Real-time in vivo confocal laser endomicroscopic imaging of equine endometrium: preliminary observations and feasibility study.

Endometrial health is vital for the reproductive efficiency of broodmares and accurate diagnostic testing is crucial for directing the best treatment options and outcomes. Confocal laser endomicroscopy (CLE) is an endoscopic technique for obtaining in-vivo, real-time microscopic imaging of tissues using a fiber optic probe. CLE relies on induced tissue fluorescence and fluorescein sodium, given intravenously, is the contrast agent most used in human medicine. This study aimed to determine the feasibility of CLE for imaging equine endometrium and determine a standard dose of fluorescein sodium to achieve optimal cellular imaging. In-vivo CLE was performed on 44 mares, and the images were compared with routine histopathological analysis of endometrial biopsies. No adverse reactions occurred after IV fluorescein sodium administration and a dose of 4mg/kg was established (0.04mL/kg of 10% fluorescein sodium solution) to achieve optimal image contrast. CLE enabled multiple regions of the endometrium to be assessed quickly. Distinct tissue architecture patterns could be appreciated using CLE, and the luminal epithelium could be assessed for integrity (ulceration) and exocytosed inflammatory cells. Endometrial gland distribution, density, shape, and epithelial height were evaluated. Blood vessels were clearly outlined, and inflammatory cells and fibrosis were discernable within the interstitium. Image quality varied between mares, and the stage of oestrous cycle may have been a factor of influence. This novel imaging modality enables collection of "virtual" biopsies and facilitates critical assessment of multiple regions of the uterus compared with the standard histopathologic assessment of a single random tissue biopsy.

Cortical Acetylcholine Response to Deep Brain Stimulation of the Basal Forebrain.

Background: Deep brain stimulation (DBS), the direct electrical stimulation of neuronal tissue in the basal forebrain to enhance release of the neurotransmitter acetylcholine, is under consideration as a method to improve executive function in patients with dementia. While some small studies indicate a positive response in the clinical setting, the relationship between DBS and acetylcholine pharmacokinetics is incompletely understood. Objective: We examined the cortical acetylcholine response to different stimulation parameters of the basal forebrain. Methods: 2-photon imaging was combined with deep brain stimulation. Stimulating electrodes were implanted in the subpallidal basal forebrain, and the ipsilateral somatosensory cortex was imaged. Acetylcholine activity was determined using the GRABACh-3.0 muscarinic acetylcholine receptor sensor, and blood vessels were imaged with Texas red. Results: Experiments manipulating pulse train frequency demonstrated that integrated acetylcholine induced fluorescence was insensitive to frequency, and that peak levels were achieved with frequencies from 60 to 130 Hz. Altering pulse train length indicated that longer stimulation resulted in higher peaks and more activation with sublinear summation. The acetylcholinesterase inhibitor donepezil increased the peak response to 10s of stimulation at 60Hz, and the integrated response increased 57% with the 2 mg/kg dose, and 126% with the 4 mg/kg dose. Acetylcholine levels returned to baseline with a time constant of 14 to 18 seconds in all experiments. Conclusions: These data demonstrate that acetylcholine receptor activation is insensitive to frequency between 60 and 130 Hz. High peak responses are achieved with up to 900 pulses. Donepezil increases total acetylcholine receptor activation associated with DBS but did not change temporal kinetics. The long time constants observed in the cerebral cortex add to the evidence supporting volume in addition to synaptic transmission.

Light-Driven Microrobots for Targeted Drug Delivery.

As a new micromanipulation tool with the advantages of small size, flexible movement and easy manipulation, light-driven microrobots have a wide range of prospects in biomedical fields such as drug targeting and cell manipulation. Recently, microrobots have been controlled in various ways, and light field has become a research hotspot by its advantages of noncontact manipulation, precise localization, fast response, and biocompatibility. It utilizes the force or deformation generated by the light field to precisely control the microrobot, and combines with the drug release technology to realize the targeted drug application. Therefore, this paper provides an overview of light-driven microrobots with drug targeting to provide new ideas for the manipulation of microrobots. Here, this paper briefly categorizes the driving mechanisms and materials of light-driven microrobots, which mainly include photothermal, photochemical, and biological. Then, typical designs of light-driven microrobots with different driving mechanisms and control strategies for multiple physical fields are summarized. Finally, the applications of microrobots in the fields of drug targeting and bioimaging are presented as well as the future prospects of light-driven microrobots in the biomedical field are demonstrated.

