For measurements of the point spread function, 28 nm diameter fluorescent carboxylate-modified polystyrene spheres (Thermo Fisher cat. #F8787), with peak excitation and emission wavelengths 505 and 515 nm, respectively, were dispersed in oil with a similar index of refraction as water (Zeiss Immersol W 2010) inside the imaging capillaries. Three-dimensional scans were taken, with a z-spacing of 0.5 μm.
Immersol w 2010
Manufactured by Zeiss
Sourced in Germany
Immersol W 2010 is a water-based immersion medium used with microscopy applications. It is designed to optimize image quality and performance when using water-immersion objectives. The product provides refractive index matching to enhance optical resolution and contrast.
Automatically generated - may contain errors
Lab products found in correlation
Zen blue software, by Zeiss (2 mentions)
Ixon3 888 emccd camera, by Oxford Instruments (1 mentions)
Csu w1, by Yokogawa (1 mentions)
Plan apochromat 63 1.4 na oil lens, by Zeiss (1 mentions)
Spcm aqrh 14 tr, by Excelitas (1 mentions)
Uplsapo60xw, by Olympus (1 mentions)
Ix73 microscope, by Olympus (1 mentions)
A1r point scanning confocal microscope, by Nikon (1 mentions)
Imaris 8, by Oxford Instruments (1 mentions)
Sir actin, by Cytoskeleton (1 mentions)
8 protocols using immersol w 2010
1
Measuring Point Spread Function with Fluorescent Spheres
2
Drosophila Pupal Wing Imaging Protocol
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To prepare the Drosophila pupal wing samples for image collection, pupae at appropriate ages were fixed to double-sided tape and the pupal case above the left wing was removed. The pupae were then placed on a small drop of water or Immersol W 2010 (Zeiss 444969-0000-000) in a glass bottom dish with the left side facing downward6 (link),7 (link),46 (link),66 (link). The fixed time-point images other than Fig. 3a, b and time-lapse images shown in Fig. 6l, m were acquired using an inverted confocal microscope (A1R; Nikon) equipped with a ×60/NA1.2 Plan Apochromat water-immersion objective at 25 °C. Other images were acquired using an inverted confocal spinning disk microscope (Olympus IX83 combined with Yokogawa CSU-W1) equipped with an iXon3 888 EMCCD camera (Andor), an Olympus ×60/NA1.2 SplanApo water-immersion objective, and a temperature control chamber (TOKAI HIT), using IQ 2.9.1 (Andor)66 (link). After imaging, we confirmed that the pupae survived to at least the pharate stage.
3
High-Resolution Imaging of Aged Brain Samples
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Before imaging, fresh imaging buffer was prepared by dissolving 0.7 M N-Acetyl-Cysteine in 0.2 M phosphate buffer, using 10 M NaOH to adjust the pH to 7.40. Approximately 500 µl of the imaging buffer was added to each well. All 4i images were taken on a Zeiss LSM800 confocal laser-scanning microscope. Images from tile scans were exported using Carl Zeiss ZEN Blue software and stitched using Fiji (Schindelin et al., 2012 (link)). For the aging study, DG image stacks were taken with a 20× magnification objective with a 0.8 numerical aperture and composed of tile regions with 10 tiles (5×2), with a pixel resolution of 1024×1024 per tile for a scale of 3.2055 pixels/µm with 16-bit pixel depth. Image stacks consisted of 40 frames acquired with a z-step interval of 1 µm and were used for all quantifications. High-resolution close-ups were acquired with a 40× magnification objective with a 1.1 numerical aperture in a water immersion (Zeiss Immersol W 2010) at a pixel resolution of 2048×2048 with a z-step size of 0.5 µm for 80 frames. These 40× images were used as example images in figures. All acquisitions were done with bi-directional scanning with a pixel dwell time of 2.06 µs. Within each cycle, all samples were labeled with the same antibodies, and imaged with identical microscopy settings for laser power, gain, digital offset, pinhole diameter, and z-step.
