Fluorescence imaging was carried out on an inverted microscope (Nikon Instruments, Eclipse Ti) with the Perfect Focus System, applying an objective-type TIRF configuration with an oil-immersion objective (Nikon Instruments, Apo SR TIRF ×100, numerical aperture 1.49, Oil). A 561 nm (200 mW, Coherent Sapphire) laser was used for excitation. The laser beam was passed through cleanup filters (Chroma Technology, ZET561/10) and coupled into the microscope objective using a beam splitter (Chroma Technology, ZT561rdc). Fluorescence light was spectrally filtered with an emission filter (Chroma Technology, ET600/50m, and ET575lp) and imaged on a sCMOS camera (Andor, Zyla 4.2 Plus) without further magnification, resulting in an effective pixel size of 130 nm (after 2 × 2 binning).
Zt561rdc
Manufactured by Chroma Technology
The ZT561rdc is a compact and versatile lab equipment product offered by Chroma Technology. It is designed to perform core functions required for various laboratory applications. The product specifications and technical details are available upon request.
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Lab products found in correlation
Zet561 10, by Chroma Technology (12 mentions)
Perfect focus system, by Nikon (9 mentions)
Apo sr tirf 100, by Nikon (8 mentions)
Et600 50m, by Oxford Instruments (7 mentions)
Zyla 4.2 plus, by Oxford Instruments (7 mentions)
Et575lp, by Chroma Technology (7 mentions)
Eclipse ti2, by Nikon (7 mentions)
Et575lp, by Oxford Instruments (4 mentions)
Eclipse ti, by Nikon (4 mentions)
Et525 50m, by Chroma Technology (3 mentions)
Et600 50m, by Chroma Technology (3 mentions)
Prism type tirf microscope, by Nikon (2 mentions)
Ixon emccd, by Oxford Instruments (2 mentions)
Zet 640 10, by Chroma Technology (2 mentions)
Zt640rdc, by Chroma Technology (2 mentions)
Et700 75m, by Oxford Instruments (2 mentions)
Cfi apo tirf 100, by Nikon (2 mentions)
Zt647rdc, by Chroma Technology (2 mentions)
Et500lp, by Chroma Technology (1 mentions)
Orca fusion bt, by Hamamatsu Photonics (1 mentions)
16 protocols using zt561rdc
1
TIRF Microscopy Imaging Setup
2
Fluorescence Imaging of Cellular Structures
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Fluorescence imaging was carried out on an inverted Nikon Eclipse Ti microscope (Nikon Instruments) with the Perfect Focus System, applying an objective-type TIRF configuration with an oil-immersion objective (Apo SR TIRF 100×, NA 1.49, Oil). Two lasers were used for excitation: 488 nm (200 mW, Toptica iBeam smart) or 561 nm (200 mW, Coherent Sapphire). The laser beam was passed through a cleanup filter (ZET488/10x or ZET561/10x, Chroma Technology) and coupled into the microscope objective using a beam splitter (ZT488rdc or ZT561rdc, Chroma Technology). Fluorescence light was spectrally filtered with two emission filters (ET525/50 m and ET500lp for 488 nm excitation and ET600/50 and ET575lp for 561 nm excitation, Chroma Technology) and imaged on a sCMOS camera (Andor Zyla 4.2) without further magnification, resulting in an effective pixel size of 130 nm after 2 × 2 binning.