In vivo detection of endogenous toxic phenolic compounds of intestine.

Phenol and p-cresol are two common toxic small molecules related to various diseases. Existing reports confirmed that high L-tyrosine in the daily diet can increase the concentration of phenolic compounds in blood and urine. L-tyrosine is a common component of protein-rich foods. Some anaerobic bacteria in the gut can convert non-toxic l-tyrosine into these two toxic phenolic compounds, phenol and p-cresol. Existing methods have been constructed for measuring the concentration of phenolic compound in feces. However, there is still a lack of direct visual evidence to measure the phenolic compounds in the intestine. In this study, we aimed to construct a whole-cell biosensor for phenolic compounds detection based on the dmpR, the regulator from the phenol metabolism cluster. The commensal bacterium Citrobacter amalonaticus PS01 was selected and used as the chassis. Compared with the biosensor based on ECN1917, the biosensor PS01[dmpR] could better implant into the mouse gut through gavage and showed a higher sensitive to phenolic compound. And the concentration of phenolic compounds in the intestines could be observed with the help of in vivo imaging system using PS01[dmpR]. This paper demonstrated endogenous phenol synthesis in the gut and the strategy of using commensal bacteria to construct whole-cell biosensors for detecting small molecule compounds in the intestines.

Monitoring RVFV Infection Using Bioluminescent Reporter Viruses In Vivo.

Rift Valley fever virus is able to infect multiple organs and cell types, and the course of infection varies between viral strains and between individuals in particular according to age, genetic background, and physiological status. Studies on viral and host factors involve detecting and quantifying viral load at multiple time points and in multiple tissues. While this is classically performed by genome quantification or viral titration, in vivo imaging techniques using recombinant viruses expressing a bioluminescent or fluorescent protein allow noninvasive longitudinal studies on the same group of mice over the entire course of disease and the detection of unsuspected sites of infection. Here, we describe the protocol to monitor and characterize mouse infection with Rift Valley fever virus by in vivo imaging using recombinant viruses expressing light-emitting reporter genes.

Inflammatory response toward a Mg-based metallic biomaterial implanted in a rat femur fracture model.

The immune system plays an important role in fracture healing, by modulating the pro-inflammatory and anti-inflammatory responses occurring instantly upon injury. An imbalance in these responses can lead to adverse outcomes, such as non-union of fractures. Implants are used to support and stabilize complex fractures. Biodegradable metallic implants offer the potential to avoid a second surgery for implant removal, unlike non-degradable implants. However, considering our dynamic immune system it is important to conduct in-depth studies on the immune response to these implants in living systems. In this study, we investigated the immune response to Mg and Mg-10Gd in vivo in a rat femur fracture model with external fixation. In vivo imaging using liposomal formulations was used to monitor the fluorescence-related inflammation over time. We combine ex vivo methods with our in vivo study to evaluate and understand the systemic and local effects of the implants on the immune response. We observed no significant local or systemic effects in the Mg-10Gd implanted group compared to the SHAM and Mg implanted groups over time. Our findings suggest that Mg-10Gd is a more compatible implant material than Mg, with no adverse effects observed in the early phase of fracture healing during our 4-week study. STATEMENT OF SIGNIFICANCE: Degradable metallic implants in form of Mg and Mg-10Gd intramedullary pins were assessed in a rat femur fracture model, alongside a non-implanted SHAM group with special respect to the potential to induce an inflammatory response. This pre-clinical study combines innovative non-invasive in vivo imaging techniques associated with multimodal, ex vivo cellular and molecular analytics. The study contributes to the development and evaluation of degradable biometals and their clinical application potential. The study results indicate that Mg-10Gd did not exhibit any significant harmful effects compared to the SHAM and Mg groups.

Multispectral label-free in vivo cellular imaging of human retinal pigment epithelium using adaptive optics fluorescence lifetime ophthalmoscopy improves feasibility for low emission analysis and increases sensitivity for detecting changes with age and eccentricity.

