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Metamorph 7.6.5.0 image analysis software

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MetaMorph 7.6.5.0 is an image analysis software. It is designed to process and analyze digital images obtained from various imaging techniques.

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Lab products found in correlation

Eclipse ti s microscope, by Nikon (8 mentions) Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions) Fluorescent mounting medium, by Agilent Technologies (1 mentions) α btx, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Sc 515751, by Santa Cruz Biotechnology (1 mentions) Hoechst 33258, by Thermo Fisher Scientific (1 mentions)

8 protocols using metamorph 7.6.5.0 image analysis software

1

Immunofluorescence Staining of Glial Markers

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Cells were fixed in 4% paraformaldehyde (ChemCruz) for 15 min and washed in PBS. After cell permeabilization with 0.2% Triton X-in PBS, cells were blocked (1% BSA in PBS) for 1 h, at RT, and incubated overnight at 4 °C with Iba1 and GFAP antibody (1:700; Wako) diluted in PBS with 0.1% BSA. Cells were washed three times in PBS and incubated with the fluorophore-conjugated secondary antibodies (1:2000, Catalog#A11012, A-21202, Invitrogen) for 45 min. After three washes in PBS, nuclei were counterstained with Hoechst 33258 for 5 min and cells washed in PBS. Coverslips were mounted on dishes using the Fluorescent Mounting Medium (DakoCytomation). Images were digitized using a CoolSNAP camera (Photometrics) coupled to an ECLIPSE Ti-S microscope (Nikon) and processed using MetaMorph 7.6.5.0 image analysis software (Molecular Device).
Palomba N.P., Martinello K., Cocozza G., Casciato S., Mascia A., Di Gennaro G., Morace R., Esposito V., Wulff H., Limatola C, & Fucile S. (2021). ATP-evoked intracellular Ca2+ transients shape the ionic permeability of human microglia from epileptic temporal cortex. Journal of Neuroinflammation, 18, 44.
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2

Neuromuscular Junction Fragmentation Analysis

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Mice treated as above were deeply anaesthetized before being transcardially perfused with PBS. Tibialis anterior muscles were dissected out and snap-frozen in cooled isopentane. Muscles were gently stretched on a Sylgard coated Petri dish and fixed with paraformaldehyde 4% in PBS for 60 min at RT. For AChR staining, the fixed muscles were incubated with rhodamine-conjugated alpha-bungarotoxin (6 mg/ml, Molecular Probes, B13423) in FBS-supplemented DMEM 20% for 50 min at 37°C. Muscle were extensively washed with FBS-supplemented DMEM 20%, then with PBS. Twenty-micrometer serial longitudinal cryosections were collected on poly-lysine coated objective slides (VWR International, Radnor USA) for the quantitative evaluation of NMJ status. Images were digitized using a CoolSNAP camera (Photometrics) coupled to an ECLIPSE Ti-S microscope (Nikon) and processed using MetaMorph 7.6.5.0 image analysis software (Molecular Device). Images of acetylcholine receptors (AChRs) immuno-stained with α-BTX were used for the quantification of NMJ fragmentation. Fragmentation of NMJ was defined as several AChR clusters in small islands with round shape. Average numbers of AChR fragments per NMJ were evaluated. For the quantitative evaluation, about 130 NMJs for each group were analyzed.
Cocozza G., di Castro M.A., Carbonari L., Grimaldi A., Antonangeli F., Garofalo S., Porzia A., Madonna M., Mainiero F., Santoni A., Grassi F., Wulff H., D’Alessandro G, & Limatola C. (2018). Ca2+-activated K+ channels modulate microglia affecting motor neuron survival in hSOD1G93A mice. Brain, behavior, and immunity, 73, 584-595.
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3

Fluorescence-based Cell Quantification

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Images were digitized using a CoolSNAP camera (Photometrics) coupled to an ECLIPSE Ti-S microscope (Nikon) and processed using MetaMorph 7.6.5.0 image analysis software (Molecular Devices). Brain slices were scanned by consecutive fields of vision (×10 objective lens) to build a single image per section. The percentage of positive cells was measured as the ratio of the area occupied by fluorescent cells versus the total tumor area (by converting pixels to square millimeters). For comparison between different treatments, at least 12 coronal sections per brain around the point of injection were analyzed.
Mormino A., Bernardini G., Cocozza G., Corbi N., Passananti C., Santoni A., Limatola C, & Garofalo S. (2021). Enriched Environment Cues Suggest a New Strategy to Counteract Glioma: Engineered rAAV2-IL-15 Microglia Modulate the Tumor Microenvironment. Frontiers in Immunology, 12, 730128.
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4