3
TIRF Microscopy Protocol for Fluorescence Imaging
4
Super-Resolution Microscopy Imaging Protocol
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Imaging was carried out using an inverted
microscope (Nikon Instruments, Eclipse Ti2) and the Perfect Focus
System, by applying an objective-type total internal reflection fluorescence
(TIRF) configuration with an oil-immersion objective (Nikon Instruments,
Apo SR TIRF 100×, NA 1.49, oil). A 561 nm laser (MPB Communications
Inc., 500 mW, DPSS-system) was used for excitation and was coupled
into a single-mode fiber. The laser beam was passed through cleanup
filters (Chroma Technology, ZET561/10) and coupled into the microscope
objective using a beam splitter (Chroma Technology, ZT561rdc). Fluorescence
light was spectrally filtered with an emission filter (Chroma Technology,
ET600/50m and ET575lp) and imaged with an sCMOS camera (Andor, Zyla
4.2 Plus) without further magnification, resulting in an effective
pixel size of 130 nm after 2 × 2 binning. Camera readout sensitivity
was set to 16-bit, and readout bandwidth to 540 MHz. Imaging parameters
used in the different experiments are shown inTable S12 , and NeNA values are listed in Table S13 .
microscope (Nikon Instruments, Eclipse Ti2) and the Perfect Focus
System, by applying an objective-type total internal reflection fluorescence
(TIRF) configuration with an oil-immersion objective (Nikon Instruments,
Apo SR TIRF 100×, NA 1.49, oil). A 561 nm laser (MPB Communications
Inc., 500 mW, DPSS-system) was used for excitation and was coupled
into a single-mode fiber. The laser beam was passed through cleanup
filters (Chroma Technology, ZET561/10) and coupled into the microscope
objective using a beam splitter (Chroma Technology, ZT561rdc). Fluorescence
light was spectrally filtered with an emission filter (Chroma Technology,
ET600/50m and ET575lp) and imaged with an sCMOS camera (Andor, Zyla
4.2 Plus) without further magnification, resulting in an effective
pixel size of 130 nm after 2 × 2 binning. Camera readout sensitivity
was set to 16-bit, and readout bandwidth to 540 MHz. Imaging parameters
used in the different experiments are shown in
5
High-resolution TIRF Microscopy Imaging
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Fluorescence imaging was carried out on an inverted microscope (Nikon Instruments, Eclipse Ti2) with the Perfect Focus System, applying an objective-type TIRF configuration equipped with an oil-immersion objective (Nikon Instruments, Apo SR TIRF 100×, NA 1.49, Oil). A 561 nm and 642 nm laser (MPB Communications Inc., 2 W, DPSS-system) were used for excitation. The laser beams were passed through cleanup filters (Chroma Technology, ZET561/10, ZET 640/10) and coupled into the microscope objective using a beam splitter (Chroma Technology, ZT561rdc, ZT640rdc). Fluorescence light was spectrally filtered with an emission filter (Chroma Technology, ET600/50m and ET700/75m) and imaged on a sCMOS camera (Andor, Zyla 4.2 Plus) without further magnification, resulting in an effective pixel size of 130 nm (after 2×2 binning). Images were acquired choosing a region of interest with the size of 512×512 pixels. More detailed imaging conditions for the respective experiments are shown in Supplementary Table 8 .
6
TIRF Microscopy Protocol for Fluorescent Imaging
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The samples were measured on inverted total internal reflection fluorescence (TIRF) microscopes (Nikon Instruments, Eclipse Ti2) which are equipped with an oil-immersion objective (Nikon Instruments, Apo SR TIRF ×100/numerical aperture 1.49, oil) and a perfect focusing system. The mRuby3 signal was bleached by the 560 nm laser (MPB Communications, 1 W) by using Highly inclined and laminated optical sheet (HILO) illumination. Afterwards, the TIRF mode was established. The Cy3B imagers were excited with the 560 nm laser. The laser beam was cleaned with a filter (Chroma Technology, no. ZET561/10) and coupled into the microscope with a beam splitter (Chroma Technology, no. ZT561rdc). The fluorescent signal was filtered with an emission filter (Chroma Technology, nos. ET600/50m and ET575lp) and projected onto a sCMOS camera (Hamamatsu, ORCA-Fusion BT) without further magnification.
The acquired region of interest has a size of 576 × 576 pixels. The resulting effective pixel size is 130 nm. The raw microscopy data was acquired via μManager.
The acquired region of interest has a size of 576 × 576 pixels. The resulting effective pixel size is 130 nm. The raw microscopy data was acquired via μManager.
