Single-particle tracking photoactivated localization microscopy of MCF and MDCK cells transfected with Cav1-mEos2 was performed on the Roper Scientific total internal reflection fluorescence (TIRF) microscope equipped with an iLas2 double-laser illuminator (Roper Technologies), a CFI Apo TIRF 100× (1.49 NA) objective (Nikon), and an Evolve512 delta EMCCD camera (Photometrics). Images were acquired using Metamorph software (version 7.78; Molecular Devices) at 50 Hz, and 16,000 frames were acquired per cell. A 405-nm laser was used to photoconvert mEos2, with simultaneous 561-nm exposure to excite the photoconverted mEos2. For stochastic photoconversion of mEos2 molecules, a low amount (3–5%) of 405-nm laser and 75–80% of 561-nm laser was used. Data analysis was performed as previously described (Bademosi et al., 2017 (link); Kasula et al., 2016 (link)) using PALM-Tracer, a plugin in Metamorph software (Molecular Devices).
Metamorph software
Manufactured by Molecular Devices
Sourced in United States, Japan, Germany, United Kingdom, Canada, France, Azerbaijan
MetaMorph software is a comprehensive image analysis platform designed for life science research. It provides advanced tools for acquiring, processing, and analyzing digital images from a variety of microscopy techniques. The software's core function is to enable researchers to quantify and extract meaningful data from their experimental images.
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Lab products found in correlation
Axio observer z1, by Zeiss (29 mentions)
Axiovert 200m, by Zeiss (20 mentions)
Lsm 700, by Zeiss (19 mentions)
Csu x1, by Yokogawa (17 mentions)
Matlab, by MathWorks (15 mentions)
Eclipse ti, by Nikon (15 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (15 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (14 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (13 mentions)
Ix81 microscope, by Olympus (13 mentions)
Lsm 710 confocal microscope, by Zeiss (13 mentions)
Ix71 microscope, by Olympus (12 mentions)
Orca flash 4, by Hamamatsu Photonics (11 mentions)
Lsm 510, by Zeiss (11 mentions)
Dmi6000b, by Leica camera (10 mentions)
Triton x 100, by Merck Group (10 mentions)
Eclipse ti e, by Nikon (10 mentions)
Lsm 780, by Zeiss (10 mentions)
Lsm 880, by Zeiss (10 mentions)
Lsm 780 confocal microscope, by Zeiss (10 mentions)
1 450 protocols using metamorph software
1
Single-Particle Tracking of Caveolins in Cells
2
Imaging Zebrafish Spinal Motor Neurons
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We limited our study to CaP motor neurons within a 4-somite window around the cloaca in order to avoid morphological and functional variability that arise between cell types and along the rostro-caudal developmental wave.
Imaging was performed on a Roper confocal spinning disk head mounted on a Zeiss upright microscope, and acquisitions were done with a CoolSNAP HQ2 CDD camera (Photometrics, USA) through the MetaMorph software (Molecular Devices, USA). Embryos were anesthetized using 0.02% tricaine (MS-222, Sigma) diluted in egg water and embedded in 1% low melting-point agarose in a glass-bottom cell tissue culture dish (Fluorodish, World Precision Instruments, USA). Acquisitions were done using water immersion long working distance lenses, at 40x magnification (W DIC PL APO VIS-IR; 421,462–9900) for z-stack images of the whole tectum and at 63x magnification (W PL APO VIS-IR (421480–9900) for single plane time-lapse imaging of linear axonal segments, and for filopodia imaging. Acquisitions were done using the MetaMorph software (Molecular Devices) and resolution in z was set at 1um for stacks. Images were assembled and analyzed in ImageJ (NIH). 6dpf z- stacks taken in two frames were stitched together using the pairwise stitching function of the Stitching plugin [46 (link)].
Imaging was performed on a Roper confocal spinning disk head mounted on a Zeiss upright microscope, and acquisitions were done with a CoolSNAP HQ2 CDD camera (Photometrics, USA) through the MetaMorph software (Molecular Devices, USA). Embryos were anesthetized using 0.02% tricaine (MS-222, Sigma) diluted in egg water and embedded in 1% low melting-point agarose in a glass-bottom cell tissue culture dish (Fluorodish, World Precision Instruments, USA). Acquisitions were done using water immersion long working distance lenses, at 40x magnification (W DIC PL APO VIS-IR; 421,462–9900) for z-stack images of the whole tectum and at 63x magnification (W PL APO VIS-IR (421480–9900) for single plane time-lapse imaging of linear axonal segments, and for filopodia imaging. Acquisitions were done using the MetaMorph software (Molecular Devices) and resolution in z was set at 1um for stacks. Images were assembled and analyzed in ImageJ (NIH). 6dpf z- stacks taken in two frames were stitched together using the pairwise stitching function of the Stitching plugin [46 (link)].
3
Liver and Lung Tissue Analysis
4
Visualizing Cellular Redox State Changes
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RoGFP was expressed in primary fibroblasts using modified pEGFP-N1 (RRID:Addgene_38120) as the expression vector and JetPei as the transfection reagent. After the cells were incubated in culture medium treated with or without HU or APH for 72 h at 37 °C, the cells were washed twice with Hanks’ balanced salt solution. For pEGFP-N1/roGFP1, the cells were imaged on a Zeiss Observer Z1 microscope with a Hamamatsu ORCA Flash 4LT camera. Images were acquired using MetaMorph software (Molecular Devices). For dual excitation ratio imaging, excitation filters at wavelengths of 400 nm and 488 nm were used, and an emission filter at a wavelength of 535 nm was used. The fluorescence excitation ratio was obtained by dividing the intensities of the cells using excitation filters at 400 nm and 488 nm.
