Dual-objective two-photon microscope for volumetric imaging of dense scattering biological samples by bidirectional excitation and collection

Full view observation throughout entire specimens over a prolonged period is crucial when exploring the physiological functions and system-level behaviors.Multi-photon microscopy(MPM)has been widely employed for such purposes owing to its deep penetration ability.However,the current MPM struggles with balancing the imaging depth and quality while avoiding photodamage for the exponential increasement of excitation power with the imaging depth.Here,we present a dual-objective two-photon microscope(Duo-2P),characterized by bidirectional two-photon excitation and fluorescence collection,for long-duration volumetric imaging of dense scattering samples.Duo-2P effectively doubles the imaging depth,reduces the total excitation energy by an order of magnitude for samples with a thickness five times the scattering length,and enhances the signal-to-noise ratio up to 1.4 times.Leveraging these advantages,we acquired volumetric images of a 380-μm suprachiasmatic nucleus slice for continuous 4-h recording at a rate of 1.67 s/volume,visualized the calcium activities over 4000 neurons,and uncovered their state-switching behavior.We conclude that Duo-2P provides an elegant and powerful means to overcome the fundamental depth limit while mitigating photodamages for deep tissue volumetric imaging.

Miniature Fluorescence Microscopy for Imaging Brain Activity in Freely-Behaving Animals

An ultimate goal of neuroscience is to decipher the principles underlying neuronal information processing at the molecular,cellular,circuit,and system levels.The advent of miniature fluorescence microscopy has furthered the quest by visualizing brain activities and structural dynamics in animals engaged in self-determined behaviors.In this brief review,we summarize recent advances in miniature fluorescence microscopy for neuroscience,focusing mostly on two mainstream solutions-miniature single-photon microscopy,and miniature two-photon microscopy.We discuss their technical advantages and limitations as well as unmet challenges for future improvement.Examples of preliminary applications are also presented to reflect on a new trend of brain imaging in experimental paradigms involving body movements,long and complex protocols,and even disease progression and aging.

A novel stochastic reaction-diffusion model of Ca^2＋ blink in cardiac myocytes

In cardiac myocytes,the sarcoplasmic reticulum(SR)is the main storage organelle of free Ca^(2+).The concentration of free Ca^(2+)in the SR is 0.5–1.0 mmol/L and is 2–3 orders of magnitude greater than that in the cytosol.The SR is composed of interconnected cisternae(junctional SR,i.e.,JSR)and tubules(free SR network,i.e.,FSR)that extend throughout the cytosol[1].Ca^(2+)is released from the JSR into the cytosol via Ca^(2+)release units(CRUs,

Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells

To modulate gene expression in research studies or in potential clinical therapies,transfection of exogenous nucleic acids including plasmid DNA and small interference RNA(siRNA)are generally performed.However,the cellular processing and the fate of these nucleic acids remain elusive.By investigating the cellular behavior of transfected nucleic acids using confocal imaging,here we show that when siRNA was cotransfected into cultured cells with other nucleic acids,including single-stranded RNA oligonucleotides,single and double-stranded DNA oligonucleotides,as well as long double-stranded plasmid DNA,they all aggregate in the same cytoplasmic granules.Interestingly,the amount of siRNA aggregating in granules was found not to correlate with the gene silencing activity,suggesting that assembly of cytoplasmic granules triggered by siRNA transfection may be separable from the siRNA silencing event.Our results argue against the claim that the siRNAaggregating granules are the functional site of RNA interference(RNAi).Taken together,our studies suggest that,independent of their types or forms,extraneously transfected nucleic acids are processed through a common cytoplasmic pathway and trigger the formation of a new type of cytoplasmic granules“transfection granules”.

Visualizing Seizure Propagation in Freely-moving Mice via Miniature Two-photon Microscopy

DearEditor,Due to the tight and precise associations between behaviors and neural activities,the investigation of neuronal activity in freely-movingand behavinganimals isamajor unaccomplished goal in neuroscience.Epileptiform seizures,characterized by hyper-synchronized discharges and hyper-excitation in neuronal networks,are one of the most common neurological disorders.