Cells transfected with mCherry cell were visualized with fluorescent differential interference contrast microscopy using an Olympus BX50WI microscope equipped with a 10× water immersion objective (Olympus, UMPlanFl, 10×/0.30 W) with excitation wavelength range 520–550 nm and emission at > 580 nm (Fig 1A and 1C ). Fluorescent and differential interference contrast images were overlapped with appropriate illumination intensities to allow simultaneous visualization of transfected and nontransfected cells (Fig 1C ). Then, the level of expression of mGFP-αCaMKII constructs was verified using 41025 PSTN GFP Chroma filter set with excitation wavelength at 470 ± 40 nm and emission at 515 ± 30 nm (Fig 1B and 1D ). The level of mGFP-αCaMKII expression substantially varied from cell to cell. Therefore, for the experiments we visually selected only those cells, which had comparable distinct GFP fluorescence on a background of differential interference contrast image of the slice at 10× objective using similar levels of illumination. For example, in Fig 1B we chose the far right cell because its strong GFP fluorescence is comparable to fluorescence of other studied cells. The slices, which had a low level of GFP fluorescence in every single transfected cell, were discarded. The electrophysiological experiments were performed under 60× objective (Olympus, UMPlanFl, 60×/0.90 W) (Fig 1C ).
Umplanfl
Manufactured by Olympus
Sourced in Japan
The UMPlanFl is a microscope objective lens manufactured by Olympus. It is designed for use in various microscopy applications. The lens provides a wide field of view and high numerical aperture, which are important characteristics for imaging and analysis tasks.
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Lab products found in correlation
Bx51wi, by Olympus (3 mentions)
Planc n, by Olympus (2 mentions)
Bx50wi microscope, by Olympus (1 mentions)
Vt1200, by Leica (1 mentions)
Bx50wi, by Olympus (1 mentions)
Lumplanfl n, by Olympus (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Du420a brdd, by Oxford Instruments (1 mentions)
8 protocols using umplanfl
1
Visualizing Transfected Cells with Fluorescence Microscopy
2
Optical Imaging of Neural Activity
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A T.I.L.L. Photonics imaging system (Martinsried, Germany) was used to perform in vivo optical recordings, as described elsewhere [19 (link),73 (link),74 (link)]. An epifluorescence microscope (Olympus BX51WI) was used to record activity in the different regions of the brain with either a 4× dry objective (Olympus, PlanCN; NA 0.10) for whole-brain recordings, a 10× water-immersion objective (Olympus, UMPlanFL; NA 0.3) for AL and MB recordings or a 20× water-immersion objective (Olympus, UMPlanFL; NA 0.5) for LH recordings. GCaMP6f was excited using 475 nm monochromatic light (T.I.L.L. Polychrom IV). The fluorescence signal was separated by a 505 nm dichroic filter and a long-pass 515 nm emission filter and recorded with a 640 × 480 pixels 12-bit monochrome CCD camera (T.I.L.L. Imago) cooled to −12°C with 4 × 4 binning on chip. Each measurement consisted of 100 frames recorded at a rate of 5 Hz (integration time for each frame approximately 50 ms).
3
In Vivo Calcium Imaging of Insect Brain
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A T.I.L.L. Photonics imaging system (Martinsried, Germany) was used to perform in vivo optical recordings, as described elsewhere [19, (link)73, (link)74 (link)]. An epifluorescence microscope (Olympus BX51WI) was used to record activity in the different regions of the brain with either a 4× dry objective (Olympus, PlanCN; NA 0.10) for whole-brain recordings, a 10× water-immersion objective (Olympus, UMPlanFL; NA 0.3) for AL and MB recordings or a 20x water-immersion objective (Olympus, UMPlanFL; NA 0.5) for LH recordings. GCaMP6f was excited using 475 nm monochromatic light (T.I.L.L. Polychrom IV). The fluorescence signal was separated by a 505 nm dichroic filter and a long-pass 515 nm emission filter and recorded with a 640 × 480 pixels 12-bit monochrome CCD camera (T.I.L.L. Imago) cooled to -12°C with 4 × 4 binning on chip. Each measurement consisted of 100 frames recorded at a rate of 5 Hz (integration time for each frame ~50ms).
4
Calcium Imaging of Pancreatic Islets
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Pancreatic mouse islets were transferred into 12-well plates 1 day after preparation on poly-L-lysine coated glass coverslips (12 mm diameter, 3–4 islets per coverslip). Two days after seeding, coverslips were transferred into a new 12-well plate, where islets were loaded with 5 μM fura-2 AM dissolved in standard bath solution for 60 min at 20–22 °C. The standard bath solution contained 140 mM NaCl, 10 mM Hepes, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 3 mM glucose (pH 7.4). After loading, the coverslip was transferred to a perfusion chamber (Warner Instruments) mounted on an upright microscope (Olympus BX51WI). Islets were visualized using a 20x water immersion objective (UMPlanFl, Olympus) and perfused with standard bath solution and stimulated by adding 300 nM ghrelin, 100 nM AVP, or 100 μM CCh to standard bath solution. Fura-2-based Ca2+ imaging was performed in intact pancreatic islets using a monochromator-based imaging system and the imaging software TILLvisION 4.0 (both T.I.L.L. Photonics). Emitted fluorescence at 510 nm (excited at 340 nm and 380 nm) was collected with a CCD camera (PCO Imaging), acquired at intervals of 2 s and corrected for background fluorescence.
