Following immunostaining, the brain sections were imaged using a spinning disk microscope and labeled NPC cells were counted using optical dissector stereologic technique [41] (link). All fluorescence immunostaining images were collected employing a Perkin-Elmer UltraVIEW VoX high speed spinning disk (Yokogawa CSU-X1) laser confocal microscope and Volocity software. All representative images were taken by Zeiss LSM710 and LSM780 and imported into Adobe Photoshop Version 12.0 (Adobe Systems Incorporated, San Jose, CA). Brightness, contrast, and background were adjusted using the “brightness and contrast” controls.
Ultraview vox
Manufactured by Yokogawa
The UltraVIEW VoX is a high-performance confocal microscopy system designed for live-cell imaging. It provides fast and efficient image acquisition, enabling researchers to capture dynamic cellular processes in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Vt isim, by Abcam (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Sp8 falcon, by Leica (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Airyscan, by Zeiss (2 mentions)
Lsm 780, by Adobe (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Eclipse ti e, by PerkinElmer (1 mentions)
Csu x1, by Yokogawa (1 mentions)
Du 897 bv, by Oxford Instruments (1 mentions)
Csu x1 spinning disc unit, by Oxford Instruments (1 mentions)
Dmi6000b, by Leica (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
μ slide angiogenesis, by Ibidi (1 mentions)
Spinning disc unit, by Hamamatsu Photonics (1 mentions)
C9100 50, by Hamamatsu Photonics (1 mentions)
C9100 13, by Olympus (1 mentions)
Csu x1, by Olympus (1 mentions)
7 protocols using ultraview vox
1
Quantifying Neural Progenitor Cells
2
Live Cell Imaging Protocols
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For the majority of live-imaging experiments, cells were imaged in their culture media (complete DMEM) at 37 °C and 5% CO2. A subset of short term (< 30 min) spinning-disk movies were acquired at 37 °C and ambient CO2. Overnight spinning disk time-lapse experiments as well as all experiments on the LLS microscope were performed in L-15 (Gibco, 11415–064) supplemented with 10% FBS, 100U/ml penicillin and 100mg/mL streptomycin (Sigma). Hippocampal neurons were imaged in Hibernate E (Brain bits). Live imaging experiments were performed on two spinning disk confocal microscopes (SDCM #1: Perkin Elmer UltraVIEW VOX, SDCM#2 Yokogawa CSU-X1), a Zeiss LSM 880 with Airyscan (Zeiss), a VT iSIM (Biovision), a custom grazing-incidence structured illumination microscope (GI-SIM; Lippincott-Schwartz lab), a Leica SP8 Falcon with STED, or a custom Lattice Light Sheet (Janelia AIC).
3
Visualizing Podosome Dynamics in MEF-Src Cells
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An inverted spinning-disk confocal microscope (Perkin-Elmer Ultraview VoX; Yokogawa CSU-X1) with a piezo Z stage was utilized to visualize the podosome dynamics in MEF-Src cells. An electron-multiplying charge-coupled device (EMCCD) camera (Hamamatsu C9100-23B), 100× oil immersion lens (1.45 NA), and acousto-optic tunable filter (AOTF)-controlled solid-state lasers (40–50 mW) were mounted on the microscope body (Nikon Eclipse Ti-E), and Volocity software (Perkin-Elmer) was used to control microscope acquisition. Typically, microscope settings of 100 ms exposure time, 250 EM gain, 200 nm z-step, and 10–20% of laser output were used to acquire z-stack images. An environmental chamber (37°C and 5% CO2) was attached to the microscope body for long-term time-lapse imaging.
4
Live-cell Imaging of Larval Brains
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Seventy-two to one-hundred twenty hours AEL larval brains were dissected in Schneider’s medium (Sigma-Aldrich, S0146) supplemented with 10% BGS (HyClone) and transferred to 50 μL wells (Ibidi, μ-Slide Angiogenesis) for live-cell imaging. For Fig 6 and S4 Fig , live samples were imaged on a Perkin Elmer spinning disk confocal system “Ultra View VoX” with a Yokogawa spinning disc unit and 2 Hamamatsu C9100-50 frame transfer EMCCD cameras. A 63×/1.40 oil immersion objective mounted on a Leica DMI 6000B was used. Live-cell imaging data shown in Figs 2 , 3 and 7 and S2 and S5 Figs was obtained with an Andor revolution spinning disc confocal system, consisting of a Yokogawa CSU-X1 spinning disc unit and 2 Andor iXon3 DU-897-BV EMCCD cameras. Either a 60×/1.4 NA or 100×/1.4 NA oil immersion objective mounted on a Nikon Eclipse Ti microscope was used.
5
Live Cell Imaging Protocols
6
Measuring Nuclear-to-Cytoplasmic Ratio using Confocal Imaging
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Confocal imaging utilized a PerkinElmer UltraVIEW VoX with a Yokogawa CSU-X1 spinning disk head, a 100× 1.46 NA Olympus Plan Apo oil objective, and CCD (ORCA-R2) and EMCCD (C9100-13) cameras. All confocal microscopy was performed at room temperature (22–23°C). GFP/mCherry images were taken using a 488-nm laser (for GFP) or a 561-nm laser (for mCherry), with alternating excitation. Images were collected using the Volocity imaging software.
To measure the intensity of NLS-GFP, sum projections of the entire z-stack were created using ImageJ. After background subtraction, a region of interest (ROI) was manually drawn around each nucleus and the integrated fluorescence intensity was divided by the area of the ROI. This ROI was then used to measure the integrated fluorescence intensity of the cytoplasm of that cell. The integrated fluorescence intensity of the nucleus over the cytoplasm gave a N:C ratio for each cell. The average of this ratio for 100 cells was determined.
To measure the intensity of NLS-GFP, sum projections of the entire z-stack were created using ImageJ. After background subtraction, a region of interest (ROI) was manually drawn around each nucleus and the integrated fluorescence intensity was divided by the area of the ROI. This ROI was then used to measure the integrated fluorescence intensity of the cytoplasm of that cell. The integrated fluorescence intensity of the nucleus over the cytoplasm gave a N:C ratio for each cell. The average of this ratio for 100 cells was determined.
7
Multimodal Microscopic Imaging of Microcapsules
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For the light microscopy imaging of the microcapsules, YS2-H optical, Nikon (Tokyo, Japan) was used, while for the scanning electron microscopy, and confocal imaging as well as the surface composition measurements Zeiss Neon 40EsB FIBSEM (Tescan, Brno, Czech Republic), UltraVIEW Vox, Perkin Elmer (Waltham, MA, USA), and Oxford Instruments, Aztec X-Act (Abingdon, U.K) were used as per our well-established procedures [32 (link),33 (link),34 (link)]. In brief, for the YS2-H optical imaging, dry microcapsules were placed on a glass slide and multiple images taken at different angles. The best image with best resolution was selected and presented. For the Zeiss Neon 40EsB FIBSEM scanning electron microscopic imaging, microcapsules were dried then coated with platinum, and using laser-guided pen, multiple images were taken. The best images with clear morphology relevant to specific desired magnifications were presented. For the UltraVIEW Vox confocal imaging complemented and equipped with a Yokogawa CSU-X1 confocal scanning unit, microcapsules with stained cells were imaged and multiple images taken, with the best resolution being presented.
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