Two-photon Microscopy Research Articles

Dendritic spines of layer 5 pyramidal neurons of the aging somatosensory cortex exhibit reduced volumetric remodeling.

Impairments in synaptic dynamics and stability are observed both in neurodegenerative disorders and in the healthy aging cortex, which exhibits elevated dendritic spine turnover and decreased long-term stability of excitatory connections at baseline, as well as an altered response to plasticity induction. In addition to the discrete gain and loss of synapses, spines also change in size and strength both during learning and in the absence of neural activity, and synaptic volume has been associated with stability and incorporation into memory traces. Furthermore, intrinsic dynamics, an apparently stochastic component of spine volume changes, may serve as a homeostatic mechanism to prevent stabilization of superfluous connections. However, the effects of age on modulation of synaptic weights remain unknown. Using two-photon excitation (2PE) microscopy of spines during chemical plasticity induction in vitro and analyzing longitudinal in vivo 2PE images after a plasticity-inducing manipulation, we characterize the effects of age on volumetric changes of spines of the apical tuft of layer 5 pyramidal neurons of mouse primary somatosensory cortex. Aged mice exhibit decreased volumetric volatility and delayed rearrangement of synaptic weights of persistent connections, as well as greater susceptibility to spine shrinkage in response to chemical long-term depression. These results suggest a deficit in the aging brain's ability to fine-tune synaptic weights to properly incorporate and retain novel memories. This research provides the first evidence of alterations in spine volumetric dynamics in healthy aging and may support a model of impaired processing and learning in the aged somatosensory system.Significance Statement Aging is known to impact cognitive functions and sensory processing, yet the underlying mechanisms at the synaptic level remain unclear. This study investigates dendritic spine dynamics of layer 5 pyramidal neurons in the aging somatosensory cortex. By employing two-photon excitation microscopy and analyzing spine volume changes during plasticity induction, we reveal that aged mice exhibit decreased volumetric volatility and delayed synaptic weight rearrangement. These alterations may impair the brain's ability to fine-tune synaptic weights, which is crucial for incorporating and retaining new memories. This research provides the first evidence of altered spine volumetric dynamics in healthy aging, suggesting a potential mechanism for impaired processing and learning in the aged cortex. Understanding these changes could aid in mitigation of age-related cognitive decline.

Membrane Associated RNA-Containing Vesicles Regulate Cortical Astrocytic Microdomain Calcium Transients in Awake Ischemic Stroke Mice.

Astrocytic processes minutely regulate neuronal activity via tripartite synaptic structures. The precision-tuning of the function of astrocytic processes is garnering increasing attention because of its significance in promoting brain repair following ischemic stroke. Microdomain calcium (Ca2+) transients in astrocytic processes are pivotal for the functional regulation of these processes. However, the understanding of the alterations and regulatory mechanism of microdomain Ca2+ transients during stroke remains limited. In the present study, a fast high-resolution, miniaturized two-photon microscopy is used to show that the levels of astrocytic microdomain Ca2+ transients are significantly reduced in the peri-infarct area of awake ischemic stroke mice. This finding correlated with the behavioral deficits shown by these mice under freely-moving conditions. Mitochondrial Ca2+ activity is an important factor driving the microdomain Ca2+ transients. DEAD Box 1 (DDX1) bound to circSCMH1 (a circular RNA involved in vascular post-stroke repair) facilitates the formation of membrane-associated RNA-containing vesicles (MARVs) and enhances the activity of astrocytic microdomain Ca2+ transients, thereby promoting behavioral recovery. These results show that targeting astrocytic microdomain Ca2+ transients is a potential therapeutic approach in stroke intervention.

Advanced deep-tissue imaging and manipulation enabled by biliverdin reductase knockout.

