a hallmark of PD pathology. NE depletion is generally associated with increased PD pathology in neurotoxin-based PD models. The effect of NE depletion in other models of PD-like α-synuclein-based models is largely unexplored. In PD models and in human patients, β-adrenergic receptors’ (AR) signaling is associated with a reduction of neuroinflammation and PD pathology. However, the effect of NE depletion in the brain and the extent of NE and β-ARs signaling involvement in neuroinflammation, and dopaminergic neuron survival is poorly understood.MethodsTwo mouse models of PD, a 6OHDA neurotoxin-based model and a human α-synuclein (hα-SYN) virus-based model of PD, were used. DSP-4 was used to deplete NE levels in the brain and its effect was confirmed by HPLC with electrochemical detection. A pharmacological approach was used to mechanistically understand the impact of DSP-4 in the hα-SYN model of PD using a norepinephrine transporter (NET) and a β-AR blocker. Epifluorescence and confocal imaging were used to study changes in microglia activation and T-cell infiltration after β1-AR and β2-AR agonist treatment in the hα-SYN virus-based model of PD.ResultsConsistent with previous studies, we found that DSP-4 pretreatment increased dopaminergic neuron loss after 6OHDA injection. In contrast, DSP-4 pretreatment protected dopaminergic neurons after hα-SYN overexpression. DSP-4-mediated protection of dopaminergic neurons after hα-SYN overexpression was dependent on β-AR signaling since using a β-AR blocker prevented DSP-4-mediated dopaminergic neuron protection in this model of PD. Finally, we found that the β-2AR agonist, clenbuterol, reduced microglia activation, T-cell infiltration, and dopaminergic neuron degeneration, whereas xamoterol a β-1AR agonist showed increased neuroinflammation, blood brain barrier permeability (BBB), and dopaminergic neuron degeneration in the context of hα-SYN-mediated neurotoxicity.ConclusionsOur data demonstrate that the effects of DSP-4 on dopaminergic neuron degeneration are model specific, and suggest that in the context of α-SYN-driven neuropathology, β2-AR specific agonists may have therapeutic benefit in PD.

Supercritical angle fluorescence for enhanced axial resolution in classical and super-resolution microscopy.

Supercritical angle fluorescence (SAF) is an intrinsic component of the emission of any fluorescent molecule. SAF emission corresponds to the near field components of any fluorophore placed in the vicinity of the glass/cell interface which becomes propagative and emitted at supercritical angles. It thus intrinsically presents in the sample, can represent up to 50% of the emission of the fluorophore. This SAF emission sharply decays with the fluorophore/interface distance z over a characteristic length of about 150 nm. We showed that this emission can be used as a sectioning strategy in classical microscopy for live cells imaging, or allow one to localize single molecule event with an absolute 15 nm axial precision in super-resolution microscopy. To decipher SAF emission from undercritcal one, so far physical masks where inserted in the back focal plane of the objective, thus discarding part of the emission which impacts SNR in classical microscopy and localization precision in single molecule localization microscopy (SMLM). This limitation can be overcome by implementing a PSF engineering strategy, thanks to a phase mask which induces a π-shift between SAF and under-critical emissions and allows to benefit of all photons. In classical microscopy, the simultaneous acquisition of an epifluorescence image and a π-shifted SAF, permits real time dual depth imaging of epifluorescence and ϕSAF image, allowing a complete following of receptors trafficking. In combination with SMLM, this permits to detect all emitted photons and thus improve the accessible localization precision. This ϕSAF/SMLM will be presented on various cytoskeleton protein imaging in fixed cells, but also live cells thanks to self blinking dyes.

Flexible and open-source programs for quantitative image analysis in microbial ecology

Epifluorescence microscopy is an essential tool for obtaining reliable estimates of the abundance of marine microorganisms including viruses. However, computational analysis is required to gain consistent and quantitative data from digital microscopy images. Many imaging programs are proprietary and cost-prohibitive. The currently available free imaging programs are often platform specific and/or lack the flexibility to analyze microscopy images from natural samples, such as the planktonic environment, which can contain challenges such as debris and high background signals. Here we describe two MATLAB-based open-source image analysis programs that work across computer platforms and provide the tools to analyze a range of image types and cell sizes with a user-friendly interface. The Microbial Image Analysis (MiA) program aims to provide flexibility for the selection, identification, and quantification of cells that vary in size and fluorescence intensity within natural microbial communities. The Viral Image Analysis (ViA) program aims to provide an effective means for quantifying viral abundances from epifluorescence images as well as enumerating the intensity of a primary and secondary stain. In this paper, we provide an overview of the functionality of the MiA and ViA programs and highlight specific program features through several microbial image case studies.

Fluorescently Guided Optical Photothermal Infrared Microspectroscopy for Protein-Specific Bioimaging at Subcellular Level.

Infrared spectroscopic imaging is widely used for the visualization of biomolecule structures, and techniques such as optical photothermal infrared (OPTIR) microspectroscopy can achieve <500 nm spatial resolution. However, these approaches lack specificity for particular cell types and cell components and thus cannot be used as a stand-alone technique to assess their properties. Here, we have developed a novel tool, fluorescently guided optical photothermal infrared microspectroscopy, that simultaneously exploits epifluorescence imaging and OPTIR to perform fluorescently guided IR spectroscopic analysis. This novel approach exceeds the diffraction limit of infrared microscopy and allows structural analysis of specific proteins directly in tissue and single cells. Experiments described herein used epifluorescence to rapidly locate amyloid proteins in tissues or neuronal cultures, thus guiding OPTIR measurements to assess amyloid structures at the subcellular level. We believe that this new approach will be a valuable addition to infrared spectroscopy providing cellular specificity of measurements in complex systems for studies of structurally altered protein aggregates.