Significance: Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE). Aim: To quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO. Approach: AOFLIO was performed on six subjects at seven eccentricities. Four imaging channels ( λ ex / λ em ) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter. Results: A repeatability analysis revealed < 11.8 % change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters. Conclusions: Multispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.

Live Imaging of the Shoot Apical Meristem of Intact, Soil-Grown, Flowering Arabidopsis Plants.

All aerial organs in plants originate from the shoot apical meristem, a specialized tissue at the tip of a plant, enclosing a few stem cells. Understanding developmental dynamics within this tissue in relation to internal and external stimuli is of crucial importance. Imaging the meristem at the cellular level beyond very early stages requires the apex to be detached from the plant body, a procedure that does not allow studies in living, intact plants over longer periods. This protocol describes a new confocal microscopy method with the potential to image the shoot apical meristem of an intact, soil-grown, flowering Arabidopsis plant over several days. The setup opens new avenues to study apical stem cells, their interconnection with the whole plant, and their responses to environmental stimuli. Key features • Novel dissection and imaging method of the shoot apical meristem of Arabidopsis. • Procedure performed with intact, soil-grown, flowering plants. • Possibility of long-term live imaging of the shoot apical meristem. • Protocol can be adapted to different plant species.

Hard X-ray imaging and tomography at the Biomedical Imaging and Therapy beamlines of Canadian Light Source.

The Biomedical Imaging and Therapy facility of the Canadian Light Source comprises two beamlines, which together cover a wide X-ray energy range from 13 keV up to 140 keV. The beamlines were designed with a focus on synchrotron applications in preclinical imaging and veterinary science as well as microbeam radiation therapy. While these remain a major part of the activities of both beamlines, a number of recent upgrades have enhanced the versatility and performance of the beamlines, particularly for high-resolution microtomography experiments. As a result, the user community has been quickly expanding to include researchers in advanced materials, batteries, fuel cells, agriculture, and environmental studies. This article summarizes the beam properties, describes the endstations together with the detector pool, and presents several application cases of the various X-ray imaging techniques available to users.

Half-curcumin-based chemiluminescence probes and their applications in detecting quasi-stable oxidized protein.

Numerous methods have been reported for detecting ROS/RNS in vitro and in vivo; however, detecting methods for the secondary products of the ROS/RNS reactions, particularly quasi-stable oxidized products, have been much less explored. In this report, we observed that half-curcumins could generate chemiluminescence. In contrast to other chemiluminescence scaffolds, the distinguishing feature of a half-curcumin is the formation of a carbanion intermediate of its acetylacetone moiety, opening unique avenues for applications. In this study, we designed a series of half-curcumins CRANAD-Xs and found that CRANAD-164 could be used to detect quasi-stable oxidized proteins (QSOP) in vivo and in patient serum samples. We illustrated that CRANAD-164 could be used to monitor the responses of taurine, an amino acid with newly reported anti-aging capacity, in an inflammatory mouse model. Remarkably, we further demonstrated that the QSOP levels were much higher in the disease serum samples, including Alzheimer's disease, compared to the samples from healthy controls. Moreover, our results revealed that the sera chemiluminescence intensities were higher in aged healthy controls compared to young healthy subjects, suggesting that CRANAD-164 can be used to monitor the increase of QSOP during aging.

Visualization of Blood-Brain Barrier Disruption in Septic Mice with the New Method Based on in Vivo Imaging Technology.

