For in vivo GCaMP analysis to visualize CIII, CIV, and Ch neurons, 19-12-Gal4 (Class III) and UAS-GCaMP6m were used in combination with ppk1.9-Gal4 (Class IV) or iav-Gal4 (Chordotonal) respectively. in vivo calcium imaging was performed as previously described [26 (link), 59 (link)]. Briefly, third instar larvae were mounted on a microscope slide with minimal water to prevent desiccation and placed on a Peltier stage (Linkam PE120) for time lapse imaging. The following temperature regimen was used during time lapse imaging: 1 minute at 25°C, ramp down to 6°C at 20°C/minute, hold at 6°C for 10 seconds, ramp up to 25°C at 20°C/minute, and hold at 25°C for one minute. Images were recorded at 212.55 μm x 212.55 μm resolution and 307.2 ms per frame. Raw time-lapse files were motion corrected in Fiji using the Stack Reg function. A region of interest was manually drawn around the cell body and mean fluorescence intensity across time was collected. ΔF/F0 was calculated as previously described [26 (link)].
Pe120
Manufactured by Linkam
Sourced in United Kingdom
The PE120 is a compact thermal analysis stage designed for use with optical microscopes. It allows for the controlled heating and cooling of small samples within a temperature range of -196°C to 600°C. The PE120 features a temperature accuracy of ±0.1°C and a temperature stability of ±0.01°C, providing a stable environment for thermal analysis and characterization.
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Lab products found in correlation
Ds qi2, by Nikon (1 mentions)
Eclipse lv100, by Nikon (1 mentions)
Tu plan fluor epi, by Nikon (1 mentions)
Usb4000, by OceanOptics (1 mentions)
Coolsnap hq, by Teledyne (1 mentions)
Dmrxa2, by Leica (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Dme optical microscope, by Leica (1 mentions)
Mc190 hd camera, by Leica (1 mentions)
T95 pe, by Linkam (1 mentions)
Jem 7600f, by JEOL (1 mentions)
Jem 2010, by JEOL (1 mentions)
11 protocols using pe120
1
In vivo GCaMP Imaging of Sensory Neurons
2
Top-View Condensation Imaging Techniques
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Top view condensation experiments were conducted on a customized top‐view optical microscopy setup implemented with a temperature controllable cold‐stage. A high‐resolution camera (DS‐Qi2, Nikon) was attached to an upright microscope (Eclipse LV100, Nikon) equipped with a 20X (TU Plan Fluor EPI, Nikon) objective lens and records condensing droplet images at 1–10 fps. Test samples were horizontally mounted onto a cold‐stage (PE‐120, Linkam) and the sample surface temperature was set to 1 ± 0.5 °C for condensation experiments. All the experiments were performed in ambient laboratory temperature. Relative humidity of environment was controlled by a commercial humidifier and the humidity level was recorded and logged by a temperature and relative humidity transmitter (HX93BDV0, Omega). A more detailed description of the experimental setup could be found in previous works.[57 , 58 ]
3
Optical Microscopy of Liquid Crystals
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Optical microscopy was carried out
in transmission on an Olympus BX61. The following objectives were
used: 5× (UMPlanFI, NA 0.15), 10× (UMPlanFI, NA 0.30), and
20× (UMPlanFI, NA 0.46). The samples were temperature controlled
with a Peltier-driven hot stage (Linkam, PE120). The effective temperature
within the LC cell was determined by a calibration run.
in transmission on an Olympus BX61. The following objectives were
used: 5× (UMPlanFI, NA 0.15), 10× (UMPlanFI, NA 0.30), and
20× (UMPlanFI, NA 0.46). The samples were temperature controlled
with a Peltier-driven hot stage (Linkam, PE120). The effective temperature
within the LC cell was determined by a calibration run.
4
Polarized Light Microscopy of Dynamic Crystallization
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The morphology of the samples was studied by PLM (Zeiss Scope. A1 Pol). 10 μL of the sample was pipetted to a preheated microscope slide and carefully, the coverslip was placed on the top of the molten sample to avoid air bubble formation. These slides were stored at 22 °C for 24 h prior to PLM. The pictures were captured after 24 h for each sample and the result of dynamic crystallization was studied by using a temperature profile similar to the one used in DSC analysis. The heating and cooling were attained by a Peltier plate setup (Linkam, model PE120). The sample was heated to 90 °C with a 2 K/min rate and kept isothermally for 60 min to erase the crystal memory. As the heating systems are different in DSC and PLM, longer isothermal heating time at 90 °C was used as compared to DSC. Afterwards, the sample was cooled down to 10 °C with a 2 K/min rate and at this temperature, the pictures were captured by using objectives 10 and 20x. All measurements were carried out in duplicates.
5
Precise Temperature Control for BPII Nucleation
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The sample was settled on a precisely controlled hot stage (PE120, Linkam). The heating process consists of step increments of the temperature of the samples at a rate of 0.2°C/min, usually starting from the cholesteric phase (25°C) to reaching the isotropic phase at 42.9°C. For cooling, we used a one-step quench process. The temperature was reduced from the isotropic temperature of 42.9° to ~40.9°C (i.e., 0.2°C above TBPI-BPII), where the desired BPII primarily nucleates and grows over the chemical pattern area and stays at certain temperature for different times.
