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colocalization and integrated morphometric analysis applications  (universal imaging inc)

 
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    universal imaging inc colocalization and integrated morphometric analysis applications
    Impaired mitochondrial fusion in the soma and motor axons of G93A SOD1 motor neurons. A, Cell bodies from non-transgenic control and G93A SOD1 motor neurons containing mitoDendra-labeled mitochondria before (−1 min) and after (0 min) photo-activation. Subsets of mitochondria (green and red fluorescence) were followed over time (10, 40, 70, and 100 min) by live imaging microscopy. Note that the appearance of yellow fluorescence, as a result of the mixing between green and red mitochondria (i.e. fusion, indicated by arrows), was delayed in mutant SOD1 motor neurons. Scale bar, 10 μm. B, Fusion rates were obtained in single optical z-sections by measuring the <t>colocalization</t> (in %) of red over green fluorescent mitochondria at the indicated time points. The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. C, In the inset, example of a time-lapse recording of axonal mitochondria (numbers indicate time in min) showing a fusion event (arrow). Scale bar, 5 μm. The graph shows the analysis of fusion (% of fusion events of total moving mitochondria) in control and G93A SOD1 motor axons. n (axons) = 24 non-transgenic, 28 WT and 16 G93A axonal segments. *P<0.05 versus non-transgenic. D, Time-lapse microscopy of mitochondrial transport in the soma of non-transgenic and G93A SOD1 motor neurons. All mitochondria in a ROI of the soma were photo-converted, while only non-photo-converted (green fluorescent) mitochondria were followed over time. Note a decrease of mobile mitochondria towards the ROI in mutant SOD1 motor neurons compared to controls. Scale bar, 10 μm. E, Analysis of the transport of green mitochondria over the photo-activated area (no green mitochondria present). The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. F, Analysis of the transport of red mitochondria over the non-photo-activated area (no red mitochondria present). The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. All data obtained from 3–5 independent experiments. The error bars represent ± SE.
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    Images

    1) Product Images from "Mitochondrial Dynamics and Bioenergetic Dysfunction Is Associated with Synaptic Alterations in Mutant SOD1 Motor Neurons"

    Article Title: Mitochondrial Dynamics and Bioenergetic Dysfunction Is Associated with Synaptic Alterations in Mutant SOD1 Motor Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1233-11.2012

    Impaired mitochondrial fusion in the soma and motor axons of G93A SOD1 motor neurons. A, Cell bodies from non-transgenic control and G93A SOD1 motor neurons containing mitoDendra-labeled mitochondria before (−1 min) and after (0 min) photo-activation. Subsets of mitochondria (green and red fluorescence) were followed over time (10, 40, 70, and 100 min) by live imaging microscopy. Note that the appearance of yellow fluorescence, as a result of the mixing between green and red mitochondria (i.e. fusion, indicated by arrows), was delayed in mutant SOD1 motor neurons. Scale bar, 10 μm. B, Fusion rates were obtained in single optical z-sections by measuring the colocalization (in %) of red over green fluorescent mitochondria at the indicated time points. The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. C, In the inset, example of a time-lapse recording of axonal mitochondria (numbers indicate time in min) showing a fusion event (arrow). Scale bar, 5 μm. The graph shows the analysis of fusion (% of fusion events of total moving mitochondria) in control and G93A SOD1 motor axons. n (axons) = 24 non-transgenic, 28 WT and 16 G93A axonal segments. *P<0.05 versus non-transgenic. D, Time-lapse microscopy of mitochondrial transport in the soma of non-transgenic and G93A SOD1 motor neurons. All mitochondria in a ROI of the soma were photo-converted, while only non-photo-converted (green fluorescent) mitochondria were followed over time. Note a decrease of mobile mitochondria towards the ROI in mutant SOD1 motor neurons compared to controls. Scale bar, 10 μm. E, Analysis of the transport of green mitochondria over the photo-activated area (no green mitochondria present). The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. F, Analysis of the transport of red mitochondria over the non-photo-activated area (no red mitochondria present). The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. All data obtained from 3–5 independent experiments. The error bars represent ± SE.
    Figure Legend Snippet: Impaired mitochondrial fusion in the soma and motor axons of G93A SOD1 motor neurons. A, Cell bodies from non-transgenic control and G93A SOD1 motor neurons containing mitoDendra-labeled mitochondria before (−1 min) and after (0 min) photo-activation. Subsets of mitochondria (green and red fluorescence) were followed over time (10, 40, 70, and 100 min) by live imaging microscopy. Note that the appearance of yellow fluorescence, as a result of the mixing between green and red mitochondria (i.e. fusion, indicated by arrows), was delayed in mutant SOD1 motor neurons. Scale bar, 10 μm. B, Fusion rates were obtained in single optical z-sections by measuring the colocalization (in %) of red over green fluorescent mitochondria at the indicated time points. The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. C, In the inset, example of a time-lapse recording of axonal mitochondria (numbers indicate time in min) showing a fusion event (arrow). Scale bar, 5 μm. The graph shows the analysis of fusion (% of fusion events of total moving mitochondria) in control and G93A SOD1 motor axons. n (axons) = 24 non-transgenic, 28 WT and 16 G93A axonal segments. *P<0.05 versus non-transgenic. D, Time-lapse microscopy of mitochondrial transport in the soma of non-transgenic and G93A SOD1 motor neurons. All mitochondria in a ROI of the soma were photo-converted, while only non-photo-converted (green fluorescent) mitochondria were followed over time. Note a decrease of mobile mitochondria towards the ROI in mutant SOD1 motor neurons compared to controls. Scale bar, 10 μm. E, Analysis of the transport of green mitochondria over the photo-activated area (no green mitochondria present). The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. F, Analysis of the transport of red mitochondria over the non-photo-activated area (no red mitochondria present). The correlation coefficient (r) for each group is indicated. n (somas) = 8 non-transgenic, and 7 G93A. *P<0.05 by ANOVA with repeated measurements. All data obtained from 3–5 independent experiments. The error bars represent ± SE.