For pEGFP-N1/roGFP-NLS (nuclear localization), the cells were incubated with DAPI (1 μg/ml) and imaged using a microscope. The images were captured using the 63x oil immersion objective of a motorized Axio Imager Z2 epifluorescence microscope (Carl Zeiss) equipped with a high-sensitivity cooled interline CCD camera (Cool SNAP HQ2; Roper Scientific) and a PIEZO stage (Physik Instrumente). Images were acquired using MetaMorph software (Molecular Devices). In each case, 300–500 cells were analyzed per condition.
For pEGFP-N1/roGFP-NLS (nuclear localization), the cells were incubated with DAPI (1 μg/ml) and imaged using a microscope. The images were captured using the 63x oil immersion objective of a motorized Axio Imager Z2 epifluorescence microscope (Carl Zeiss) equipped with a high-sensitivity cooled interline CCD camera (Cool SNAP HQ2; Roper Scientific) and a PIEZO stage (Physik Instrumente). Images were acquired using MetaMorph software (Molecular Devices). In each case, 300–500 cells were analyzed per condition.
5
Live-cell Fluorescence Imaging of AChR
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Fluorescence imaging of live and fixed cell cultures was performed on an inverted fluorescence microscope (IX70, Olympus). Digital images were acquired using a cooled CCD camera (ORCA II, Hamamatsu) that was controlled using MetaMorph software (Molecular Devices). Time-lapse images of AChR vesicles were captured at an interval of 500 ms for 3 min or 3 s for 30 min under the control of an optical filter changer (Lambda 10-2; Sutter Instrument). Data were analyzed using ImageJ (National Institute of Health) and MetaMorph software (Molecular Devices). Data are shown as mean ± SEM and the statistical significance of differences between the control and experimental groups was assessed using Student’s t test.
6
Single Particle Tracking PALM of Cav1-mEos2
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Single particle tracking photoactivated localization microscopy (sptPALM) of MCF and MDCK cells transfected with Cav1-mEos2 was carried out on the Roper Scientific TIRF microscope equipped with an iLas 2 double laser illuminator (Roper technologies), a CFI Apo TRIF 100×
(1.49-NA) objective (Nikon) and an Evolve512 delta EMCCD camera (Photometrics). Images were acquired using Metamorph software (version 7.78; Molecular Devices) at 50 Hz and 16000 frames were acquired per cell. A 405 nm laser was used to photoconvert mEos2, with simultaneous 561 nm exposure to excite the photoconverted mEos2. For stochastic photoconversion of mEos2 molecules, low amount (3-5%) of 405 nm laser and 75-80% of 561 nm laser was used. Data analysis was carried out as previously described 47,48 using PALM-Tracer, a plugin in Metamorph software (Molecular Devices).
(1.49-NA) objective (Nikon) and an Evolve512 delta EMCCD camera (Photometrics). Images were acquired using Metamorph software (version 7.78; Molecular Devices) at 50 Hz and 16000 frames were acquired per cell. A 405 nm laser was used to photoconvert mEos2, with simultaneous 561 nm exposure to excite the photoconverted mEos2. For stochastic photoconversion of mEos2 molecules, low amount (3-5%) of 405 nm laser and 75-80% of 561 nm laser was used. Data analysis was carried out as previously described 47,48 using PALM-Tracer, a plugin in Metamorph software (Molecular Devices).
7
Live-cell imaging of mitotic progression
8
Quantifying Bone Tumor Burden and PTHrP Expression
9
Neurite Morphology Analysis of Dopaminergic Neurons
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For morphological analysis, the TH-immunostained images were analyzed with MetaMorph software (Molecular Devices, CA, USA). A morphological analysis of DN was performed as previously described [19 (link)]. Briefly, neurite length and number of branches were measured from 100 TH-positive cells in 20 non-overlapping TH-immunostained images selected randomly from three experiments using the Neurite Outgrowth and Multi-Wavelength Cell Scoring module of MetaMorph software (Molecular Devices). Next, the cells were classified into 4 stages based on neurite length and cell diameter as previously described [19 (link)].
10
Effects of NAMPT and FK866 on hPASMC Apoptosis
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Example 9
Materials and Methods
hPASMCs were cultured on a coverslip in a 6-well cell culture plate. H2O2 (100 μM, Sigma, Cat. #ab66110) was used as an apoptosis stimulus for hPASMCs.
When cell confluence reached 95%, cells were starved for 3 hrs and stimulated by H2O2, FK866 (10 μM), H2O2+rhNAMPT (20 μg/ml) or H2O2+rhNAMPT (20 μg/ml) with FK866 (10 μM) for 24 hrs. Cell apoptosis was examined using an in situ BrdU-Red DNA fragmentation (TUNEL) kit. The cells on the coverslips were examined using a Nikon Eclipse E800 fluorescence microscope, and the images were processed by MetaMorph software (Molecular Devices, Inc.). Approximately ten images were taken from each condition, and over 500 cells were counted according to DAPI staining.
Results
The data from the TUNEL assay revealed that the NAMPT protein inhibits hPASMC apoptosis, and FK866 promotes cell apoptosis. The number of TUNEL positive cells was quantified, as shown in
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