Fluorescence imaging was carried out using Nikon Ti-E microscope with CFI Apo TIRF objective with an NA of 1.49. The microscope was enclosed by a custom-made chamber that was pre-heated overnight and kept at 26-27 °C. For excitation of sfGFP or NirFP signal, cells were illuminated by Nikon-Intensilight illumination lamp through a GFP filter cube (λex / λbs / λem = 450-490 / 495 / 500-550 nm) or a RFP filter cube (λex / λbs / λem = 540-580 / 585 / 592 - 668). The fluorescence signal was recorded by an Andor iXon EMCCD camera. The MinD dynamics in this study was imaged with a frame time-interval of 5 seconds for 25 frames.
Cfi apo tirf objective
Manufactured by Nikon
The CFI Apo TIRF objective is a high-performance objective lens designed for Total Internal Reflection Fluorescence (TIRF) microscopy. It features a high numerical aperture and specialized optics to optimize performance for TIRF applications.
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6 protocols using cfi apo tirf objective
1
Fluorescence Imaging of MinD Dynamics
2
Optical Microscopy of Magnetic Colloids
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Optical microscopy images were obtained using a Nikon Eclipse Ti-E inverted microscope equipped with a DMK 23UX174 Digital Camera (The Imaging Source Europe GmbH, Bremen, Germany). A Nikon CFI Apo TIRF objective (100× magnification, N.A. 1.49) was used. Pictures were recorded in bright field mode. Samples were prepared by mixing desired amounts of stock solutions of superparamagnetic silica spheres, PEO and salt in vials and hollow glass tubes (0.1 × 2 × 50 mm, VitroCom) were filled with these mixtures by capillary action. The tubes were sealed with optical adhesive (Norland 81) that was cured with UV-light (wavelength of 365 nm, 6 W UVP UVGL-58 lamp) and sealed with a layer of nail polish to prevent solvent evaporation. Samples consisted of varying concentrations superparamagnetic colloids and PEO, and 10 mm sodium chloride. Sodium chloride was occasionally (partially) replaced by sodium azide (while keeping the total ionic strength constant) to suppress bacterial growth.
3
Multicolor Fluorescence Imaging Microscopy
4
Fluorescence Imaging of MinD Dynamics
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Fluorescence imaging was carried out using Nikon Ti-E microscope with CFI Apo TIRF objective with an NA of 1.49. The microscope was enclosed by a custom-made chamber that was pre-heated overnight and kept at 26-27 °C. For excitation of sfGFP or NirFP signal, cells were illuminated by Nikon-Intensilight illumination lamp through a GFP filter cube (λex / λbs / λem = 450-490 / 495 / 500-550 nm) or a RFP filter cube (λex / λbs / λem = 540-580 / 585 / 592 - 668). The fluorescence signal was recorded by an Andor iXon EMCCD camera. The MinD dynamics in this study was imaged with a frame time-interval of 5 seconds for 25 frames.
5
Visualizing Lysozyme Fibrils with Fluorescence Microscopy
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To visualize the fibrils in fluorescence microscopy experiments,
either labeled or unlabeled BSA was added to 1 mM lysozyme fibrils
(equivalent monomer concentration) in Tris buffer, pH7.4, to a final
BSA concentration of 10 μM. Fibrils were stained with 5 μM
thioflavin T (ThT, Sigma-Adrich). Images of the fibrils were obtained
using a Nikon Ti-E microscope with a 100× CFI APO TIRF objective
(Nikon) and an iXon 3 DU-870 EMCCD camera (Andor). ThT fluorescence
was excited with an argon laser using the 457 nm line. We made use
of a 455 nm excitation filter with a 10 nm bandpass and a 485 nm emission
filter with a 30 nm bandpass.
either labeled or unlabeled BSA was added to 1 mM lysozyme fibrils
(equivalent monomer concentration) in Tris buffer, pH7.4, to a final
BSA concentration of 10 μM. Fibrils were stained with 5 μM
thioflavin T (ThT, Sigma-Adrich). Images of the fibrils were obtained
using a Nikon Ti-E microscope with a 100× CFI APO TIRF objective
(Nikon) and an iXon 3 DU-870 EMCCD camera (Andor). ThT fluorescence
was excited with an argon laser using the 457 nm line. We made use
of a 455 nm excitation filter with a 10 nm bandpass and a 485 nm emission
filter with a 30 nm bandpass.
6
Microscopic Monitoring of Bacterial Colonies
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For imaging, colonies of various sizes and diameters were randomly chosen and imaged using an inverted microscope (Ti-E Nikon) equipped with a CSU-X1 (Yokogawa) spinning disk unit, a 100× CFI Apo Tirf objective (Nikon), an EMCCD camera (iXon 897 X-11662, Andor), and 488 nm and 561 nm excitation lasers. If not stated otherwise, colonies were imaged at their equator once every minute (acquisition time 100 ms) in the brightfield, green, and red channel (laser power 3% and 2%, respectively).
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