5
Calcium Imaging of LH Slices
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Brain was extracted after mice were decapitated under isoflurane anaesthesia at least 3 weeks after virus injection. LH slices were dissected to a thickness of 250 μm using a vibratome (VT1200S, Leica, Nussloch, Germany) in ice-cold standard artificial cerebrospinal fluid (ACSF) containing the following (in mM): 125 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose, bubbled with 95% O2 and 5% CO2. For recovery, the slices were incubated at 32 °C for 15 min and then further incubated for 1 h at room temperature. The slices were then transferred to the recording chamber and perfused with ACSF at 32 °C during imaging. Calcium measurements were performed using a CMOS camera (Photometrics, Tucson, AZ) attached to an upright microscope (BX50WI, Olympus, Tokyo, Japan) with a 40X or 10X water-immersion objective (NA 0.8 or 0.3, LUMPlanFl N or UMPlanFl; Olympus) at 10 frames per second. A broad white light source (pE-340 Fura, CoolLED, Andover, UK) was passed through an excitation filter (450–480 nm) and collected through an emission filter (525/50 nm). Fluorescence images were acquired using VisiView software (Visitron Systems GmbH, Puchheim Germany).
6
Femtosecond Laser Perforation for Cell Wall Analysis
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Laser pulses from a regeneratively amplified Ti:Sapphire femtosecond laser (800 ± 5 nm, 100 fs, < 1 mJ/pulse, 32 Hz) (Spectra-Physics, Solstice Ace) were introduced to the microscope through a × 20 objective lens (Olympus, UMPlan FL, NA = 0.46). The pulse was focused on the cell wall. The laser focal position was adjusted to the center of the cell along the short axis and at 50 µm away from the AFM probe position along the long axis, so that the perforation would not interfere with detection. Perforation was controlled by a mechanical shutter (Sigma Koki, Σ-65GR) with a gate time of 1/32 s. The laser pulse energy was tuned from 50 to 200 nJ/pulse using a half-wave plate, a polarizer, and a neutral density (ND) filter in the optical path. AFM measurements were performed before and after LP on the cell wall as shown in Fig. 1 B. Deformation of the cell wall was monitored using a CMOS camera (WRAYMER, FLOYD-100).
7
Calcium Imaging of Hippocampal Cultures
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Calcium imaging experiments were performed on hippocampal cultures (11-14 DIV) as previously described (Tinning et al., 2018) . Prior to imaging, cells were loaded with Fura-2 AM (1 M; Invitrogen, UK) for 1 hour in a humidified incubator at 37°C/5% CO2, while simultaneously being treated with either vehicle or rIL-16 (300 pg mL -1 , #RP8610; Thermo Fisher, UK). Cultures were then washed three times in HEPES-buffered saline (HBS) consisting of the following (in mM): NaCl 140, KCl 5, MgCl2 2, CaCl2 2, HEPES 10, D-glucose 10, pH 7.4 ± 0.02, 310 ± 2 mOsm. Throughout imaging, cultures were perfused with HBS at a rate of 3-3.5 mL min -1 with all drug solutions being added via the perfusate. Cultures were placed in a perfusion bath under a 20×/0.5 water dipping objective lens (Olympus UMPlanFl, Tokyo, Japan) on an upright widefield epifluorescence microscope (Olympus BXW51, Japan) with ratiometric images (350/380 nm; OptoLED, Cairn Research, UK) taken at 0.5 Hz from the neuronal soma and astrocytes using WinFluor imaging software (J. Dempster, University of Strathclyde). Cells were identified as neurons and astrocytes based on their morphological characteristics and their response to ADP and/or high extracellular KCl.
8
Raman Microspectroscopy for Cell Analysis
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Raman spectra were recorded using a custom-built Raman microspectroscopy system, as previously described [22, (link)23] (link). Briefly, this system employed a 150 mW laser with a wavelength of 532 nm (Laser Quantum, Torus), spectrograph (Andor, Shamrock 500) operating with 600 lines/mm grating, and a CCD camera (Andor; DU420A-BR-DD) cooled to -80 °C. A 50× microscope objective (MO), with numerical aperture of 0.8 (Olympus, UMPlanFl), was used to image the spectral irradiance to a 100 μm confocal aperture, which isolates the signal from the cell nucleus, and minimises background noise from the sample substrate, as well as from optical elements in the system [22, (link)23] (link).
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