two photon laser scanning microscope  (Scientifica)

Journal: bioRxiv

Article Title: A neurorecording toolkit for longitudinal assessments of transplanted human cortical organoids in vivo

doi: 10.64898/2025.12.20.695690

Figure Lengend Snippet: A. Angiogram generated with OCT imaging two months post-transplantation shown as (top) 500-µm maximum intensity projection along the Z axis (MIP Z ) and (bottom) as 100-µm maximum intensity projection along the X axis (MIP X ). The yellow dashed line indicates xenograft borders with the host cortex. The blue dashed line indicates the image segment corresponding to the bottom MIP X image. Schematic created in BioRender . Abbreviations: m , medial, c , caudal. B. Angiogram generated with two-photon (2P) imaging after intravenous injection of fluorescent Alexa-680 Dextran two months post-transplantation for the same animal as shown in panel A. The yellow dashed line indicates xenograft borders with the host cortex. C. Magnified 500-µm MIP Z angiograms showing capillaries within the xenograft as captured by OCT (top) and 2P microscopy (bottom) demonstrating strong agreement between the two modalities. D. Quantification of capillary density within the xenograft using 1 mm x 1 mm x 500 µm MIP Z OCT images at 1, 2, and 3 months after xenotransplantation. The plot shows the mean ± s.e.m. for three animals. E. Volumetric analysis of xenograft size 1, 2, and 3 months after xenotransplantation. (top) Logarithm-normalized, raw intensity, 100-µm MIP Z OCT images. The yellow dashed line indicates xenograft borders with the host cortex. (bottom) Manually segmented, 3D representations of the xenograft and the corresponding estimate of the xenograft volume.

Article Snippet: Two-photon imaging was performed in awake, head-fixed animals on commercial two-photon laser scanning microscope systems (Bruker Ultima, Bruker Ultima Investigator Plus) with Coherent Chameleon Ultra II or Coherent Chameleon Discovery Ti:Sapphire lasers tuned to 920-950 nm for excitation of GCaMP6s or 8s.

Techniques: Generated, Imaging, Transplantation Assay, Injection, Microscopy

Journal: bioRxiv

Article Title: A neurorecording toolkit for longitudinal assessments of transplanted human cortical organoids in vivo

doi: 10.64898/2025.12.20.695690

Figure Lengend Snippet: A. Two-photon (2P) images from xenografts labelled by either AAV-hSyn-GCaMP8s (top) or LV-hSyn-GCaMP8s (bottom) in live mice one and three months after xenotransplantation. Images were generated from standard deviations of 3-minute time-series recordings. B. Immunohistochemical (IHC) staining for GCaMP-expressing cells with a GFP antibody and human cells with a human nuclear antigen (HNA) antibody. The tissue was isolated three months after xenotransplantation. C. IHC-based quantification of GCaMP labelling density between LV and AAV-labelled xenografts. The bar chart shows mean ± s.e.m. for N = 3 mice per condition. *, P = 0.0002 (two-tailed t-test). D. Representative calcium traces (shown as baseline normalized ΔF/F) recorded with 2P imaging of GCaMP8s-expressing human neurons within the xenograft; hCOs were transduced with AAV (top) or LV (bottom) before transplantation. Animals were recorded while being awake and head-fixed without presentation of external stimuli.

Article Snippet: Two-photon imaging was performed in awake, head-fixed animals on commercial two-photon laser scanning microscope systems (Bruker Ultima, Bruker Ultima Investigator Plus) with Coherent Chameleon Ultra II or Coherent Chameleon Discovery Ti:Sapphire lasers tuned to 920-950 nm for excitation of GCaMP6s or 8s.

Techniques: Generated, Immunohistochemical staining, Immunohistochemistry, Expressing, Isolation, Two Tailed Test, Imaging, Transduction, Transplantation Assay

Journal: bioRxiv

Article Title: A neurorecording toolkit for longitudinal assessments of transplanted human cortical organoids in vivo

