Embryos labeled by in situ hybridization were photographed on a Zeiss Axioplan 2 microscope, equipped with a MicroPublisher 5.0 RTV camera using Volocity software (Improvision). Fluorescent immunostained embryos were photographed on a Zeiss LSM780 confocal microscope using a 63x/1.15 W C-APO objective. For time-lapse imaging, embryos were imaged on a Nikon Eclipse Ti spinning disk microscope equipped with a 40x/1.15 WI Apo LWD objective. Approximately 100 um z-stacks were captured at 0.5 um intervals every 5 minutes for 8 hours. ImageJ/Fiji was used for image processing. Cell contours were hand-drawn in ImageJ and measured for length-width ratio (LWR) and orientation. Each cell was divided into 4 quadrants to determine MTOC position. Cell orientation and MTOC position were plotted as rosette diagrams and Watson's U2 tests for significance were conducted using Vector Rose (PAZ software). In Fat3-MO or Dchs2-MO embryos, LWR and orientation were recorded within a 3-to-4 cell-thick presumptive palatoquadrate (pq) region bordered posteriorly by the mandibular aorta, dorsally by the adductor mandibularis muscle (amm) and anteriorly by the presumptive jaw joint – at amm mid-length.
Apo lwd
Manufactured by Nikon
Sourced in United Kingdom
The Apo LWD is a high-performance laboratory objective lens manufactured by Nikon. It is designed to provide excellent optical performance with a wide field of view. The Apo LWD lens features apochromatic correction, which helps to minimize chromatic aberrations and deliver sharp, clear images.
Automatically generated - may contain errors
Lab products found in correlation
Plan apo lambda, by Nikon (2 mentions)
Plan apo vc, by Nikon (2 mentions)
Labram hr evolution, by Horiba (2 mentions)
Axioplan 2 microscope, by Zeiss (1 mentions)
Micropublisher 5.0 rtv camera, by PerkinElmer (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Ez c1, by Nikon (1 mentions)
Plan apo, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Eclipse ti e, by Nikon (1 mentions)
8 protocols using apo lwd
1
Quantitative Analysis of Craniofacial Morphogenesis
2
Confocal and Live Imaging of Cell Structures
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Confocal Imaging was performed in a Nikon Eclipse TE300 inverted microscope converted for spinning disc confocal microscopy (Andor Technologies, United Kingdom) using a 40× Nikon Apo LWD lenses. Images were acquired with an electron-multiplied cooled charge-coupled device camera (DV887 1004X1002; Andor Technologies) driven by Andor IQ2 imaging software. All images were processed further using Photoshop and Illustrator (Adobe, San Jose, CA), and QuickTime Pro (Apple, San Jose, CA) software.
Live imaging of HPAF II cysts was taken with an OLYMPUS 1X71 inverted microscope using a 20× lens. The images were taken every 5 min for 12 hr. Temperature was controlled by a Weather station connected to the microscope.
For live imaging, FAK inhibitor treated S1P2 morpholino fish were anesthetized with 0.02% Tricaine in E3, mounted in 1% low melt agarose and imaged on a spinning disc confocal at 20×, capturing a z-series every 2 min (Eisenhoffer and Rosenblatt, in press (link)) for 3–6 hr.
Live imaging of HPAF II cysts was taken with an OLYMPUS 1X71 inverted microscope using a 20× lens. The images were taken every 5 min for 12 hr. Temperature was controlled by a Weather station connected to the microscope.
For live imaging, FAK inhibitor treated S1P2 morpholino fish were anesthetized with 0.02% Tricaine in E3, mounted in 1% low melt agarose and imaged on a spinning disc confocal at 20×, capturing a z-series every 2 min (Eisenhoffer and Rosenblatt, in press (link)) for 3–6 hr.
3
Spectral Imaging of Biological Samples
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All spectral images were captured using a custom modified microscope (A1R or C1si; Nikon) equipped with a spectral detector allowing a one-shot acquisition of a given spectral range with a multi-anode photomultiplier tube capable of capturing 32 spectral bins. Spectral images were typically acquired with 10-nm resolution covering the range of 430 to 750 nm. The C1si was equipped with a 10× Air lens (NA 0.45, Plan Apo; Nikon), and images were acquired using EZ-C1 software (Nikon). The A1R was equipped with a 25× lens (NA 1.1, Apo LWD; Nikon) with water or PBS as an imaging medium, and images were acquired using NIS Elements (Nikon). Images were imported into ImageTrak (written by P.K. Stys; see Image analysis); emission spectra were determined within a given region of interest using ImageTrak.
