The detection system was set up on an epi-fluorescence microscope (Nikon Eclipse Ti2). A multi-laser engine (Toptica Photonics, Munich, Germany) was used for selective fluorescence excitation of CFP, GFP, YFP, RFP, and Alexa-647/Cy5 at 405, 488, 516, 561, and 640 nm, respectively. The samples were illuminated in total internal reflection (TIR) configuration (Nikon Ti-LAPP) using a 60x oil immersion objective (NA = 1.49, APON 60XO TIRF). After appropriate filtering using standard filter sets, the fluorescence was imaged onto a sCMOS camera (Zyla 4.2, Andor, Northern Ireland). The samples were mounted on an x-y-stage (CMR-STG-MHIX2-motorized table, Märzhäuser, Germany), and scanning of the larger areas was supported by a laser-guided automated Perfect Focus System (Nikon PFS).
Multi laser engine
Manufactured by Toptica
Sourced in Germany
The Multi-laser engine is a compact and versatile laser system that combines multiple laser sources into a single output. It is designed to provide researchers and scientists with a flexible and reliable platform for various applications. The core function of the Multi-laser engine is to deliver precise and stable laser light, enabling users to easily integrate it into their experimental setups.
Automatically generated - may contain errors
4 protocols using multi laser engine
1
Multicolor Fluorescence Microscopy Setup
2
Quantitative Live-Cell Imaging Protocol
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For live cell experiments, respective wells were incubated with 100 μl streptavidin solution (50 μg/ml; Sigma Aldrich) for 30 min at room temperature, followed by rigorous washing with PBS. Subsequently, biotinylated anti‐GFP antibody (10 μg/ml; antibodies‐online) was incubated for a further 30 min. Prior to cell seeding, wells were washed again with PBS. Cells were allowed to attach to the antibody‐patterned surface for at least 3–4 h. Total internal reflection fluorescence (TIRF) microscopy was carried out on an epi‐fluorescence microscope (Nikon Eclipse Ti2), where the samples were illuminated in TIR configuration (Nikon Ti‐LAPP) using a 60× oil immersion objective (NA = 1.49, APON 60XO TIRF). A multi‐laser engine (Toptica Photonics) was used for selective fluorescence excitation of CFP and YFP at 405 and 516 nm, respectively. After appropriate filtering using standard filter sets, the fluorescence was imaged onto a sCMOS camera (Zyla 4.2, Andor). The samples were mounted on an x‐y‐stage (CMR‐STG‐MHIX2‐motorized table, Märzhäuser), and scanning of the larger areas was supported by a laser‐guided automated Perfect Focus System (Nikon PFS). Quantitation of fluorescence contrast was carried out as previously.38
3
Multimodal Fluorescence Imaging Setup
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The detection system
was set up on
an epi-fluorescence microscope (Nikon Eclipse Ti2). A multilaser engine
(Toptica Photonics, Munich, Germany) was used for selective fluorescence
excitation of CFP, GFP, RFP, and Cy5 at 405, 488, 561, and 640 nm,
respectively. The samples were illuminated in TIR configuration (Nikon
Ti-LAPP) using a 60× oil immersion objective (NA = 1.49, APON
60XO TIRF). After appropriate filtering using standard filter sets,
the fluorescence was imaged onto a sCMOS camera (Zyla 4.2, Andor,
Northern Ireland). The samples were mounted on an x-y-stage (CMR-STG-MHIX2-motorized table, Märzhäuser,
Germany), and scanning of the larger areas was supported by a laser-guided
automated Perfect Focus System (Nikon PFS).
was set up on
an epi-fluorescence microscope (Nikon Eclipse Ti2). A multilaser engine
(Toptica Photonics, Munich, Germany) was used for selective fluorescence
excitation of CFP, GFP, RFP, and Cy5 at 405, 488, 561, and 640 nm,
respectively. The samples were illuminated in TIR configuration (Nikon
Ti-LAPP) using a 60× oil immersion objective (NA = 1.49, APON
60XO TIRF). After appropriate filtering using standard filter sets,
the fluorescence was imaged onto a sCMOS camera (Zyla 4.2, Andor,
Northern Ireland). The samples were mounted on an x-y-stage (CMR-STG-MHIX2-motorized table, Märzhäuser,
Germany), and scanning of the larger areas was supported by a laser-guided
automated Perfect Focus System (Nikon PFS).
4
Single-Molecule Imaging of R. sphaeroides
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R. sphaeroides cultures containing the appropriate PAmCherry fusions on pIND4 were grown photoheterotrophically under high and low light in the presence of 1 mM IPTG. Cells were placed on 1% agarose pads and imaged on a custom-built PALM/TIRF microscope at Micron Advanced Imaging Consortium, University of Oxford. The microscope was equipped with an Andor iXon 897 ultra electron multiplying CCD (EMCCD) camera and a Toptica Multi Laser Engine with 405 nm, 488 nm, 561 nm, and 640 nm lasers.
Bright field images of the targeted cells from a 64 × 64 pixels field of view (FOV) were recorded before imaging. The 564-nm laser was used to minimize the cellular autofluorescence. After the prebleach step, the cells were imaged with a 405-nm (336 μW) laser for photoactivation and a 70% (9.5 mW) 564-nm laser for excitation of PAmCherry. The intensity of the 405-nm laser was increased gradually as the PAmCherry molecules in the cells started to photobleach over time. Around 6 movies (800 frames each) were recorded for each FOV. Exposure time was 7 ms, and a cycle time was 7.75 ms. PAmCherry filter set ET600/50 (575–625 nm) was used.
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Bright field images of the targeted cells from a 64 × 64 pixels field of view (FOV) were recorded before imaging. The 564-nm laser was used to minimize the cellular autofluorescence. After the prebleach step, the cells were imaged with a 405-nm (336 μW) laser for photoactivation and a 70% (9.5 mW) 564-nm laser for excitation of PAmCherry. The intensity of the 405-nm laser was increased gradually as the PAmCherry molecules in the cells started to photobleach over time. Around 6 movies (800 frames each) were recorded for each FOV. Exposure time was 7 ms, and a cycle time was 7.75 ms. PAmCherry filter set ET600/50 (575–625 nm) was used.
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