S. pombe cells with mEos3.1-tagged proteins were imaged with a custom-built inverted microscope (Olympus IX71) fitted with a motorized stage (Prior H117E1I4), using a 561-nm imaging laser (Cobolt, Jive) and a 405-nm activation laser (LaserBoxx, Oxxius). Each laser line displayed a quarter-wave plate (Thorlabs WPQ05M-405 and -561) and a low pass filter (Semrock FF01-417/60-25 and FF01-561/14-25). Both laser beams were expanded and collimated with a custom-built beam expander constituted of two matching lenses (Thorlabs LC1975 and LA1986), and coupled using a dichroic mirror (Semrock FF552-Di02-25). The resulting beams were focused to the back focal plane of an apochromatic 1.45 NA, 60× TIRF objective (Olympus, UIS2 APON 60× OTIRF) using a coated plane convex lens (Thorlabs LA1253-A). A multi-band dichroic mirror (Semrock Di01-R405/488/561/635-25 36), a band-pass filter (Semrock FF01-580/14-25) and a longpass filter (Semrock BLP02-561R-25) were used to separate fluorescence signal from the laser emission. The emission beam was further enlarged by a 2.5 beam expander, leading to an optimized pixel size of 107 nm/pixel after projection onto the EMCCD camera (Photometrics Evolve 512).
Ff01 417 60 25
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The FF01-417/60-25 is a lab equipment product manufactured by IDEX Corporation. It is designed for use in scientific and research applications. The core function of this product is to filter and purify fluid samples, but a more detailed description cannot be provided while maintaining an unbiased and factual approach.
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2 protocols using ff01 417 60 25
1
Super-Resolution Imaging of S. pombe
2
Optimized Fluorescence Microscopy Setup
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The microscope used is an Olympus BX51, with a Chroma filter cube “Qdot set with long pass emission filter“(set 32013 Exciter E460SPUVv2; emitter E500lp; Dichroic 475dcxru). For the comparison of LED illumination sources in the 2nd part of the study, the set was improved by changing the excitation filter with a Semrock FF01-417/60-25 (laser2000) (average transmission >90% over 60nm compared with 70% for E460SPUVv2) (S1 File ). The data presented was captured using a x40 objective (Olympus UplanFl 40x/0.75 ∞/0.17); brighter objectives (40x Olympus UPlanSApo 40x/0.95 ∞/0.11–0.23/FN26.5 and 20x Olympus UPlanSApo 20x/0.75 ∞/0.17/FN26.5) reduced exposure times by 30–50% (S2 Table ).
The Cy5 filter was Cy5-4040C (Semrock) and Cy3 filter Cy3-4040C (Semrock) adapted with a reduced band emission filter FF01- 543/22 (Semrock) to prevent bleed through).
The Cy5 filter was Cy5-4040C (Semrock) and Cy3 filter Cy3-4040C (Semrock) adapted with a reduced band emission filter FF01- 543/22 (Semrock) to prevent bleed through).
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