We developed near-infrared (NIR) photoacoustic and fluorescence probes, as well as optogenetic tools from bacteriophytochromes, and enhanced their performance using biliverdin reductase-A knock-out model (Blvra-/-). Blvra-/- elevates endogenous heme-derived biliverdin chromophore for bacteriophytochrome-derived NIR constructs. Consequently, light-controlled transcription with IsPadC-based optogenetic tool improved up to 25-fold compared to wild-type cells, with 100-fold activation in Blvra-/- neurons. In vivo , light-induced insulin production in Blvra-/- reduced blood glucose in diabetes by ∼60%, indicating high potential for optogenetic therapy. Using 3D photoacoustic, ultrasound, and two-photon fluorescence imaging, we overcame depth limitations of recording NIR probes. We achieved simultaneous photoacoustic imaging of DrBphP in neurons and super-resolution ultrasound localization microscopy of blood vessels ∼7 mm deep in the brain, with intact scalp and skull. Two-photon microscopy provided cell-level resolution of miRFP720-expressing neurons ∼2.2 mm deep. Blvra-/- significantly enhances efficacy of biliverdin-dependent NIR systems, making it promising platform for interrogation and manipulation of biological processes.

Two-Photon Microscopy to Measure Calcium Signaling in the Living Brain.

Two-photon microscopy enables imaging of calcium signaling at cellular or subcellular resolution up to hundreds of microns deep in the living brain. Changes in the brightness of fluorescent calcium indicators provide a readout of calcium levels over time, affording information about neuronal activity and/or calcium-dependent subcellular signaling. Here, we describe a protocol for repeated two-photon imaging of calcium signals in mice expressing a genetically encoded calcium indicator that have been implanted with a chronic cranial window.

Effects of oil-water interfacial properties on protein structuring and droplet deformation in high moisture meat analogues containing oil

Adding oil to high moisture meat analogues (HMMA) can increase juiciness, and can be achieved by incorporating emulsion droplets during extrusion. Since these droplets can coalesce when subjected to high shear, selecting appropriate emulsion stabilisers is important. For several commercial plant-protein emulsion stabilisers, it was investigated how oil-water interfacial mechanical properties affect droplet deformation and protein structuring in extrusion of HMMAs. Emulsions with 10 wt% or 15 wt% oil, stabilised by potato protein isolates (POPI-1 (Rich in patatin) and POPI-2 (rich in protease inhibitor)) and pea protein isolate PPI, were used to make extrudates with 5.7 wt% and 8.5 wt% oil, respectively. In 8.5 wt%-extrudates, POPI-2 had the most oil leakage from the cooling die, while PPI had the smallest amount despite having softer and more stretchable interfaces. Blade-cutting tests showed the highest maximum force for 8.5 wt%-extrudates with POPI-1, likely because POPI-1 formed the stiffest interfaces. Tensile stress testing showed the largest fracture strain in 8.5% wt%-extrudates with PPI, corresponding to its longer wedge length. Multiphoton excitation microscopy was used to visualise the extrudates protein structure and oil droplets. This showed that droplets near the surface of the extrudate were less deformed than droplets in the centre. There were only small differences between protein stabilisers regarding oil droplet deformation, indicating droplet deformation was dominated by deformation of the protein matrix. The O/W interfacial properties significantly affected oil leakage, cutting force, and tensile strength of extrudates. These results are important to consider when designing emulsions for HMMAs processed by extruders.

Biological Scaffolds in 3D Cell Models: Driving Innovation in Drug Discovery.

The discipline of 3D cell modeling is currently undergoing a surge of captivating developments that are enhancing the realism and utility of tissue simulations. Using bioinks which represent cells, scaffolds, and growth factors scientists can construct intricate tissue architectures layer by layer using innovations like 3D bioprinting. Drug testing can be accelerated and organ functions more precisely replicated owing to the precise control that microfluidic technologies and organ-on-chip devices offer over the cellular environment. Tissue engineering is becoming more dynamic with materials that can modify their surroundings with the advent of hydrogels and smart biomaterials. Advances in spheroids and organoids are not only bringing us towards more effective and customized therapies, but they are also improving their ability to resemble actual human tissues. Confocal and two-photon microscopy are examples of advanced imaging methods that provide precise images of the functioning and interaction of cells. Artificial Intelligence models have applications for enhanced scaffold designs and for predicting the response of tissues to medications. Furthermore, via strengthening predictive models, optimizing data analysis, and simplifying 3D cell culture design, artificial intelligence is revolutionizing this field. When combined, these technologies are improving our ability to conduct research and moving us toward more individualized and effective medical interventions.

