Zebrafish myofibers were imaged on a custom-built widefield inverted microscope (Axiovert 200, Zeiss, Göttingen, Germany) with single-molecule sensitivity47 (link), equipped with a C-Apochromat, × 63/1.2 W Corr objective (Zeiss), multiple excitation lasers (405, 473 and 561 nm), an image splitter (Optosplit, Cairn Research Ltd, Faversham, UK) and an EMCCD camera (Ixon Ultra 897, Andor, Belfast, Northern Ireland). Embryos (4–5 dpf) anesthetized with 0.02% MESAB were immobilized on cover glass surfaces in 1% LMP agarose; another cover slip on top held the embryos closely to the bottom surface. Muscle cell membranes were damaged by focusing 405-nm laser light (5 mW on the specimen) for 2–4 s. mEosFP was photoconverted to its red emitting form by 405 nm light and excited at 561 nm. Image stacks were analysed using a-livePALM software48 (link) (Supplementary Methods ).
Optosplit
Manufactured by Cairn Research
Sourced in United Kingdom
The Optosplit is a high-performance optical beam splitter designed for use in various scientific and research applications. It is capable of precisely dividing an input light beam into multiple output beams with adjustable intensity ratios. The Optosplit is a versatile and reliable instrument that can be used to support a wide range of experimental setups and scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200, by Zeiss (4 mentions)
Apochromat na1, by Zeiss (2 mentions)
Ixon du 897 dv em ccd camera, by Oxford Instruments (2 mentions)
Ibeam smart 200 mw, by Spectra-Physics (2 mentions)
C apochromat 63 1.2 w corr objective, by Zeiss (1 mentions)
Lsm 700 confocal system, by Zeiss (1 mentions)
Tcs nt confocal system, by Leica (1 mentions)
Ixon ultra 897, by Oxford Instruments (1 mentions)
Axiocam mrm, by Zeiss (1 mentions)
Hxp 120, by Zeiss (1 mentions)
Plan apochromat 63, by Zeiss (1 mentions)
Axiophot, by Zeiss (1 mentions)
6 protocols using optosplit
1
Zebrafish Myofiber Imaging and Analysis
2
Multimodal Microscopy for Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To image cAMP levels, the FRET-sensor H187 was excited at 405 nm and fluorescence was measured at 480 ± 10 nm and 530 ± 10 nm in xy-mode on a Zeiss LSM700 confocal system. To image NO levels, DAR-4M was AM-loaded into cells (10 µM DAR-4M AM, for 10 min), and its fluorescence was excited at 514 nm and emission detected at 580 ± 20 nm on a Leica TCS NT system.47 (link) To image Ca2+ waves, Fluo3 was AM-loaded into myocytes (5 µM Fluo3, for 10 min), and its fluorescence was excited at 488 nm and detected >520 nm on a Leica TCS NT confocal system in xt mode. To measure pHi, myocytes were AM-loaded with cSNARF1 (10 µM, for 10 min), and fluorescence was excited at 530 nm and detected simultaneously at 580 ± 10 nm and 640 ± 10 nm on an inverted Olympus microscope with an Orca 05G CCD Camera (Hamamatsu, Japan) and Optosplit (Cairn Research, UK).
3
Multi-Color Microscopy Setup and Techniques
4
Multi-Color Microscopy Setup and Techniques
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All experiments were performed on a modified Zeiss Axiovert 200 inverted microscope equipped with a 100 × oil-immersion objective (Zeiss Apochromat NA1.46). The setup was equipped with a 640 nm diode laser (Toptica iBeam smart 200 mW), a 532 nm diode-pumped solid state (DPSS) laser (Spectra physics Millennia 6s) and a near UV light 405 nm ion laser (Coherent Innova 90C). Intensity modulation and timings were controlled either directly or with an acousto-optic modulator (AOM) using custom-written Labview software. Laser lines were overlaid with an OBIS Galaxy beam combiner (Coherent). Emission light was filtered using appropriate filter sets (Chroma) and recorded on an IXON DU 897-DV EM-CCD camera (Andor). Multi-color imaging was performed using an emission light splitter (Optosplit; Cairn Research) adapted to the spectral characteristics of the used fluorophores. Total internal reflection fluorescence (TIRF) illumination was achieved by shifting the excitation beam in parallel to the optical axis with a mirror mounted on a motorized movable table. For ratiometric Fura-2 imaging, we used a polychromatic Xenon light source combined with a monochromator (polychrome V; TILL photonics) that provided light at 340 nm and 380 nm.
5
Fluorescence Imaging of DNA Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All images were acquired using an inverted Nikon Eclipse Ti microscope (model TI-DH Nikon Corporation, Tokyo, Japan) with an electron-multiplying (EMCCD) camera (iXon 897-DU Andor Technology, Belfast, Northern Ireland) and SOLA light engineTM (6-LCR-SB, Lumencor Inc, Beaverton, OR, USA) with FITC or Cy5 filter cubes. Objectives 2× (Nikon Plan UW, NA 0.06, Field of View (FoV) of 4096 µm), 10× (Nikon Plan Apo λ, NA 0.45, FoV of 819 μm) and 100× (Nikon Plan Apo VC, Oil Immersion, NA 1.4, FoV of 82 μm) were used and videos were captured at 10 to 201 frames per second. For dual-colour imaging and polarization microscopy, an Optosplit (Cairn Research Ltd., Kent, UK) was used with the appropriate filter sets.
The plotted lateral distributions of the DNA are based on integrating the fluorescence intensity at the inlet and outlet regions. They have been normalized so that the total area under each curve is the same for each flow velocity for each experiment. In this way they can be interpreted as probability distributions. The full lateral range (0 to 1) corresponds to the full width of the microfluidic channel. SeeSection S3 of the Supplementary Materials for more details on the image processing.
The plotted lateral distributions of the DNA are based on integrating the fluorescence intensity at the inlet and outlet regions. They have been normalized so that the total area under each curve is the same for each flow velocity for each experiment. In this way they can be interpreted as probability distributions. The full lateral range (0 to 1) corresponds to the full width of the microfluidic channel. See
6
Multimodal Fluorescence Microscopy Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All GFP/mCherry fluorescence images were captured using an upright microscope (Axiophot, Zeiss) equipped with an oil immersion objective lens [Plan-Apochromat, 63×, numerical aperture (NA) 1.4, Zeiss], a charge-coupled device (CCD) camera (AxioCam MRm, Zeiss), and a mercury arc lamp (HXP 120, Zeiss). Images were processed and analyzed using Zen software (Zeiss). All other conventional fluorescence and superresolution cell images were obtained by an inverted microscope (Axiovert 200, Zeiss) equipped with a high-NA water immersion objective (C-Apochromat, 63×, NA 1.2, Zeiss), multiple excitation laser lines (405, 473, and 561 nm), an image splitter (Optosplit, Cairn Research Ltd.), and an electron-multiplying CCD camera (Ixon Ultra 897, Andor). Recorded image stacks were analyzed using Fiji (46 (link)) for conventional fluorescence data and custom-written Matlab software, a-livePALM (47 (link)). For details, see the Supplementary Materials, PALM Imaging section.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!