Dual imaging was performed using an upright microscope (BX51WI; Olympus) and a 10 x objective (UplanSApo, NA 0.4; Olympus). For bright-field imaging, light emanating from a near-infrared (780 nm) LED (M780LP1 and driver LEDD1B; Thorlabs) was filtered using a (785/62 BrightLine HC; Semrock) and projected onto the sample via the bright-field illumination condenser. To excite fluorescence, the Teal line from an LED lamp (Spectra X light engine; Lumencor) was filtered (513/17 BrightLine HC; Semrock) and projected onto the sample using a 520 nm long-pass dichroic (FF520-Di02; Semrock). Transmitted and emitted light were filtered using a 532 long-pass filter (BLP01-532R; Semrock). To simultaneously record images in bright-field and fluorescence, a dual-camera device was used (DC2; Photometrics). Light was split into two channels using a 695 long-pass dichroic mirror (695DCXRUV; Photometrics) and images were projected into two cameras (acA3088-57um; BASLER). Fluorescent light was band-pass filtered (550/49 Brightline HC; Semrock) before reaching the camera sensor. The exposure time (6ms) of one camera served to synchronize the acquisition of the second camera and the Lumencor light engine. Individual worms were manually tracked using a 3-axis motorized stage (X-LSM150A; Zaber).
Ff520 di02
Manufactured by IDEX Corporation
The FF520-Di02 is a laboratory equipment product manufactured by IDEX Corporation. It serves as a digital flow rate controller capable of precisely regulating liquid or gas flow rates. The device utilizes advanced digital technology to provide accurate and reliable flow control functionality for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ff01 452 45, by IDEX Corporation (2 mentions)
H7422 40 gaasp, by Hamamatsu Photonics (2 mentions)
1.0 na water immersion objective, by Olympus (2 mentions)
Blp01 532r, by IDEX Corporation (1 mentions)
Aca3088 57um, by Basler (1 mentions)
Spectra x light engine, by Lumencor (1 mentions)
Brightline hc, by IDEX Corporation (1 mentions)
Bx51wi, by Olympus (1 mentions)
Uplansapo, by Olympus (1 mentions)
Loflo ynb, by Formedium (1 mentions)
Ff01 562 40, by IDEX Corporation (1 mentions)
Ff605 di02, by IDEX Corporation (1 mentions)
Ff01 542 27, by IDEX Corporation (1 mentions)
Ff01 647 57, by IDEX Corporation (1 mentions)
4 protocols using ff520 di02
1
Dual Imaging of Fluorescent Worms
2
Intravital Imaging of Somatosensory Cortex
3
Intravital Imaging of Somatosensory Cortex
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Live animal imaging was performed as previously described 7 (link), 33 (link), 56 (link). Briefly, a Sutter Movable Objective Microscope (MOM) equipped with a pulsed femtosecond Ti:Sapphire laser (Chameleon Ultra II, Coherent) with two fluorescence detection channels was used for imaging (dichroic beamsplitter: FF520-Di02 (Semrock); blue emission filter: FF01-452/45 (Semrock); green emission filter: ET525/70M (Chroma); photomultiplier tubes: H7422-40 GaAsP (Hamamatsu)). Laser excitation wavelength was set to 830nm. Average laser power was <10-15mW at the tissue surface and adjusted with depth as needed to compensate for signal loss due to scattering and absorption. An Olympus 20× 1.0-NA water immersion objective was used for light delivery and collection. Z-stacks included up to 350 images, acquired at 1μm axial step size, used a 2-frame average, 512 × 512 pixel resolution, and 2.0x-10x zoom (corresponding to 350μm-72μm fields of view). Time-lapse recordings typically included 60–70 images/stack, acquired at 1.0–1.2 μm axial step size, used a 2-frame average, 60 stack repeats (corresponding to approximately 94 min total recording duration), 512 × 512 pixel resolution, and 3.3x-5x zoom (corresponding to 212 μm - 142 μm fields of view). Up to thirteen z-stacks, and four to seven time-lapse recordings were acquired per animal in layers 1 and 2 of the somatosensory cortex.
4
Yeast Protein Tagging and Imaging
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Standard yeast media and protocols were used to manipulate yeast strains listed in Supplementary Table 1. The C-terminal tagging and deletion of yeast proteins was made by homologous recombination of respective genes with PCR cassettes previously described (Janke et al., 2004) or constructed in the lab. For microscopy, strains were grown to a log phase in a low fluorescence SD-Trp, -Ura medium (prepared from LoFlo YNB; Formedium) containing 1 M sorbitol where indicated. To compare behaviour of Sla2 force sensors, Sla2-mNeonGreen and Abp1-mScarlet-I at endocytic sites, 4 min movies were acquired with 1 s frame rate by filters set FF01-504/12 (excitation), FF520-Di02 (dicroic) and FF01-542/27 (emission) for mNeonGreen, and with 0.5 s frame rate by FF01-562/40 (excitation), FF605-Di02 (dichroic) and FF01-647/57 (emission; all Semrock) for mScarlet-I fluorescence.
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