Application Exploration of the Photon Chip Technology in Real time Processing of Biomedical Images

In the biomedical field, photonic chip technology is gradually becoming the key to improving the speed and accuracy of medical imaging. The development of this technology has given us new hope in early disease diagnosis and cell monitoring. Recent research has not only sorted out the hotspots and future trends of biomedical photonics, but also paid special attention to the application of photon technology in these fields. Specifically, photonic chips have demonstrated their unique capabilities in multi-color two-photon excitation fluorescence microscopy imaging, two-photon fluorescence lifetime imaging, and two-photon fiber endoscopic imaging. These applications not only improve the clarity of imaging, but also accelerate processing speed, which is crucial for rapid diagnosis and treatment. At the end of the article, the author summarized the contributions of photonic chip technology in the field of biomedical imaging and provided prospects for future development directions.

PD-1 blockade does not improve efficacy of EpCAM-directed CAR T-cell in lung cancer brain metastasis

BackgroundLung cancer brain metastasis has a devastating prognosis, necessitating innovative treatment strategies. While chimeric antigen receptor (CAR) T-cell show promise in hematologic malignancies, their efficacy in solid tumors, including brain metastasis, is limited by the immunosuppressive tumor environment. The PD-L1/PD-1 pathway inhibits CAR T-cell activity in the tumor microenvironment, presenting a potential target to enhance therapeutic efficacy. This study aims to evaluate the impact of anti-PD-1 antibodies on CAR T-cell in treating lung cancer brain metastasis.MethodsWe utilized a murine immunocompetent, syngeneic orthotopic cerebral metastasis model for repetitive intracerebral two-photon laser scanning microscopy, enabling in vivo characterization of red fluorescent tumor cells and CAR T-cell at a single-cell level over time. Red fluorescent EpCAM-transduced Lewis lung carcinoma cells (EpCAM/tdtLL/2 cells) were implanted intracranially. Following the formation of brain metastasis, EpCAM-directed CAR T-cell were injected into adjacent brain tissue, and animals received either anti-PD-1 or an isotype control.ResultsCompared to controls receiving T-cell lacking a CAR, mice receiving EpCAM-directed CAR T-cell showed higher intratumoral CAR T-cell densities in the beginning after intraparenchymal injection. This finding was accompanied with reduced tumor growth and translated into a survival benefit. Additional anti-PD-1 treatment, however, did not affect intratumoral CAR T-cell persistence nor tumor growth and thereby did not provide an additional therapeutic effect.ConclusionCAR T-cell therapy for brain malignancies appears promising. However, additional anti-PD-1 treatment did not enhance intratumoral CAR T-cell persistence or effector function, highlighting the need for novel strategies to improve CAR T-cell therapy in solid tumors.

Optogenetic estimation of synaptic connections in brain slices

BackgroundDetection of synaptic connections is essential for understanding neural circuits. By using optogenetics, current injection, and glutamate uncaging to activate presynaptic cells and simultaneously recording the subsequent response of postsynaptic cells, the presence of synaptic connections can be confirmed. However, these methods present throughput challenges, such as the need for simultaneous multicellular patch-clamp recording and two-photon microscopy. These challenges lead to a trade-off between sacrificing resolution and experimental throughput. New methodWe adopted the laser, typically used for local field ablation, and combined this with post hoc analysis. We successfully approximated the synaptic connection probabilities using only an epi-fluorescence microscope and single-cell recordings. ResultsWe sequentially stimulated the channelrhodopsin 2-expressing cells surrounding the recorded cell and approximated the synaptic connection probabilities. This probability value was comparable to that obtained from simultaneous multi-cell patch-clamp recordings, which included more than 600 pairs. Comparison with existing methodsOur setup allows us to estimate connection probabilities within 100 s, outperforming existing methods. We successfully estimated synaptic connection probabilities using only the optical path typically used by an epi-fluorescence microscope and single-cell recordings. It may also be suitable for dendritic ablation experiments. ConclusionsThe proposed method simplifies the estimation of connection probabilities, which is expected to advance the study of neural circuits in conditions such as autism and schizophrenia where connection probabilities vary. Furthermore, this approach is applicable not only to local circuits but also to long-range connections, thus increasing experimental throughput.

