photon microscope  (Nikon)

Journal: bioRxiv

Article Title: nELAVL phosphorylation by CDKL5 regulates inter-condensates composition and communication to promote experience-dependent maturation of the visual cortex

doi: 10.64898/2026.04.03.716270

Figure Lengend Snippet: (A) Graphical design of the visual cliff task with measurements. Animals were placed on a platform (3 cm height) in the middle of the open field (left). Percentage of descents from the cliff side per mouse (5 trials per mouse) between WT and Cdkl5 KO mice (right). WT: n=16 mice, Cdkl5 KO: n=16 mice. Mann-Whitney U test (two-tailed). (B) Head-fixed wildtype control (WT) mice and Cdkl5 KO were presented, either to one eye or both eyes, with visual stimuli of different directional grating patterns. In the binocular visual cortex (bV1), calcium signal in layer 2/3 (L2/3) neurons were live-imaged with two-photon laser microscopy. (C) Field of views (FOVs) showing grating and natural scene responsive neurons in WT (left) and Cdkl5 KO (right) mice. Scale bar, 20 μm. (D) Traces showing grating responsive neurons in WT and Cdkl5 KO mice across 3 viewing conditions, which are binocular (Binoc), contralateral (Contra) and ipsilateral (Ipsi) viewing relatively to the brain hemisphere imaged. Yellow regions indicate grating presentation periods. Gray lines represent the Z-score from all trials and the colored lines show trial average. (E) Boxplots showing the orientation selective index (OSI) of Cdkl5 KO mice were reduced across Binoc, Contra and Ipsi conditions under grating (s.f. 0.08cpd). Binoc OSI: 870 neurons from 6 WT and 698 neurons from 6 Cdkl5 KO mice. Contra OSI: 794 neurons from 6 WT and 693 neurons from 6 Cdkl5 KO. Ipsi OSI: 629 neurons from 6 WT and 476 neurons from 6 Cdkl5 KO. The centerlines represent median values, and the whiskers connect the nonoutlier minimum and maximum values to 0.25 and 0.75 quartiles respectively. Outliers are values greater than 1.5 interquartile range away from the quartiles. Mann-Whitney U test (two-tailed). (F) Scatter plots comparing OSI and correlation of grating (s.f. 0.08cpd) responsive neurons in WT mice and Cdkl5 KO mice. Color bar shows the correlation between tuning curves. 301 neurons from 6 WT and 211 neurons from 6 Cdkl5 KO. Mann-Whitney U test (two-tailed). (G) Boxplots showing fraction of neurons per FOV within the blue region (OSI > 0.5) and red region (OSI < 0.5) from E. 26 FOV from 6 WT mice and 21 FOV from 6 Cdkl5 KO mice. Mann-Whitney U test (two-tailed). (H) Distribution of Contra and Ipsi eye correlation between WT and Cdkl5 KO under grating (s.f. 0.08cpd) stimuli. 301 neurons from 6 WT and 211 neurons from 6 Cdkl5 KO. Mann-Whitney U test (two-tailed). (I) Distribution of difference in preferred orientation between WT and Cdkl5 KO under grating (s.f. 0.08cpd) stimuli. 301 neurons from 6 WT and 211 neurons from 6 Cdkl5 KO. Mann-Whitney U test (two-tailed). (J) Traces showing natural scene responsive neurons in WT and Cdkl5 KO mice across 3 viewing conditions. Pink regions indicate natural scene presentation periods. Gray lines represent the Z-score from all trials and the colored lines show trial average. (K) Distribution of contra and ipsi eye correlation between WT and Cdkl5 KO under natural scene stimuli. 529 neurons from 6 WT and 274 neurons from 6 Cdkl5 KO. Mann-Whitney U test (two-tailed). (L) Mice were virally injected to express GCaMP6f (Ctrl), GCaMP6f and nELAVL (WT), GCaMP6f and nELAVL-SE (SE) or GCaMP6f and nELAVL-SA (SA). Head-fixed mice were presented, either to one eye or both eyes, with visual stimuli of different directional grating patterns and natural scene. In the binocular visual cortex (bV1), calcium signal in layer 2/3 (L2/3) neurons were live-imaged with two-photon laser microscopy. (M) Boxplots showing OSI were reduced in WT, SE and SA mice across Binoc, Contra and Ipsi conditions under grating (s.f. 0.08cpd). Binoc OSI: 870 neurons from 6 Ctrl, 670 neurons from 5 WT injected, 532 neurons from 4 SE injected and 782 neurons from 5 SA injected mice. Contra OSI: 794 neurons from 6 Ctrl, 715 neurons from 5 WT injected, 500 neurons from 4 SE injected and 852 neurons from 5 SA injected mice. Ipsi OSI: 629 neurons from 6 Ctrl, 574 neurons from 5 WT injected, 384 neurons from 4 SE injected and 613 neurons from 5 SA injected mice. Kruskal-Wallis test followed by Tukey’s multiple comparisons test. (N) Boxplots showing Contra and Ipsi correlation under grating (s.f. 0.08cpd) stimuli in Ctrl, WT injected, SE injected and SA injected mice. 301 neurons from 6 Ctrl, 253 neurons from 5 WT injected, 159 neurons from 4 SE injected and 275 neurons from 5 SA injected mice. Kruskal-Wallis test followed by Tukey’s multiple comparisons test. (O) Boxplots showing difference in preferred orientation under grating (s.f. 0.08cpd) stimuli in Ctrl, WT injected, SE injected and SA injected mice. 301 neurons from 6 Ctrl, 253 neurons from 5 WT injected, 159 neurons from 4 SE injected and 275 neurons from 5 SA injected mice. Kruskal-Wallis test followed by Tukey’s multiple comparisons test. (P) Boxplots showing Contra and Ipsi correlation under natural scene stimuli in Ctrl, WT injected, SE injected and SA injected mice. 529 neurons from 6 Ctrl, 185 neurons from 5 WT injected, 428 neurons from 4 SE injected and 443 neurons from 5 SA injected mice. Kruskal-Wallis test followed by Tukey’s multiple comparisons test. Data are presented as mean ± SEM, * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001.