Assessment of Tissue Viability by Functional Imaging of Membrane Potential.

Electrical activity plays a key role in physiology, in particular for signaling and coordination. Cellular electrophysiology is often studied with micropipette-based techniques such as patch clamp and sharp electrodes, but for measurements at the tissue or organ scale, more integrated approaches are needed. Epifluorescence imaging of voltage-sensitive dyes ("optical mapping") is a tissue non-destructive approach to obtain insight into electrophysiology with high spatiotemporal resolution. Optical mapping has primarily been applied to excitable organs, especially the heart and brain. Action potential durations, conduction patterns, and conduction velocities can be determined from the recordings, providing information about electrophysiological mechanisms, including factors such as effects of pharmacological interventions, ion channel mutations, or tissue remodeling. Here, we describe the process for optical mapping of Langendorff-perfused mouse hearts, highlighting potential issues and key considerations.

PROTOCOL DEVELOPMENT FOR SINGLE-NUCLEUS RNA SEQUENCING OF HUMAN HEART TISSUE

PROTOCOL DEVELOPMENT FOR SINGLE-NUCLEUS RNA SEQUENCING OF HUMAN HEART TISSUE

Light-field micro-endoscopy using a fiber bundle: a snapshot 3D epi-fluorescence endoscope

Micro-endoscopes are widely used for detecting and visualizing hard-to-reach areas of the human body and for in vivo observation of animals. A micro-endoscope that can realize 3D imaging at the camera framerate could benefit various clinical and biological applications. In this work, we report the development of a compact light-field micro-endoscope (LFME) that can obtain snapshot 3D fluorescence imaging, by jointly using a single-mode fiber bundle and a small-size light-field configuration. To demonstrate the real imaging performance of our method, we put a resolution chart in different z positions and capture the z-stack images successively for reconstruction, achieving 333-μm-diameter field of view, 24 μm optimal depth of field, and up to 3.91 μm spatial resolution near the focal plane. We also test our method on a human skin tissue section and HeLa cells. Our LFME prototype provides epi-fluorescence imaging ability with a relatively small (2-mm-diameter) imaging probe, making it suitable for in vivo detection of brain activity and gastrointestinal diseases of animals.

Far-Red Fluorescent Murine Glioma Model for Accurate Assessment of Brain Tumor Progression.

Simple SummaryThe creation of stable fluorescent glioma cell lines opens prospects for the detailed characterization of the molecular mechanisms of glioma origin and pathogenesis, and the development of breakthrough therapeutic approaches to achieve maximum glioma destruction and minimize the risk of future metastases. Herein, we generated and characterized a novel fluorescent glioma GL261-kat cell line stably expressing a far-red fluorescent protein, TurboFP635 (Katushka). Using the orthotopic mouse glioma model and epi-fluorescence imaging, we demonstrate the detection of fluorescent glioma GL261-kat cells in mice, and observe an increase in the fluorescence signal during glioma progression, which is accompanied by a gradual development of neurological deficit and behavioral alterations in mice. We show that GL261-kat cells can be a useful tool for studying glioma biology, because they can accurately and non-invasively monitor the characteristics of glioma growth in brain tissue in orthotopic mouse models.Glioma is the most common brain tumor, for which no significant improvement in life expectancy and quality of life is yet possible. The creation of stable fluorescent glioma cell lines is a promising tool for in-depth studies of the molecular mechanisms of glioma initialization and pathogenesis, as well as for the development of new anti-cancer strategies. Herein, a new fluorescent glioma GL261-kat cell line stably expressing a far-red fluorescent protein (TurboFP635; Katushka) was generated and characterized, and then validated in a mouse orthotopic glioma model. By using epi-fluorescence imaging, we detect the fluorescent glioma GL261-kat cells in mice starting from day 14 after the inoculation of glioma cells, and the fluorescence signal intensity increases as the glioma progresses. Tumor growth is confirmed by magnetic resonance imaging and histology. A gradual development of neurological deficit and behavioral alterations in mice is observed during glioma progression. In conclusion, our results demonstrate the significance and feasibility of using the novel glioma GL261-kat cell line as a model of glioma biology, which can be used to study the initialization of glioma and monitor its growth by lifetime non-invasive tracking of glioma cells, with the prospect of monitoring the response to anti-cancer therapy.

Computational single-objective scanning light sheet (cSOLS)

Single-objective scanning light sheet (SOLS) imaging has fueled major advances in volumetric bioimaging because it supports low phototoxic, high-resolution imaging over an extended period. The remote imaging unit in the SOLS does not use a conventional epifluorescence image detection scheme (a single tube lens). In this paper, we propose a technique called the computational SOLS (cSOLS) that achieves light sheet imaging without the remote imaging unit. Using a single microlens array after the tube lens (lightfield imaging), the cSOLS is immediately compatible with conventional epifluorescence detection. The core of cSOLS is a Fast Optical Ray (FOR) model. FOR generates 3D imaging volume (40 × 40 × 14 µm3) using 2D lightfield images taken under SOLS illumination within 0.5 s on a standard central processing unit (CPU) without multicore parallel processing. In comparison with traditional lightfield retrieval approaches, FOR reassigns fluorescence photons and removes out-of-focus light to improve optical sectioning by a factor of 2, thereby achieving a spatial resolution of 1.59 × 1.92 × 1.39 µm3. cSOLS with FOR can be tuned over a range of oblique illumination angles and directions and, therefore, paves the way for next-generation SOLS imaging. cSOLS marks an important and exciting development of SOLS imaging with computational imaging capabilities.