Acne-related UVA-induced facial fluorescence: An exploratory study from physiological properties to tissue structure information

UVA-induced facial fluorescence (UVAF) is recognized as an objective measurement technique to quantify the severity of acne. However, notable inconsistencies in quantitative outcomes have been observed in various studies, possibly due to the fact that different colors of fluorescence represent different pathophysiological implications. This study investigated the pathophysiological importance of UVAF color differences and improved its reliability in assessing acne severity. MIDOO Smart Skin Imager was used to capture UVAF and analyze the correlation between fluorescence colors and acne lesions. Techniques such as two-photon excited fluorescence microscopy, scanning electron microscopy, western blot, and high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) were used to examine the biochemical composition and structure of comedonal plugs and follicular casts associated with different fluorescence colors. We found that green fluorescence correlates with non-inflammatory acne lesions (comedones), while orange-red fluorescence shows no correlation with either type of lesion. Green fluorescence is associated with higher levels of keratin, indicating keratinization, while orange-red fluorescence is associated with porphyrin from S. epidermidis. UVAF color differences - orange-red are from porphyrins and green from keratin. This distinction helps to understand the structural and physiological bases of facial fluorescence, with potential implications for clinical evaluations of acne.

Effect of diabetes on microvascular morphology and permeability of rat skeletal muscle: in vivo imaging using two-photon laser scanning microscopy.

This investigation evaluated the microvascular permeability and ultrastructure of skeletal muscle capillaries in the skeletal muscle of diabetic (DIA) rats using two-photon laser scanning microscopy (TPLSM) and transmission electron microscopy (TEM). Microvascular permeability was assessed in the tibialis anterior muscle of control (CON) and DIA (streptozocin) male Wistar rats (n = 20, 10-14 wk) by in vivo imaging using TPLSM after fluorescent dye intravenous infusion. Fluorescent dye leakage was quantified to determine microvascular permeability. The ultrastructure was imaged by TEM ex vivo to calculate the size and number of intercellular clefts between capillary endothelial cells and also intracellular vesicles. Compared with control, the volumetrically determined interstitial fluorescent dye leakage, the endothelial cell thickness, and the number of intercellular clefts per capillary perimeter were significantly higher, and the cleft width was significantly narrower in tibialis anterior (TA) of DIA (interstitial fluorescent dye leakage, 2.88 ± 1.40 vs. 10.95 ± 1.41 µm3 × min × 106; endothelial thickness, 0.28 ± 0.02 vs. 0.45 ± 0.03 µm; number of intercellular clefts per capillary perimeter, 6.3 ± 0.80 vs. 13.6 ± 1.7/100 µm; cleft width, 11.92 ± 0.95 vs. 8.40 ± 1.03 nm, CON vs. DIA, respectively, all P < 0.05). The size of intracellular vesicles in the vascular endothelium showed an increased proportion of large vesicles in the DIA group compared with the CON group (P < 0.05). Diabetes mellitus enhances the microvascular permeability of skeletal muscle microvessels due, in part, to a higher density and narrowing of the endothelial intercellular clefts, and larger intracellular vesicles.NEW & NOTEWORTHY Microvascular permeability in diabetic muscle was investigated using our original two-photon scanning laser microscopy method. Compared with controls, the leakage volume was increased in diabetic muscle, which was atrophic with smaller capillary diameter, endothelial cell thickening, and the appearance of more endothelial intercellular gaps or clefts, and large vesicles. Hyperpermeability was closely related to ultrafine structural changes of the capillary endothelial cell junctions.