Article Snippet: Mice were head-fixed, and the left V1 was imaged by a two-photon microscope (Scientifica, U.K.) with a resonant scanning module controlled by ScanImage built-in Matlab software.

Techniques: MANN-WHITNEY, Two Tailed Test, Control, Microscopy, Injection

Journal: Nature Communications

Article Title: Distinct roles of cortical layer 5 subtypes in associative learning

doi: 10.1038/s41467-026-68307-5

Figure Lengend Snippet: a Schematic showing the genetic and viral intersectional strategy, targeting L5 IT neurons in Tlx3-Cre mice and ET neurons in Sim1-Cre mice. b Images from coronal sections from S1 of a Tlx3-Cre mouse (top) and Sim1-Cre mouse (bottom) injected with AAV-Flex-GFP, showing the injection site in S1 and labeled axons in the secondary somatosensory cortex (S2), corpus callosum (CC), striatum (Str), and posterior medial thalamic nucleus (POm). c Two-photon calcium imaging from Tlx3-Cre and Sim1-Cre mice injected with AAV-Flex-jGCaMP8m in S1. Inset, example FOVs showing GCaMP-expressing IT neuronal dendrites in a Tlx3-Cre mouse (left) and ET dendrites in a Sim1-Cre mouse (right). d Heatmaps of IT neuronal responses to whisker stimuli (left, CS+; middle, CS−) and to water reward (right) in naïve mice ( n = 5 mice). ROIs in each heatmap are sorted by their mean response amplitudes within 1.5 s of stimulus or reward onset. e Left, pie-chart showing the fraction of IT neurons responding to each CS, and their average responses. Dashed line, stimulus onset and offset. Right, pie-chart showing the fraction of IT neurons responding to rewards, and their average responses. Dashed line, reward onset. f, g Same as ( d , e ) but for ET neurons ( n = 6 mice). h SVM decoder performance in classifying the CS identity (CS+ or CS−) based on IT neuronal responses (purple; n = 5 mice) or ET neuronal responses (orange; n = 6 mice; two-sided Student’s t -test). Data were presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Imaging from behaving mice was performed with a resonant-scanning two-photon microscope (Thorlabs) equipped with GaAsP photomultiplier tubes (Hamamatsu). jGCaMP8m was excited at 940 nm with a Ti:Sapphire laser (Mai Tai eHP DeepSee, Spectra-Physics) and imaged through a 16×, 0.8 NA water-immersion objective (Nikon).

Techniques: Injection, Labeling, Imaging, Expressing, Whisker Assay

Journal: bioRxiv

Article Title: A Three-dimensional Analytical Framework for Retinal Microvasculature Reveals Layer-associated Vulnerability in Development and Neovascular Remodeling

doi: 10.64898/2026.03.16.711909

Figure Lengend Snippet: (a) Retinal vasculature segmentation. High-resolution two-photon excitation microscopy Z-stacks spanning the choroid (Ch) and the neurosensory retina (Rt) were acquired. The volumetric data were segmented to generate three-dimensional representations for analysis. (b) Whole-structure vascular quantification. Global analyses of vessel morphology and network architecture were performed across the entire vascular volume or within user-defined regions of interest. Quantified parameters include vessel length, diameter, branching angle, tortuosity, vessel volume density, branching topology, network connectivity, and fractal dimension. (c) Plexus-resolved analyses. The retinal vasculature was separated into the superficial layer (SL), middle layer (ML), and deep layer (DL) vascular plexuses. Plexus-specific quantification was then performed to characterize layer-dependent vascular architecture. (d) Inter-plexus connectivity and parallelism. Cross-plexus relationships were analyzed by characterizing direct inter-plexus connections between SL, ML, and DL and assessing deviation in inter-plexus parallelism.

Article Snippet: Labeled eye specimens were mounted in 1% agarose and imaged using a custom two-photon excited fluorescence microscope (modified Bergamo II, ThorLabs) using 1238 nm excitation light (Insight X3, SpectraPhysics) and a high numerical aperture water-dipping lens (Nikon 25× Apo LWD, 1.10 NA, 2.0 mm WD; THN25X-APO-MP1300) to acquire image data with subcellular resolution.

Techniques: Microscopy