All zebrafish experiments were approved by the Stony Brook University Institutional Animal Use and Care Committee. Two-day-old kdrl:RFP transgenic embryos were anesthetized with 0.016% tricaine and transferred to embryo medium containing 4% Ficoll. Fifty to 100 cells (control, STAT3 knockout, STAT3 Y640F, and STAT3 S727E) were injected into the common cardinal vein of the embryos using a CellTram Vario (Eppendorf) and transferred to a 33°C incubator. Embryos were imaged every 2 d after injection using a Leica DMI6000B inverted microscope and, on the eighth day after injection, with a spinning disc confocal microscope (Yokogawa, CSU-10; Carl Zeiss,AxioImager; Hamamatsu Photonics, EM-CCD camera).
Dmi6000b inverted microscope
Manufactured by Yokogawa
Sourced in Germany
The DMI6000B is an inverted microscope manufactured by Yokogawa. It is designed for a wide range of applications in life science research and industrial inspection. The DMI6000B provides high-quality imaging and precise control of microscope settings to support various observation techniques.
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Lab products found in correlation
Imagem em ccd, by Hamamatsu Photonics (4 mentions)
Csu x1 scan head, by Hamamatsu Photonics (4 mentions)
Hc pl apo 63x 1.4 0 6 oil objective, by Leica camera (3 mentions)
Visiscope spinning disk confocal microscope system, by Visitron (2 mentions)
Visiscope, by Visitron (2 mentions)
Celltram vario, by Eppendorf (1 mentions)
Em ccd camera, by Hamamatsu Photonics (1 mentions)
Axioimager, by Hamamatsu Photonics (1 mentions)
Csu 10, by Zeiss (1 mentions)
Polystyrene microsphere, by Polysciences (1 mentions)
Polystyrene, by Polysciences (1 mentions)
Hc pl apo 63 1.4 0 6 oil objective, by Leica camera (1 mentions)
Volocity, by Quorum Technologies (1 mentions)
Hcx pl apo 63x, by Leica camera (1 mentions)
Ixon emccd camera, by Oxford Instruments (1 mentions)
Fluo 4, by Thermo Fisher Scientific (1 mentions)
Csu x1 spinning disk unit, by Yokogawa (1 mentions)
9 protocols using dmi6000b inverted microscope
1
Zebrafish Xenotransplantation of STAT3 Variants
2
Compression Microscopy of C. elegans Embryos
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Embryos were dissected from gravid hermaphrodites, mounted in 2.5 μl of an M9 buffer suspension containing either 25, 20, or 15 μm poly-styrene microspheres (Polyscience, Warrington, PA, United States), and sealed between two coverslips (Roth, Karlsruhe, Germany) with vaseline. Details of compression under these conditions have been published previously (Singh et al., 2019 (link)). Microscopy was performed with a VisiScope spinning disk confocal microscope system (Visitron Systems, Puchheim, Germany) based on a Leica DMI6000B inverted microscope, a Yokogawa CSU X1 scan head, and a Hamamatsu ImagEM EM-CCD as described earlier (Dutta et al., 2015 ). All acquisitions were performed at 21–23°C using a Leica HC PL APO 63x/1.4-0.6 oil objective.
3
Embryo Mounting and Cortex Ablation
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Embryo preparation and mounting has been described elsewhere (Pohl and Bao, 2010 (link); Dutta et al., 2015 (link)). Mounting was modified by using differently sized polystyrene (15 µm, 20 µm, 25 µm; Polysciences, Hirschberg, Germany) and polymethylmethacrylate spheres (12 µm and 13.5 µm, PolyAn, Berlin, Germany). Microscopy was performed with a VisiScope spinning disk confocal microscope system (Visitron Systems, Puchheim, Germany) based on a Leica DMI6000B inverted microscope, a Yokogawa CSU X1 scan head, and a Hamamatsu ImagEM EM-CCD. All acquisitions were performed at 21–23°C using a Leica HC PL APO 63×/1.4-0.6 oil objective. Cell cortex ablations were performed using a pulsed 355 nm UV laser mounted on the same microscope. One ablation cycle was performed per acquisition with a residence time per pixel of 3.5 ms. Acquisitions pre and post ablation were performed at 200-ms intervals.
4
Imaging Acetate-Induced Cellular Changes
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Cells were seeded into a 12-well plate containing poly-L-lysine-coated coverslips in complete medium and grown for 48 h in the presence of 20 mM sodium acetate for 48 h, induced with 1 μg/ml tetracycline for 24 h, then incubated with dFBS or complete FBS for 1 h. Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and then stained for Kac, GFP, or phalloidin as detailed in Supplementary Table S1B . Image stacks were acquired on a Leica DMI 6000 B inverted microscope with a Yokogawa CSU10 confocal unit at 63×. Z stacks were collected, deconvolved using Volocity (Quorum Technologies), and shown as intensity projections. Images were cropped using Adobe Photoshop. For all quantitatively compared images, identical imaging conditions (including exposure times) were used.