    Techniques Used: Transgenic Assay, Control, Labeling, Activation Assay, Fluorescence, Imaging, Microscopy, Mutagenesis, Time-lapse Microscopy



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    universal imaging inc computer-based morphometric analysis image 1
    Third harmonic generation (THG) brain imaging in groups of Danionella dracula 3 to 90 dpf. A . Left: Side view of a male. Scale bar indicates 3mm. Right: Head-on view of male extending its hypertrophied lower jaw during mid-lunge at another male. B . Dorsal views of fish imaged from 90-3 dpf (left to right). Scale bar left indicates 500 μm for all images. Top insert at 90 dpf is zoom-in on fangs (indicated by arrows; dashed outline on left fang), which are first apparent in 90 dpf males along with hypertrophied lower jaw. C . Schematic of imaging apparatus (see Materials and Methods). Left: profile drawing of animal placement under objective. Right: dorsal view of adult placed in putty holder with perfusion mouthpiece. Fish imaged less than 60 dpf were imaged using an alternative setup than pictured (see Materials and Methods, Fish stabilization for imaging). D. 30, 21, and 14 dpf example fish depicting changes in swim bladder (SB) separation into anterior and posterior chambers. Scale bar indicates 500 μm for all images. E . Schematic of representative D. dracula brain; illustrations based on pictures of dissected whole brains. Brain regions of <t>morphometric</t> analysis indicated in lateral and dorsal views; green, OB (olfactory bulb); blue, telencephalon; orange, TL (torus longitudinalis); pink, TeO (optic tectum). Insert indicates horizontal slice in midbrain depicting TeO (pink), periventricular gray zone (PGZ) of TeO (purple, included in TeO measurement), depicted for indication of boundary between TeO and TS (yellow, torus semicircularis). F . Scatterplot of age in dpf of animals in this study and gross brain volume in μm 3 . Boxplots indicate median, upper and lower quartiles and 1.5× interquartile range for each Age. Gross brain volume calculation is described in and Materials and Methods. Different ages depicted by different colored dots as shown in legend: red, 5 dpf; yellow, 14 dpf; green, 21 dpf; teal, 30 dpf; blue, 60 dpf; purple, 90 dpf. As is described in Materials and Methods, we performed the best fitting quadratic regression (F 2,30= 120.8, R 2 = 0.8821, p<0.001) on log-transformed data to normalize the residuals. The green line displayed here represents the quadratic regression line based on the original (non-transformed) data, for best visual clarity and understanding.
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    Third harmonic generation (THG) brain imaging in groups of Danionella dracula 3 to 90 dpf. A . Left: Side view of a male. Scale bar indicates 3mm. Right: Head-on view of male extending its hypertrophied lower jaw during mid-lunge at another male. B . Dorsal views of fish imaged from 90-3 dpf (left to right). Scale bar left indicates 500 μm for all images. Top insert at 90 dpf is zoom-in on fangs (indicated by arrows; dashed outline on left fang), which are first apparent in 90 dpf males along with hypertrophied lower jaw. C . Schematic of imaging apparatus (see Materials and Methods). Left: profile drawing of animal placement under objective. Right: dorsal view of adult placed in putty holder with perfusion mouthpiece. Fish imaged less than 60 dpf were imaged using an alternative setup than pictured (see Materials and Methods, Fish stabilization for imaging). D. 30, 21, and 14 dpf example fish depicting changes in swim bladder (SB) separation into anterior and posterior chambers. Scale bar indicates 500 μm for all images. E . Schematic of representative D. dracula brain; illustrations based on pictures of dissected whole brains. Brain regions of morphometric analysis indicated in lateral and dorsal views; green, OB (olfactory bulb); blue, telencephalon; orange, TL (torus longitudinalis); pink, TeO (optic tectum). Insert indicates horizontal slice in midbrain depicting TeO (pink), periventricular gray zone (PGZ) of TeO (purple, included in TeO measurement), depicted for indication of boundary between TeO and TS (yellow, torus semicircularis). F . Scatterplot of age in dpf of animals in this study and gross brain volume in μm 3 . Boxplots indicate median, upper and lower quartiles and 1.5× interquartile range for each Age. Gross brain volume calculation is described in and Materials and Methods. Different ages depicted by different colored dots as shown in legend: red, 5 dpf; yellow, 14 dpf; green, 21 dpf; teal, 30 dpf; blue, 60 dpf; purple, 90 dpf. As is described in Materials and Methods, we performed the best fitting quadratic regression (F 2,30= 120.8, R 2 = 0.8821, p<0.001) on log-transformed data to normalize the residuals. The green line displayed here represents the quadratic regression line based on the original (non-transformed) data, for best visual clarity and understanding.