4
Monitoring Cellulose Hydrolysis by Two-Photon Microscopy
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The two‐photon microscope was used for monitoring hydrolysis of individual particles. To perform this measurement, a custom windowed chamber was developed. The pCS sample was diluted in a 0.9 ml of 0.2 M acetate buffer at pH 5 and an appropriate amount of hydrolytic enzyme mixture containing cellulases and hemicellulases (DSM, Delft, Netherlands) was added. The sample was placed inside the chamber and under the microscope on a heating plate (50 °C) and was monitored for 18 hours. This time period is enough to convert most of the cellulose to sugars. Brightfield, TPM, and FLIM images were acquired before and after 18 hours of hydrolysis. Since the objective is water immersion, in order to avoid evaporation during imaging, a mineral oil‐based medium (Immersol W 2010, Zeiss, Germany) with refractive index similar to water was used instead.
5
Imaging of Fixed Immunofluorescence Samples
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Fixed cells on slides after immunofluorescence were imaged at 21°C using an inverted Zeiss 880 microscope fitted with an Airyscan detector using ZEN black software. The system was equipped with Plan-Apochromat ×63/1.4-NA oil lens, with an immersion oil (Immersol W 2010, Carl Zeiss; refractive index of 1.518). 488 nm argon and 405, 561, and 633 nm solid-state diode lasers were used to excite fluorophores. Z-sections with 0.37-μm-thick intervals were collected. The oil objective was covered with an immersion oil (ImmersolT 518F, Carl Zeiss) with a refractive index of 1.518.
Microscopy images with CZI file format were analyzed using ImageJ (bundled with Java 1.8.0_172) software. Scoring of nuclear morphology was done after maximum intensity projection image processing.
Microscopy images with CZI file format were analyzed using ImageJ (bundled with Java 1.8.0_172) software. Scoring of nuclear morphology was done after maximum intensity projection image processing.
6
Fluorescence Microscopy Imaging Protocol
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Fluorescence measurements were performed on a Microtime 200 device (PicoQuant) with an integrated laser line at 488 nm in pulsed mode (20 MHz), along with an Olympus IX73 microscope and a detection unit containing two single-photon avalanche diodes (SPCM-AQRH-14-TR; Excelitas). To detect signal from different flanks of the spectrum two bandpass filters at 520 nm (10 nm) and 530 nm (10 nm) were used in combination with a high numerical aperture (1.2), 60× water immersion objective (UPLSAPO60XW; Olympus). Instead of water an oil substitute with similar spectral properties as water was used (Immersol W2010; Zeiss) to avoid evaporation of the immersion fluid during the measurement cycle. For cell as well as vesicle measurements a chamber (FCS2; Bioptechs) was used which is closable by a coverslip containing the sample. The coverslip as part of the chamber was coupled to the immersion objective. The chamber was connected to a heating bath (Lauda Eco Silver). Outer pipes ensure a flow around the sample with heating bath fluids and allow temperature control of the sample. Temperature was measured with a thermometer (TMD-56; Amprobe) coupled to the chamber with an accuracy of 0.1 °C. However, absolute temperature values may be affected by the coupling of the chamber to the nonheated immersion objective depending on the extent of the deviation between sample and room temperature.
7
High-Resolution Confocal Imaging of Organoids
8
Imaging Adherent Platelets on Saphenous Vein
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Surplus surgically prepared human saphenous vein was collected from consenting patients (Ethics number: REC14/EE/109). Segments of vein were dissected in endothelial cell growth medium: veins were opened along their longitudinal axis and 5–10 mm transverse segments cut. In Sylgard resin-coated petri dishes, the four corners of the saphenous vein segments were pinned down onto mesh using minute pins and cultured in 5 mL of endothelial cell growth medium for 24 h at 37°C, 5% CO2 in a humidified atmosphere. Vein segments were then co-cultured with 5 mL of activated and BCECF-AM-labeled platelets (prepared as described in Section “2.7 Washed platelet preparation”) for 10 min at 37°C, 5% CO2 in a humidified atmosphere. Non-adherent platelets were removed by gently washing twice with Tyrode’s buffer supplemented with 2 mM CaCl2 and 1 mM MgCl2. Vein segments were coated with Immersion medium Immersol W 2010 (Zeiss; 444969), mounted onto glass slides with the luminal side facing downward then imaged immediately on the Zeiss AxioObserver Z1 fluorescent miscroscope. Z-stack images were taken of 6 randomly selected fields. Adherent platelets were counted manually.
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