GCaMP6f trafficking variants that were found to localize predominantly in the soma of cultured neurons (Figure 1 , Figure 3 and Supplemental Table 1 ) were imaged with an LED (X-Cite XLED1, Excelitas Tecnologies) mounted on a microscope for wide-field illumination (Leica 3000B), through a Leica HCX APO L 40x objective (air, NA=0.6). Imaging was performed with a Hamamatsu Orca Flash 4.0 camera using a 480 nm LED and GFP-3035D filter cube (Semrock) for GFP fluorescence (power, 34.84 mW/mm2).
Gfp 3035d filter cube
Manufactured by IDEX Corporation
The GFP-3035D filter cube is a laboratory equipment component designed for fluorescence microscopy applications. It functions as a combination of optical filters that selectively transmit specific wavelengths of light, facilitating the observation and analysis of fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Hcx apo l 20 objective, by Leica (4 mentions)
Brightline single band bandpass filter, by IDEX Corporation (2 mentions)
543 nm 11 nm excitation filter, by IDEX Corporation (2 mentions)
Hcx apo l 40 objective, by Leica (2 mentions)
Orca flash 4, by Hamamatsu Photonics (2 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (2 mentions)
X cite xled1, by Excelitas (2 mentions)
Nis elements br 4, by Nikon (1 mentions)
4 protocols using gfp 3035d filter cube
1
Imaging GCaMP6f Trafficking Variants
2
Fluorescent Protein Imaging in Neurons
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GFP-fusions with trafficking sequences, opsin-GFP with trafficking sequences, and cytosolic mCherry expressed in cultured neurons were imaged with a LED mounted (X-Cite XLED1, Excelitas Technologies) on a microscope for widefield illumination (Leica 3000B), through either a Leica HCX APO L 40× objective (air, NA=0.6) or a Leica HCX APO L 20× objective (air, NA=0.5). Imaging was performed with a Hamamatsu Orca Flash 4.0 camera under identical illumination conditions throughout: a 480 nm LED using GFP-3035D filter cube (Semrock) for GFP fluorescence (34.84 mW/mm2) and a 540 nm LED with 543 nm ± 11 nm excitation filter (Semrock) for mCherry fluorescence. Images were taken with an exposure time of 300 ms.
Cultured neurons expressing CoChR-GFP, KA2-GFP or KA2(1–150)-GFP were imaged using similar parameters: fluorescence was excited with a 480 nm LED filtered by a 472/30 nm BrightLine single-band bandpass filter (Semrock) and focused on the sample through a Leica HCX APO L 20× objective (air, NA = 0.6), with a power of 25.19 mW/mm2. Images were acquired with a Hamamatsu Orca Flash 4.0 with an exposure time of 300 ms.
Cultured neurons expressing CoChR-GFP, KA2-GFP or KA2(1–150)-GFP were imaged using similar parameters: fluorescence was excited with a 480 nm LED filtered by a 472/30 nm BrightLine single-band bandpass filter (Semrock) and focused on the sample through a Leica HCX APO L 20× objective (air, NA = 0.6), with a power of 25.19 mW/mm2. Images were acquired with a Hamamatsu Orca Flash 4.0 with an exposure time of 300 ms.
3
Fluorescent Protein Imaging in Neurons
4
Visualizing Labeled Lipid Nanoparticles
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Transmitted light microscopy was performed using an inverted Nikon Ti-S microscope equipped with an Andor Zyla 3.2 camera; the magnification was 100×.
Fluorescence was detected using the same microscope and a Semrock GFP-3035D filter cube. Image alignment and contrast correction were performed using the Nikon NIS Elements BR 4.0 software.
LNPs containing a fluorescent label were obtained using the base lipid composition (S1) with the addition of 0.6% tetramethyl-BODIPY-C3 acid (3-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl) propionic acid, as provided by Dr. I. A. Boldyrev, Institute of Bioorganic Chemistry, Russian Academy of Sciences). Lipoplexes with the labeled LNPs were prepared as described above.
Fluorescence was detected using the same microscope and a Semrock GFP-3035D filter cube. Image alignment and contrast correction were performed using the Nikon NIS Elements BR 4.0 software.
LNPs containing a fluorescent label were obtained using the base lipid composition (S1) with the addition of 0.6% tetramethyl-BODIPY-C3 acid (3-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl) propionic acid, as provided by Dr. I. A. Boldyrev, Institute of Bioorganic Chemistry, Russian Academy of Sciences). Lipoplexes with the labeled LNPs were prepared as described above.
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