6
Optical Characterization of Cholesteric Liquid Crystals
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The PMMAZO brush thickness was determined by a Woollam VUV-VASE32 variable angle spectroscopic ellipsometer. Optical characterization was performed using cross-polarized and reflection mode with an Olympus BX60 microscope with 10×/50× objectives, Bertrand lens, and 405-nm monochromic light filter. Samples were heated up to the isotropic phase using a Linkam PE120 temperature controller controlling the hot stage at different rates. Chemical patterns were observed by scanning electron microscopy (SEM; ZEISS MERLIN). BP type and crystal orientation were detected by Kossel diagrams (19 (link), 30 ). A spectrophotometer (USB4000, Ocean Optics) was used to measure ultraviolet-visible spectra of different BP films. The laser source with output wavelength of 445 nm/40 nW was used to detect the diffraction of the binary pattern array. A laser light with 445-nm wavelength was converted to circular polarized light after successively passing through a linear polarizer and a quarter wave plate. The circularly polarized light impinges on the sample by passing through beam splitters, generating the diffraction pattern. The diffractive light was projected on a black screen.
7
Time-lapse Imaging of C. elegans Embryonic Development
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Time-lapse DIC images were captured with a Leica DMRXA2 (40× objective) coupled to a Coolsnap HQ (Roper Scientific) camera and a temperature-controlled stage (Linkam PE-120). Embryos were dissected out, washed in M9 and transferred to a 5% agarose pad; the coverslip was sealed with paraffin oil. Stacks of 25 images were acquired every 5 min.
For hatchling measurements, 5-10 hermaphrodites laid eggs overnight. Mothers and larvae were washed away the next day. Hatchlings were mounted in M9 (+0.5 mM levamisole) every 30 min.
Spinning-disc confocal images were acquired 3-4 h after egg laying, with an inverted Zeiss Observer Z1 microscope (100× objective) coupled with an Evolve camera (Photometrics) and a Yokogawa spinning-disc head, monitored by MetaMorph (Molecular Devices).
For hatchling measurements, 5-10 hermaphrodites laid eggs overnight. Mothers and larvae were washed away the next day. Hatchlings were mounted in M9 (+0.5 mM levamisole) every 30 min.
Spinning-disc confocal images were acquired 3-4 h after egg laying, with an inverted Zeiss Observer Z1 microscope (100× objective) coupled with an Evolve camera (Photometrics) and a Yokogawa spinning-disc head, monitored by MetaMorph (Molecular Devices).
8
Dynamic Crystallization Analysis of Samples
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The morphology of the samples was studied by PLM (Zeiss Scope, A1 Pol). A 10 µL of sample was pipetted to a preheated microscope slide, and carefully, the coverslip was placed on the top of the molten sample to avoid air bubble formation. These slides were stored at 22 °C for 24 h prior to PLM. The pictures were captured after 24 h for each sample, and the result of dynamic crystallization was studied by using a temperature profile similar to the one used in DSC analysis. The heating and cooling were attained by a Peltier plate setup (Linkam, model PE120). The sample was heated to 90 °C with a 2 K/minute rate and kept isothermally for 60 min to erase the crystal memory. As the heating systems are different in DSC and PLM, longer isothermal heating time at 90 °C was used as compared to DSC. Afterward, the sample was cooled down to 10 °C with a 2 K/minute rate, and at this temperature, the pictures were captured by using objective 20×. All measurements were carried out in duplicate.
9
Polarized Microscopy of Aerated Dispersions
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A Leica DME optical microscope mounted with a Leica MC190 HD camera was used to capture optical micrographs of crystal dispersions in oil and air-in-oil foams. A polarizer (13596080) and an analyzer were used to cross-polarize the transmitted light. Images were acquired by Leica Application Suite 4.12.0. A small amount of sample was transferred carefully by spatula onto the middle of a glass slide (76 mm × 26 mm) possessing a single cavity (15 mm), and then gently covered with a thin coverslip (24 mm × 24 mm). The temperature of the glass slide was controlled by a hot stage (Linkam PE120), which was connected to an ECP water circulator and controlled by a Linkam T95PE controller. A moderate flow rate of dry compressed air was applied over the coverslip during imaging to avoid condensation. Both polarized and non-polarized microscope images were taken for the same sample under the same condition at fixed time intervals during both whipping (foams) and storage. All glassware and spatulas were pre-cooled at the whipping or storage temperature before use. The micrographs were analyzed with ImageJ 1.47V software which was calibrated with a Pyser-sgi limited graticule. The number average bubble diameter D [1,0] of the aerated samples was calculated from at least 100 representative bubbles of different samples: (1) where d i is the bubble diameter and n is the number of bubbles.
10
Polarized Light Microscopy Photomicrography
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Polarising light microscopy (PLM) photomicrographs were obtained using a CCD camera (Flea3, Point Grey, Richmond, BC, Canada) coupled to a polarizing light microscope (Kozo XJP 300 or Nikon Ci-S). Temperature control was achieved using a peltier temperature stage (Linkam Scientific PE120) coupled to a recirculating water bath, with an accuracy of ±0.1 • C.
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