BACKGROUND: Dynamic monitoring of the blood-brain barrier (BBB) functional status in septic mice can help to explore the pathological mechanisms. Therefore, we proposed a new method for monitoring BBB permeability and applied it to the detection of sepsis models. METHODS: The new method involves the construction of an optical cranial window and in vivo imaging. We performed dynamic monitoring of BBB permeability and cerebral blood flow (CBF) in cecal ligation puncture (CLP) and endotoxemia (lipopolysaccharide [LPS]) mice. RESULTS: The sensitivity and accuracy of this method were higher than those of Evans blue evaluation. The increase of BBB permeability in the group of CLP mice was relatively mild and correlated with overall survival, and the damage was irreversible. Contrarily, BBB damage in the LPS group was more acute and severe, unrelated to overall survival, but recoverable. The CBF decreased significantly in both model mouse groups 24 h after modeling, but only the CBF proportion decrease in the LPS group was significantly correlated with an increase in BBB permeability. Within 24 h after both models were established, the decrease in blood flow in the digestive organs occurred earlier than in the brain and kidneys, and the decrease in small intestine blood flow in the LPS group progressed faster. CONCLUSIONS: We have successfully demonstrated the feasibility of our novel method to detect BBB permeability in mice. Our results revealed a significant difference in the BBB permeability change trend between the CLP and LPS model mice when survival curves were consistent. Notably, the CLP-model mice demonstrated a closer resemblance to clinical patients. Our findings suggest that early-stage brain tissue hypoperfusion has a greater impact on BBB function damage in endotoxemia mice, which is related to the faster progression of blood flow redistribution.

A novel method for measurements of sleep/wake states, feeding and drinking behaviors using the tracking technique of 3D positions in freely moving mice.

We have previously developed a 3D video tracking system which enables us to analyze long-term quantitative analysis of gene expression in freely moving mice. In the present study, we improved 3D video tracking and developed a system that analyzes more detailed behavioral data. We succeeded in simultaneously analyzing sleep-wake, feeding, and drinking behavior rhythms in the same individual using our tracking system. This system will make it possible to measure gene expression in each tissue in vivo in real time in relation to the various behavioral rhythms mentioned above.

A dual-emission fluorescent probe with independent polarity and viscosity responses: The synthesis, spectroscopy and bio-imaging applications.

Viscosity and polarity are essential parameters that play critical roles in various physiological processes. Thus, dual-emission fluorescent probes that respond to both polarity and viscosity are highly sought-after tools for studying these processes. In addressing this need, a novel fluorescent probe (L), with dual emissions centered at 460 nm and 780 nm, which can sensitively respond to polarity and viscosity respectively, has been developed. Probe (L) is constructed through rational molecular design, utilizing two conjugated synthons connected by a π-bond to form a D-π-A system. The twisted intramolecular charge transfer (TICT) state is dominant in low-viscosity environments, resulting in weak near-infrared (NIR) fluorescence. Conversely, the intramolecular charge transfer (ICT) state is expected to prevail in high-viscosity environments, leading to strong NIR fluorescence. The polarity-sensitive fluorescence centered at 460 nm can be attributed to the emission of the coumarin unit. Moreover, probe (L) exhibits low cytotoxicity and primarily targets mitochondria. By leveraging the dual-emission properties of probe (L), real-time imaging of polarity and viscosity fluctuations within cells has been achieved. Additionally, probe (L) can be used for in situ and in vivo imaging of rheumatoid arthritis (RA) with good imaging resolution.

Tetrandrine activates STING/TBK1/IRF3 pathway to potentiate anti-PD-1 immunotherapy efficacy in non-small cell lung cancer.

The efficacy of PD-1 therapy in non-small cell lung cancer (NSCLC) patients remains unsatisfactory. Activating the STING pathway is a promising strategy to improve PD-1 inhibitor efficacy. Here, we found tetrandrine (TET), an anti-tumor compound extracted from a medicinal plant commonly used in traditional Chinese medicine, has the ability to inhibit NSCLC tumor growth. Mechanistically, TET induces nuclear DNA damage and increases cytosolic dsDNA, thereby activating the STING/TBK1/IRF3 pathway, which in turn promotes the tumor infiltration of dendritic cells (DCs), macrophages, as well as CD8+ T cells in mice. In vivo imaging dynamically monitored the increased activity of the STING pathway after TET treatment and predicted the activation of the tumor immune microenvironment. We further revealed that the combination of TET with αPD-1 monoclonal antibody (αPD-1 mAb) yields significant anti-cancer effects by promoting CD8+ T cell infiltration and enhancing its cell-killing effect, which in turn reduced the growth of tumors and prolonged survival of NSCLC mice. Therefore, TET effectively eliminates NSCLC cells and enhances immunotherapy efficacy through the activation of the STING pathway, and combining TET with anti-PD-1 immunotherapy deserves further exploration for applications.

A new GRAB sensor reveals differences in the dynamics and molecular regulation between neuropeptide and neurotransmitter release.