Endplate Morphology Evaluation in Tibialis Anterior

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To evaluate the shape of the endplate, tibialis anterior muscles were fixed in 4% paraformaldehyde and snap frozen in isopentane at −80 °C. Longitudinal cryostat sections (20 μm) were incubated with rhodamine-conjugated alpha-bungarotoxin (α-BTX; Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA; B13423; 6 mg/mL) in DMEM plus 20% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA) for 50 min at 37 °C. Thereafter, slices were extensively washed with the same medium, then with PBS. Images of AChRs stained by α-BTX were obtained using a system integrated by Crisel Instruments (Rome, Italy), comprising a CoolSNAP camera (Photometrics, Tucson, AZ, USA) coupled to an ECLIPSE Ti-S microscope (Nikon, Tokio Japan) and processed using MetaMorph 7.6.5.0 image analysis software (Molecular Device, San Jose, CA, USA), after background subtraction. A deconvolution algorithm was applied on z-stacked images. Junctions were defined as continuous if AChRs formed a continuous ribbon with 0–2 interruptions, or fragmented if 3 or more AChR clusters were present [13 (link)]. 50 to 110 endplates per mouse were scored.
Morotti M., Gaeta A., Limatola C., Catalano M., Di Castro M.A, & Grassi F. (2022). Early Developmental Changes of Muscle Acetylcholine Receptors Are Little Influenced by Dystrophin Absence in mdx Mouse. Life, 12(11), 1861.
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5

Quantifying Tumor Cell Fluorescence

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Images were digitized using a CoolSNAP camera (Photometrics) coupled to an ECLIPSE Ti-S microscope (Nikon) and processed using MetaMorph 7.6.5.0 image analysis software (Molecular Device). Slices were scanned by consecutive fields of vision (x 10 objective lens) to build a single image per section. Data were expressed as area occupied by fluorescent cells versus total tumor area (by converting pixel to mm [Graeber et al., 2002 (link)]). For comparison between different treatments, at least 12 coronal sections per brain around the point of injection were analyzed.
Garofalo S., Porzia A., Mainiero F., Di Angelantonio S., Cortese B., Basilico B., Pagani F., Cignitti G., Chece G., Maggio R., Tremblay M.E., Savage J., Bisht K., Esposito V., Bernardini G., Seyfried T., Mieczkowski J., Stepniak K., Kaminska B., Santoni A, & Limatola C. (2017). Environmental stimuli shape microglial plasticity in glioma. eLife, 6, e33415.
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6

Quantifying Gli1 Protein Expression in Astrocytes

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GL261 cells (1 × 105/ well) or pure primary astrocytes were plated in 24 well plates on glass coverslip. After 48 h, cells were fixed with paraformaldehyde, permeabilized with 0.2% Triton-X-100, blocked with 1% BSA-PBS and incubated O/N at 4 °C with a mouse monoclonal antibody against mouse Gli1 in 0.1% BSA-PBS (1:200, sc-515,751, Santa Cruz Biotechnology, CA, USA). The specific protein was visualized using a secondary antibody coupled to a fluorescent marker (1:2000 Alexa anti mouse#594 in 0.1%BS-PBS, 1 h at RT). Nuclei were stained with Hoechst 33258 (Molecular Probes, Life Technologies, USA) and examined by fluorescence microscopy. The images were digitized using a CoolSNAP camera (Photometrics) coupled to an ECLIPSE Ti-S microscope (Nikon) and processed using MetaMorph 7.6.5.0 image analysis software (Molecular Device). Immunofluorescence intensity was quantified by the integrated intensity density method on automatic threshold analysis.
D’Alessandro G., Quaglio D., Monaco L., Lauro C., Ghirga F., Ingallina C., De Martino M., Fucile S., Porzia A., Di Castro M.A., Bellato F., Mastrotto F., Mori M., Infante P., Turano P., Salmaso S., Caliceti P., Di Marcotullio L., Botta B., Ghini V, & Limatola C. (2019). 1H-NMR metabolomics reveals the Glabrescione B exacerbation of glycolytic metabolism beside the cell growth inhibitory effect in glioma. Cell Communication and Signaling : CCS, 17, 108.
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7

Image Acquisition and Analysis of Fluorescent Cells

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Images were digitized using a CoolSNAP camera (Photometrics) coupled to an ECLIPSE Ti-S microscope (Nikon) and processed using MetaMorph 7.6.5.0 image analysis software (Molecular Device).
Slices were scanned by consecutive fields of vision (×10 objective lens) to build a single image per section. Data were expressed as area occupied by fluorescent cells versus total area by converting pixel to mm. For comparison between different treatments, at least 12 coronal sections per lumbar spinal cord or brain were analysed.
Garofalo S., Cocozza G., Porzia A., Inghilleri M., Raspa M., Scavizzi F., Aronica E., Bernardini G., Peng L., Ransohoff R.M., Santoni A, & Limatola C. (2020). Natural killer cells modulate motor neuron-immune cell cross talk in models of Amyotrophic Lateral Sclerosis. Nature Communications, 11, 1773.
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8

Quantitative Analysis of Fluorescent Cells

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Images were digitized using a CoolSNAP camera (Photometrics) coupled to an ECLIPSE Ti‐S microscope (Nikon) and processed using MetaMorph 7.6.5.0 image analysis software (Molecular Devices). Brain slices were scanned by consecutive fields of vision (10X objective lens) to build a single image per section. The percentage of positive cells was measured as the ratio of the area occupied by fluorescent cells versus the total tumor area (by converting pixels to square millimeters). For comparison between different treatments, at least 12 coronal sections per brain around the point of injection were analyzed.
Mormino A., Cocozza G., Fontemaggi G., Valente S., Esposito V., Santoro A., Bernardini G., Santoni A., Fazi F., Mai A., Limatola C, & Garofalo S. (2021). Histone‐deacetylase 8 drives the immune response and the growth of glioma. Glia, 69(11), 2682-2698.
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