P1.03F.01 Imaging the Nucleolus Using Two-Photon Microscopy to Distinguish Between Tumor Cells and Normal Cells

P1.03F.01 Imaging the Nucleolus Using Two-Photon Microscopy to Distinguish Between Tumor Cells and Normal Cells

Simultaneous assessment of NAD(P)H and flavins with multispectral fluorescence lifetime imaging microscopy at a single excitation wavelength of 750nm.

Autofluorescence characteristics of the reduced nicotinamide adenine dinucleotide and oxidized flavin cofactors are important for the evaluation of the metabolic status of the cells. The approaches that involve a detailed analysis of both spectral and time characteristics of the autofluorescence signals may provide additional insights into the biochemical processes in the cells and biological tissues and facilitate the transition of spectral fluorescence lifetime imaging into clinical applications. We present the experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of the reduced nicotinamide adenine dinucleotide and oxidized flavin under a single excitation wavelength aimed at understanding whether the use of multispectral detection is helpful for metabolic imaging of cancer cells. We use two-photon spectral fluorescence lifetime imaging microscopy. Starting from model solutions, we switched to cell cultures treated by metabolic inhibitors and then studied the metabolism of cells within tumor spheroids. The use of a multispectral detector in combination with an excitation at a single wavelength of 750nm allows the identification of fluorescence signals from three components: free and bound NAD(P)H, and flavins based on the global fitting procedure. Multispectral data make it possible to assess not only the lifetime but also the spectral shifts of emission of flavins caused by chemical perturbations. Altogether, the informative parameters of the developed approach are the ratio of free and bound NAD(P)H amplitudes, the decay time of bound NAD(P)H, the amplitude of flavin fluorescence signal, the fluorescence decay time of flavins, and the spectral shift of the emission signal of flavins. Hence, with multispectral fluorescence lifetime imaging, we get five independent parameters, of which three are related to flavins. The approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750nm and a fluorescence time-resolved spectral detection with the consequent global analysis of the data not only simplifies image acquisition protocol but also allows to disentangle the impacts of free and bound NAD(P)H, and flavin components evaluate changes in their fluorescence parameters (emission spectra and fluorescence lifetime) upon treating cells with metabolic inhibitors and sense metabolic heterogeneity within 3D tumor spheroids.

Differences in motor learning-related structural plasticity of layer 2/3 parvalbumin-positive interneurons of the young and aged motor cortex.

Changes to neuronal connectivity are believed to be a key factor in cognitive impairments associated with normal aging. Because of its effect on activities of daily living, deficient motor control is a critical type of cognitive decline to understand. Diminished inhibitory networks in the cortex are implicated in such motor control deficits, pointing to the connectivity of inhibitory cortical interneurons as an important area for study. Here, we used chronic two-photon microscopy to track the structural plasticity of en passant boutons (EPBs) of parvalbumin-positive interneurons in the mouse motor cortex in the first longitudinal, in vivo study of inhibitory interneuron synapses in the context of aging. Young (3-5months) and aged (23-28months) mice underwent training on the accelerating rotarod to evoke motor learning-induced structural plasticity. Our analysis reveals that, in comparison with axons from young mice, those from aged mice have fewer EPBs at baseline that also tend to be larger in size. Aged axons also express learning-related structural plasticity-like new bouton stabilization and bouton enlargement-that is less persistent than that of young axons. This study reveals striking baseline differences in young and aged axon morphology as well as differences in the deployment of learning-related structural plasticity across axons.

Recent advances in label-free imaging techniques based on nonlinear optical microscopy to reveal the heterogeneity of the tumor microenvironment

Recent advances in label-free imaging techniques based on nonlinear optical microscopy to reveal the heterogeneity of the tumor microenvironment

TWINKLE: An open-source two-photon microscope for teaching and research.