5
Live-cell Imaging of Optogenetic Activation
6
Spinning Disk Confocal Imaging of Embryo Development
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Imaging was executed with a VisiScope spinning disk confocal microscope system (Visitron Systems, Puchheim, Germany). The system consists of a Leica DMI6000B inverted microscope, a Yokogawa CSU X1 scan head, and a Hamamatsu ImagEM EM-CCD. Z-sectioning was performed with a Piezo-driven motorized stage (Applied Scientific Instrumentation, Eugene, OR, United States) using a Leica HC PL APO 63X/1,4-0,6 oil objective. All acquisitions were performed at 20–23°C.
For most experiments, we collected z-stacks with 45 steps at 1.0 μm distance each with 2, 3, 4, or 5 min intervals, respectively, for a total duration of 1–3 h for acquiring early lima bean to 1.5-fold stage embryos. For imaging of the head-on-view, we used 3–4 min intervals to avoid tipping of embryos. For lineaging, we performed long-term imaging for 250 time points at 3 min intervals and with z sampling of 1 μm over a distance of 30 μm. For acquiring of embryos for one time point before and a time-lapse series after UV laser ablation, we used 2 min intervals and z sampling at 1 μm over a distance of 40–45 μm.
For most experiments, we collected z-stacks with 45 steps at 1.0 μm distance each with 2, 3, 4, or 5 min intervals, respectively, for a total duration of 1–3 h for acquiring early lima bean to 1.5-fold stage embryos. For imaging of the head-on-view, we used 3–4 min intervals to avoid tipping of embryos. For lineaging, we performed long-term imaging for 250 time points at 3 min intervals and with z sampling of 1 μm over a distance of 30 μm. For acquiring of embryos for one time point before and a time-lapse series after UV laser ablation, we used 2 min intervals and z sampling at 1 μm over a distance of 40–45 μm.
7
Live-cell Imaging of Embryonic Development
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Appropriately staged embryos were imaged using a VisiScope spinning disk confocal microscope system (Visitron Systems, Puchheim, Germany) consisting of a Leica DMI6000B inverted microscope, a Yokogawa CSU X1 scan head, and a Hamamatsu ImagEM EM-CCD. Z-sectioning was performed with a Piezo-driven motorized stage (Applied Scientific Instrumentation, Eugene, OR, United States). All acquisitions were performed at 20–23°C using a Leica HC PL APO 63X/1.4-0.6 oil objective. For imaging/quantifying of anterior and posterior contacts of ABpl, we collected z-sections of 16 focal planes (0.5 μm apart) with 10 s intervals with a 488 and 561 nm laser at an exposure of 150 ms from the onset of ABa/ABp division, for a total duration of 10 min. While for PIV analysis, we collected 8 z-sections (0.5 μm apart) with 3 s intervals, again for a total duration of 10 min with the same laser settings as above. For imaging 12-cell stage embryos, we collected 26 z-sections (1 μm apart) with intervals of 1 min. For most imaging, laser intensities used were 5% for the 488 nm and 40 or 60% for the 561 nm laser (each 25 mW maximal output at the source).
8
Calcium Imaging of Hippocampal Neurons
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Hippocampal neurons were cultured from l day postnatal Sprague-Dawley rats. We performed time-lapse imaging for monitoring intracellular calcium in hippocampal neurons at 7 th day after plating. The neurons were loaded with 1 µM Fluo-4 (Molecular Probes) for 30 min in Hank's Balanced Salt Solution (HBSS). The cells were then washed with HBSS for 3 times and Fluo-4 intensity was monitored using excitation at 488 nm and emission at 510 nm. We performed imaging using a spinning-disk confocal imaging system comprising a Leica DMI6000B inverted microscope, a Yokogawa CSU-X1 spinning-disk units [16] . The interval between successive images, while set at 1 s, was observed to be between 0.8 s to 1 s due to inherent variabilities. During imaging, neurons were kept in the incubation chamber with the microscope maintained at 37 o C and 5% CO 2 .
9
Calcium Dynamics in Cultured Hippocampal Neurons
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We performed time-lapse confocal imaging (using a Leica DMI6000B inverted microscope fitted with a Yokogawa CSU-X1 spinning-disk unit) on hippocampal neurons, cultured from l day postnatal Sprague-Dawley rats. In particular, we monitored intracellular calcium at 7-th day after plating using excitation at 488 nm and emission at 510 nm [6] . During imaging, neurons were kept in the attached incubation chamber maintained at 37 o C and 5% CO 2 . The interval between successive images, while set at 1 s, was observed to vary between 0.8 s to 1 s. From the timelapse image data (see Fig. 1 for a representative frame), the time course of spatially resolved Fluo-4 fluorescence intensity in neuron populations was obtained using Andor software. At present, out of a population of 28 neurons, we consider 8 neurons, indexed 1-8, for analysis (time course of calcium responses are shown in Fig. 3 for neurons 1-4 and heterogeneity in such responses is visually evident here).
For each neuron, we inferred binary spike train from its time course using suitable normalization and a fast nonnegative deconvolution algorithm [5] , [7] . Such spike sequences were used for further analysis.
For each neuron, we inferred binary spike train from its time course using suitable normalization and a fast nonnegative deconvolution algorithm [5] , [7] . Such spike sequences were used for further analysis.
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