doi: 10.64898/2025.12.20.695690

Figure Lengend Snippet: A. Experimental paradigm. For data shown in this figure, hCOs were transduced with LV-hSyn-GCaMP8s and LV-EF1-mScarlet before transplantation; animals were recorded while being awake and head-fixed without presentation of external stimuli. Schematic created with BioRender . B. Representative low-magnification 2P images (maximum intensity projections along the Z axis) of the entire optical window showing vasculature (Alexa 680-Dextran, white), GCaMP8s (green), and mScarlet (red). Abbreviations: m , medial, c , caudal. C. Two-photon images of GCaMP8s-labelled neurons in the xenograft one and three months after xenotransplantation (top) with corresponding Z-scored heat maps of calcium activity during spontaneous activity (middle) and calcium activity traces averaged across all cells (bottom). The red line on the color bar denotes the activity threshold of 10 standard deviations (Z score = 10). D. Longitudinal changes in neuronal activity over three months for each animal and imaging session (left) and averaged for pairwise comparison (right). Activity of individual cells is determined by the percentage of time the calcium trace for a given cell exceeds a threshold of 10 standard deviations (Z score >10). The scatter plot (left) shows linear mixed effects model (LMM; N=5; p<0.001, r=0.452). The scatter plot (right) shows a pairwise comparison (*, p = 0.001) paired t-test. E. Longitudinal changes in event frequency over three months for each animal and imaging session (left) and averaged for pairwise comparison (right). Events for a given cell are defined as a period where the calcium trace exceeds a threshold of 10 standard deviations (Z score >10). Data from 5 animals. The scatter plot (left) shows linear mixed effects model (LMM; N=5; p=0.0058, r=0.468). The scatter plot (right) shows a pairwise comparison (ns = not significant, p=0.088) paired t-test. F. Cell-to-cell correlation of calcium activity calculated as Pearson’s correlation coefficient between individual cells shown in panel C as measurement of synchronicity. G. Longitudinal changes in cell-to-cell synchronicity over three months for each animal and imaging (left) and averaged for pairwise comparison (right). Synchronicity is calculated as average Pearson’s correlation coefficient across all cells for each field of view. Data from 5 animals. The scatter plot (left) shows linear mixed effects model (LMM; p<0.001, r=0.759). The scatter plot (right) shows the pairwise comparison (*, p=0.026) paired t-test. H. Principal component analysis (PCA) computed from individual cells with at least one calcium event during data acquisition. Data was pooled across all imaging trials (5 animals, 144 trials, 3089 cells). Features estimated for each cell include event frequency, rhythmicity (inter-event-interval CV), activity, event length, event height, and synchronicity. PC 1 represents increases in cell activity while PC 2 represents increases in synchronicity and rhythmicity. Zones 1, 2a, and 2b define differential cell activity phenotypes within the dataset. I. PCA plot binned by month after xenotransplantation with centroid shown in red. J. Representative calcium traces for cells within Zones 1, 2a, and 2b.

Article Snippet: Two-photon imaging was performed in awake, head-fixed animals on commercial two-photon laser scanning microscope systems (Bruker Ultima, Bruker Ultima Investigator Plus) with Coherent Chameleon Ultra II or Coherent Chameleon Discovery Ti:Sapphire lasers tuned to 920-950 nm for excitation of GCaMP6s or 8s.

Techniques: Transduction, Transplantation Assay, Activity Assay, Imaging, Comparison

Journal: bioRxiv

Article Title: A neurorecording toolkit for longitudinal assessments of transplanted human cortical organoids in vivo

doi: 10.64898/2025.12.20.695690

Figure Lengend Snippet: A. Experimental paradigm for simultaneous two-photon (2P) calcium imaging and ECoG recordings. In this experiment, hCOs were transduced with AAV7m8 hSyn1-GCaMP6s-p2A-NLS-tdTomato in culture before xenotransplantation into retrosplenial cortex. Animals were recorded while awake and head-fixed without presentation of external stimuli. Schematic created in BioRender . B. Representative brightfield image taken at the end of the implantation surgery of the cranial exposure showing the implanted hCO xenograft and the gMEA. The gMEA is fixed to the glass window covering the exposure with optical-grade glue. Abbreviations: m , medial; c , caudal. C. Overview of the exposure acquired with 2P microscopy after labeling the blood plasma with Alexa 680-Dextran. Xenotransplant boundaries are indicated by the yellow dotted line. Graphene electrodes of the gMEA are highlighted in blue, red, and grey boxes that correspond to their location above host cortex, xenograft, and bone, respectively. The white box highlights the location of the calcium imaging field of view (FOV) for the data shown in panels D-F. D. GCaMP6s-expressing neuron recorded in the FOV shown in panel C. E. Excerpt of recorded calcium activity (shown as ΔF/F) of the neuron shown in panel D corresponding local field potential (LFP) signal recorded at the same time in channel 3 of the gMEA. F. (left) Calcium events detected in the neuron in panel D; events are aligned by calcium event onset. (right) Corresponding LFP signals in channels above the xenograft (channels 3 and 11) and above the host cortex (channels 6, 15, and 16). G. Proportion of graphene electrodes with an impedance <5 MΩ as a function of time in vivo (top) and average impedance of electrodes (with <5 MΩ) as a function of time in vivo (bottom); individual traces from six animals are shown.

Article Snippet: Two-photon imaging was performed in awake, head-fixed animals on commercial two-photon laser scanning microscope systems (Bruker Ultima, Bruker Ultima Investigator Plus) with Coherent Chameleon Ultra II or Coherent Chameleon Discovery Ti:Sapphire lasers tuned to 920-950 nm for excitation of GCaMP6s or 8s.

Techniques: Imaging, Transduction, Microscopy, Labeling, Clinical Proteomics, Expressing, Activity Assay, In Vivo