    Journal: bioRxiv

    Article Title: Label-free multiphoton imaging reveals volumetric shifts across development in sensory-related brain regions of a miniature transparent vertebrate

    doi: 10.1101/2024.07.18.604134

    Figure Lengend Snippet: Third harmonic generation (THG) brain imaging in groups of Danionella dracula 3 to 90 dpf. A . Left: Side view of a male. Scale bar indicates 3mm. Right: Head-on view of male extending its hypertrophied lower jaw during mid-lunge at another male. B . Dorsal views of fish imaged from 90-3 dpf (left to right). Scale bar left indicates 500 μm for all images. Top insert at 90 dpf is zoom-in on fangs (indicated by arrows; dashed outline on left fang), which are first apparent in 90 dpf males along with hypertrophied lower jaw. C . Schematic of imaging apparatus (see Materials and Methods). Left: profile drawing of animal placement under objective. Right: dorsal view of adult placed in putty holder with perfusion mouthpiece. Fish imaged less than 60 dpf were imaged using an alternative setup than pictured (see Materials and Methods, Fish stabilization for imaging). D. 30, 21, and 14 dpf example fish depicting changes in swim bladder (SB) separation into anterior and posterior chambers. Scale bar indicates 500 μm for all images. E . Schematic of representative D. dracula brain; illustrations based on pictures of dissected whole brains. Brain regions of morphometric analysis indicated in lateral and dorsal views; green, OB (olfactory bulb); blue, telencephalon; orange, TL (torus longitudinalis); pink, TeO (optic tectum). Insert indicates horizontal slice in midbrain depicting TeO (pink), periventricular gray zone (PGZ) of TeO (purple, included in TeO measurement), depicted for indication of boundary between TeO and TS (yellow, torus semicircularis). F . Scatterplot of age in dpf of animals in this study and gross brain volume in μm 3 . Boxplots indicate median, upper and lower quartiles and 1.5× interquartile range for each Age. Gross brain volume calculation is described in and Materials and Methods. Different ages depicted by different colored dots as shown in legend: red, 5 dpf; yellow, 14 dpf; green, 21 dpf; teal, 30 dpf; blue, 60 dpf; purple, 90 dpf. As is described in Materials and Methods, we performed the best fitting quadratic regression (F 2,30= 120.8, R 2 = 0.8821, p<0.001) on log-transformed data to normalize the residuals. The green line displayed here represents the quadratic regression line based on the original (non-transformed) data, for best visual clarity and understanding.

    Article Snippet: Morphometric image analysis was performed using the software Imaris (BITPLANE, Oxford Instruments).

    Techniques: Imaging, Transformation Assay