The co-existence and co-transmission of neuropeptides and small molecule neurotransmitters in the same neuron is a fundamental aspect of almost all neurons across various species. However, the differences regarding their in vivo spatiotemporal dynamics and underlying molecular regulation remain poorly understood. Here, we developed a GPCR-activation-based (GRAB) sensor for detecting short neuropeptide F (sNPF) with high sensitivity and spatiotemporal resolution. Furthermore, we explore the differences of in vivo dynamics and molecular regulation between sNPF and acetylcholine (ACh) from the same neurons. Interestingly, the release of sNPF and ACh shows different spatiotemporal dynamics. Notably, we found that distinct synaptotagmins (Syt) are involved in these two processes, as Syt7 and Sytα for sNPF release, while Syt1 for ACh release. Thus, this new GRAB sensor provides a powerful tool for studying neuropeptide release and providing new insights into the distinct release dynamics and molecular regulation between neuropeptides and small molecule neurotransmitters.

Activity-dependent dendrite patterning in the postnatal barrel cortex.

For neural circuit construction in the brain, coarse neuronal connections are assembled prenatally following genetic programs, being reorganized postnatally by activity-dependent mechanisms to implement area-specific computational functions. Activity-dependent dendrite patterning is a critical component of neural circuit reorganization, whereby individual neurons rearrange and optimize their presynaptic partners. In the rodent primary somatosensory cortex (barrel cortex), driven by thalamocortical inputs, layer 4 (L4) excitatory neurons extensively remodel their basal dendrites at neonatal stages to ensure specific responses of barrels to the corresponding individual whiskers. This feature of barrel cortex L4 neurons makes them an excellent model, significantly contributing to unveiling the activity-dependent nature of dendrite patterning and circuit reorganization. In this review, we summarize recent advances in our understanding of the activity-dependent mechanisms underlying dendrite patterning. Our focus lays on the mechanisms revealed by in vivo time-lapse imaging, and the role of activity-dependent Golgi apparatus polarity regulation in dendrite patterning. We also discuss the type of neuronal activity that could contribute to dendrite patterning and hence connectivity.

[In vivo tumor imaging and therapy based on near-infrared cadmium-free quantum dots].

Near-infrared fluorescence imaging technology, which possesses superior advantages including real-time and fast imaging, high spatial and temporal resolution, and deep tissue penetration, shows great potential for tumor imaging in vivo and therapy. Ⅰ-Ⅲ-Ⅵ quantum dots exhibit high brightness, broad excitation, easily tunable emission wavelength and superior stability, and do not contain highly toxic heavy metal elements such as cadmium or lead. These advantages make Ⅰ-Ⅲ-Ⅵ quantum dots attract widespread attention in biomedical field. This review summarizes the recent advances in the controlled synthesis of Ⅰ-Ⅲ-Ⅵ quantum dots and their applications in tumor imaging in vivo and therapy. Firstly, the organic-phase and aqueous-phase synthesis of Ⅰ-Ⅲ-Ⅵ quantum dots as well as the strategies for regulating the near-infrared photoluminescence are briefly introduced; secondly, representative biomedical applications of near-infrared-emitting cadmium-free quantum dots including early diagnosis of tumor, lymphatic imaging, drug delivery, photothermal and photodynamic therapy are emphatically discussed; lastly, perspectives on the future directions of developing quantum dots for biomedical application and the faced challenges are discussed. This paper may provide guidance and reference for further research and clinical translation of cadmium-free quantum dots in tumor diagnosis and treatment.

Animal models of neglected parasitic diseases: In vivo multimodal imaging of experimental trypanosomatid infections.