7
TIRF Microscopy Protocol for Fluorescence Imaging
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Experiments were performed using a prism–type TIRF microscope (Nikon) with two back–illuminated iXon EMCCDs (Andor Technology)61 (link). Illumination was provided by a 200 mW, 488–nm laser and a 150 mW, 561–nm laser (Coherent, Inc.). Intensity at prism face was ~14 mW and ~25 mW for the 488–nm and 561–nm lasers, respectively. Fluorescence signals were separated by a filter cube equipped with a dichroic mirror (ZT561rdc), band pass filter (ET525/50m), and long pass filter (ET575lp)(Chroma Technology Corp.).
8
High-resolution DNA-PAINT Microscopy
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DNA-PAINT imaging was carried out on an inverted microscope (Nikon Instruments, Eclipse Ti2) with the Perfect Focus System, applying an objective-type total internal reflection fluorescence (TIRF) configuration equipped with an oil-immersion objective (Nikon Instruments, Apo SR TIRF 100×, NA 1.49, oil). A 488-nm (200 mW, Toptica iBeam smart) or 561-nm laser (Coherent Sapphire, 200 mW) was used for excitation and was coupled into a single-mode fiber. The laser beam was passed through cleanup filters (Chroma Technology, ZET561/10) and coupled into the microscope objective using a beam splitter (Chroma Technology, ZT561rdc). Fluorescence light was spectrally filtered with an emission filter (Chroma Technology, ET600/50 m) and imaged with a scientific complementary metal-oxide semiconductor camera (Andor, Zyla 4.2plus) without further magnification, resulting in an effective pixel size of 130 nm after 2 × 2 binning. The camera readout sensitivity was set to 16-bit, and readout bandwidth to 540 MHz. Three-dimensional imaging was performed using a cylindrical lens (Nikon Instruments, N-STORM) in the detection path.
9
DNA-PAINT Imaging with Gold Fiducials
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Before imaging, 90 nm gold nanoparticle fiducial markers were added and incubated for 5 min (diluted 1:5 in PBS + 5 mM MgCl2). After rinsing with PBS with 5 mM MgCl2, imaging buffer containing DNA-PAINT imaging strands was added. DNA-PAINT imaging was carried out on an inverted Nikon Eclipse Ti microscope (Nikon Instruments) with the Perfect Focus System, applying an objective-type TIRF configuration with an oil-immersion objective (CFI Apo TIRF 100×, NA 1.49, Oil). Samples were excited using a 561 nm laser (200 mW nominal, Coherent Sapphire). The laser beam was passed through a cleanup filter (ZET561/10, Chroma Technology) and coupled into the microscope objective using a beam splitter (ZT561rdc, Chroma Technology). Fluorescence light was spectrally filtered with two emission filters (ET600/50m and ET575lp, Chroma Technology) and imaged on an sCMOS camera (Zyla 4.2, Andor Technologies). Imaging was performed without additional magnification in the detection path and 2×2 camera binning resulting in a pixel size of 130 nm.
10
Super-Resolution Fluorescence Imaging
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Fluorescence imaging was carried out on an inverted microscope (Nikon Instruments, Eclipse Ti2) with the Perfect Focus System, applying an objective-type TIRF configuration with an oil-immersion objective (Nikon Instruments, Apo SR TIRF 100×, NA 1.49, Oil). A 561 nm and 640 nm (MPB Communications Inc, 2W, DPSS-system) laser were used for excitation. The laser beam was passed through clean-up filters (Chroma Technology, ZET561/10, ZET642/20x) and coupled into the microscope objective using a beam splitter (Chroma Technology, ZT561rdc, ZT647rdc). Fluorescence light was spectrally filtered with an emission filter (Chroma Technology, ET600/50m and ET575lp, ET705/72m and ET665lp) and imaged on a sCMOS camera (Andor, Zyla 4.2 Plus) without further magnification, resulting in an effective pixel size of 130 nm (after 2 × 2 binning).
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