Many laboratories use two-photon microscopy through commercial suppliers, or homemade designs of considerable complexity. The integrated nature of these systems complicates customization, troubleshooting as well as grasping the principles of two-photon microscopy. Here, we present "Twinkle": a microscope for Two-photon Imaging in Neuroscience, and Kit for Learning and Education. It is a fully open, high-performance and cost-effective research and teaching microscope without any custom parts beyond what can be fabricated in a university machine shop. The instrument features a large field of view, using a modern objective with a long working distance and large back aperture to maximize the fluorescence signal. We document our experiences using this system as a teaching tool in several two week long workshops, exemplify scientific use cases, and conclude with a broader note on the place of our work in the growing space of open-source scientific instrumentation.

Deep Mouse Brain Two-Photon Near-Infrared Fluorescence Imaging Using a Superconducting Nanowire Single-Photon Detector Array.

Two-photon microscopy (2PM) has become an important tool in biology to study the structure and function of intact tissues in vivo. However, adult mammalian tissues such as the mouse brain are highly scattering, thereby putting fundamental limits on the achievable imaging depth, which typically reside at around 600-800 μm. In principle, shifting both the excitation as well as (fluorescence) emission light to the shortwave near-infrared (SWIR, 1000-1700 nm) region promises substantially deeper imaging in 2PM, yet this shift has proven challenging in the past due to the limited availability of detectors and probes in this wavelength region. To overcome these limitations and fully capitalize on the SWIR region, in this work, we introduce a novel array of superconducting nanowire single-photon detectors (SNSPDs) and associated custom detection electronics for use in near-infrared 2PM. The SNSPD array exhibits high efficiency and dynamic range as well as low dark-count rates over a wide wavelength range. Additionally, the electronics and software permit a seamless integration into typical 2PM systems. Together with an organic fluorescent dye emitting at 1105 nm, we report imaging depth of >1.1 mm in the in vivo mouse brain, limited mostly by available labeling density and laser properties. Our work establishes a promising, and ultimately scalable, new detector technology for SWIR 2PM that facilitates deep tissue biological imaging.

Out-of-focus signal rejection for in vivo pO₂ measurements using two-photon phosphorescence lifetime microscopy

Out-of-focus signal rejection for in vivo pO₂ measurements using two-photon phosphorescence lifetime microscopy

Neural Basis of Number Sense in Larval Zebrafish.

Number sense, the ability to discriminate the quantity of objects, is crucial for survival. To understand how neurons work together and develop to mediate number sense, we used two-photon fluorescence light sheet microscopy to capture the activity of individual neurons throughout the brain of larval Danio rerio, while displaying a visual number stimulus to the animal. We identified number-selective neurons as early as 3 days post-fertilization and found a proportional increase of neurons tuned to larger quantities after 3 days. We used machine learning to predict the stimulus from the neuronal activity and observed that the prediction accuracy improves with age. We further tested ethanol's effect on number sense and found a decrease in number-selective neurons in the forebrain, suggesting cognitive impairment. These findings are a significant step towards understanding neural circuits devoted to discrete magnitudes and our methodology to track single-neuron activity across the whole brain is broadly applicable to other fields in neuroscience.

Photobleaching analysis of fluorescent proteins in two-photon microscopy at high repetition rates

Severe photobleaching in two-photon excitation fluorescence microscopy (TPM) has limited its potential for long-term and quantitative imaging of live cells and tissues. One solution is to excite fluorophores with a high-repetition rate which reduces the peak intensity of irradiance. However, there is a lack of knowledge about the general utility of this strategy for fluorescent proteins. Here, using simple photobleaching assay, we studied the photobleaching of EGFP and tdTomato at 80 MHz and 640 MHz repetition rates. Our analysis shows that the impact of high repetition rate excitation on reducing photobleaching in TPM is highly dependent on the excitation wavelength and fluorophores. Our results are useful for selecting optimal parameters to minimize photobleaching and photodamage in TPM.