African trypanosomiases and leishmaniases are significant neglected tropical diseases (NTDs) that affect millions globally, with severe health and socio-economic consequences, especially in endemic regions. Understanding the pathogenesis and dissemination of Trypanosoma brucei and Leishmania spp. parasites within their hosts is pivotal for the development of effective interventions. Whole-body bioluminescence and fluorescence imaging systems (BLI and FLI, respectively), are powerful tools to visualize and quantify the progression and distribution of these parasites in real-time within live animal models. By combining this technology with the engineering of stable T. brucei and Leishmania spp. strains expressing luciferase and/or fluorescent proteins, crucial aspects of the infection process including the parasites' homing, the infection dynamics, the tissue tropism, or the efficacy of experimental treatments and vaccines can be deeply investigated. This methodology allows for enhanced sensitivity and resolution, elucidating previously unrecognized infection niches and dynamics. Importantly, whole-body in vivo imaging is non-invasive, enabling for longitudinal studies during the course of an infection in the same animal, thereby aligning with the "3Rs" principle of animal research. Here, we detail a protocol for the generation of dual-reporter T. brucei and L. major, and their use to infect mice and follow the spatiotemporal dynamics of infection by in vivo imaging systems. Additionally, 3D micro-computed tomography (µCT) coupled to BLI in T. brucei-infected animals is applied to gain insights into the anatomical parasite distribution. This Chapter underscores the potential of these bioimaging modalities as indispensable tools in parasitology, paving the way for novel therapeutic strategies and deeper insights into host-parasite interactions.

Glyoxal-methyl-ethylene sulfonic acid fixative enhances the fixation of cytoskeletal structures for Förster resonance energy transfer measurements.

Förster resonance energy transfer (FRET) serves as a tool for measuring protein-protein interactions using various sensor molecules. The tension sensor module relies on FRET technology. In our study, this module was inserted within the actinin molecule to measure the surface tension of the cells. Given that the decay curve of FRET efficiency correlates with surface tension increase, precise and accurate efficiency measurement becomes crucial. Among the methods of FRET measurements, FRET efficiency remains the most accurate if sample fixation is successful. However, when cells were fixed with 4% paraformaldehyde (PFA), the actinin-FRET sensor diffused across the cytoplasm; this prompted us to explore fixation method enhancements. Glyoxal fixative has been reported to improve cytoskeletal morphologies compared to PFA. However, it was not known whether glyoxal fits FRET measurements. Glyoxal necessitates an acetic acid solution for fixation; however, acidic conditions could compromise fluorescence stability. We observed that the pH working range of glyoxal fixative aligns closely with MES (methyl-ethylene sulfonic acid) Good's buffer. Initially, we switched the acidic solution for MES buffer and optimized the fixation procedure for in vitro and in vivo FRET imaging. By comparing FRET measurements on hydrogels with known stiffness to tumor nodules in mouse lung, we estimated in vivo stiffness. The estimated stiffness of cancerous tissue was harder than the reported stiffness of smooth muscle. This discovery shed lights on how cancer cells perceive environmental stiffness during metastasis.

Two-dimensional electro-optical multiphoton microscopy.

Significance: The development of genetically encoded fluorescent indicators of neural activity with millisecond dynamics has generated demand for ever faster two-photon (2P) imaging systems, but acoustic and mechanical beam scanning technologies are approaching fundamental limits. We demonstrate that potassium tantalate niobate (KTN) electro-optical deflectors (EODs), which are not subject to the same fundamental limits, are capable of ultrafast two-dimensional (2D) 2P imaging in vivo. Aim: To determine if KTN-EODs are suitable for 2P imaging, compatible with 2D scanning, and capable of ultrafast in vivo imaging of genetically encoded indicators with millisecond dynamics. Approach: The performance of a commercially available KTN-EOD was characterized across a range of drive frequencies and laser parameters relevant to in vivo 2P microscopy. A second KTN-EOD was incorporated into a dual-axis scan module, and the system was validated by imaging signals in vivo from ASAP3, a genetically encoded voltage indicator. Results: Optimal KTN-EOD deflection of laser light with a central wavelength of 960 nm was obtained up to the highest average powers and pulse intensities tested (power: 350 mW; pulse duration: 118 fs). Up to 32 resolvable spots per line at a 560 kHz line scan rate could be obtained with single-axis deflection. The complete dual-axis EO 2P microscope was capable of imaging a 13 µm by 13 µm field-of-view at over 10 kHz frame rate with ∼0.5 µm lateral resolution. We demonstrate in vivo imaging of neurons expressing ASAP3 with high temporal resolution. Conclusions: We demonstrate the suitability of KTN-EODs for ultrafast 2P cellular imaging in vivo, providing a foundation for future high-performance microscopes to incorporate emerging advances in KTN-based scanning technology.