4
Imaging Cellular Deformation Dynamics
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An inverted microscope (Eclipse Ti-E; Nikon) with a spinning-disk confocal head (CSU-X; Yokagawa Electric) was used for all imaging. Imaging was done under magnification of a 40× 1.15 N.A. water-immersion objective (Apo LWD; Nikon) or a 60×/1.49 NA oil immersion objective (Zeiss). Images were acquired with a CMOS camera (Zyla-4.2-USB3; Andor) or a CCD camera (Coolsnap HQ2, Photometrics). Images were acquired every 1 s for Figs. 1 –5 and every 5 s for Fig. 6 . Calculation of flow vectors and deformation anisotropy was conducted as we described previously (14 ) using PIV software (www.oceanwave.jp/softwares/mpiv/ ) and custom Matlab scripts. Values of ρmyo and Pbiaxial were smoothed such that data points report the average value over overlapping windows of 50 s in Fig. 6 and 20 s in all other figures.
5
Hippocampal Subfield Imaging Protocol
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For each animal, 3–4 coronal tissue sections that included the subiculum (S) and CA1 field of the hippocampus (CA1) were imaged with a Nikon A1R HD confocal microscope using a 10× (Plan Apo Lambda, NA: 0.40), 20× (Plan Apo VC, NA: 0.75) or 40× water-submersion (Apo LWD, NA: 1.15) objective lens as indicated in the figure legends. Imaging parameters were kept constant across all sections for each set of immunofluorescent labels. All image analysis was performed using ImageJ FIJI (NIH) with semi-automated custom macros. Experimenters were blinded to treatment.
6
Raman Analysis of Lipid Droplets
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Raman spectral analysis from individual LDs was performed using a commercial confocal Raman microscope (LabRAM HR Evolution, Horiba) at room temperature. A 15 mW (after the objective), 532 nm diode laser was used to excite the sample through a 40x water immersion objective (Apo LWD, 1.15 N.A., Nikon). The total data acquisition time was 60 s using the LabSpec 6 software. For each group, at least 10 spectra from individual LDs in different locations or cells were obtained. To analyze the spectrum, the background was removed manually based on the glass background profile, and peak intensity was measured using OriginPro. The calibration curve for CE percentage in LD was generated by measuring CE/triacylglyceride emulsion at various percentages and linearly correlate it with the 704 cm-1 peak (cholesterol rings) normalized with the 1445 cm-1 peak (CH2 bending vibration). The unsaturation degree of LD is determined by the peak intensity at 1654 cm-1 (C=C stretching vibration) normalized with the 1445 cm-1 peak.
7
Raman Microscopy Analysis of Lipid Droplets
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Individual LDs in the cells were analyzed by a confocal Raman microscope (LabRAM HR Evolution, Horiba). A 40x water immersion objective (Apo LWD, 1.15 N.A., Nikon) was employed in the system. The sample was excited by a laser of 532 nm. The percentage of CE in the LDs was recorded by measuring Raman intensity at the 702 cm -1 peak, which corresponded to cholesterol ester, and the degree of unsaturated fatty acid (UFA) in the cells was recorded by measuring Raman intensity at the 1,654 cm -1 peak, which corresponded to C=C stretching vibration, indicating the degree of unsaturated fatty acid. The normalization of Raman intensity was accomplished with the area between 1,400 cm -1 to 1,500 cm -1 , which was the area for -CH2 bending vibration.
8
Hippocampal Subfield Imaging Protocol
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For each animal, 3-4 coronal tissue sections that included the subiculum (S) and CA1 eld of the hippocampus (CA1) were imaged with a Nikon A1R HD confocal microscope using a 10x (Plan Apo Lambda, NA: 0.40), 20x (Plan Apo VC, NA: 0.75) or 40x water-submersion (Apo LWD, NA: 1.15) objective lens as indicated in gure legends. Imaging parameters were kept constant across all sections for each set of immuno uorescent labels. All image analysis was performed using ImageJ FIJI (NIH) with semiautomated custom macros. Experimenters were blinded to treatment.
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