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 561 14 25
Manufactured by IDEX Corporation
Sourced in United States
The FF01-561/14-25 is a laser bandpass filter designed for use in scientific and industrial applications. It has a center wavelength of 561 nanometers and a bandwidth of 14 nanometers. The filter is housed in a 25-millimeter diameter mount.
Automatically generated - may contain errors
Lab products found in correlation
Ff01 417 60 25, by IDEX Corporation (1 mentions)
Di01 r405 488 561 635 25 36, by IDEX Corporation (1 mentions)
Blp02 561r 25, by IDEX Corporation (1 mentions)
Smz25, by Nikon (1 mentions)
Di02 r561 25 36, by IDEX Corporation (1 mentions)
Orca flash4.0 v2, by Hamamatsu Photonics (1 mentions)
Ff01 609 54 25, by IDEX Corporation (1 mentions)
Nis elements imaging software, by Nikon (1 mentions)
X10468 01, by Hamamatsu Photonics (1 mentions)
Lpvise100 a, by Thorlabs (1 mentions)
3 protocols using ff01 561 14 25
1
Super-Resolution Imaging of S. pombe
2
Ca2+ Imaging of Aspergillus nidulans
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Aspergillus nidulans expressing the genetically-encoded Ca2+ biosensor R-GECO was imaged with a motorized fluorescence stereo microscope (SMZ-25; Nikon) equipped with P2-SHR PLAN APO 1x/0.16 objective lens (Nikon) and an sCMOS camera (ORCA-Flash4.0 V2; Hamamatsu Photonics) (13 (link), 53 ). R-GECO was excited using a mercury lamp (Intensilight Hg Illuminator; Nikon), a 561/14-nm excitation filter (FF01-561/14-25, Semrock), and a 575-nm dichroic mirror (Di02-R561-25 × 36; Semrock). The red fluorescent signal passing through a 609/54-nm filter (FF01-609/54-25; Semrock) was acquired with the sCMOS camera using NIS-Elements imaging software (Nikon).
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Aspergillus nidulans expressing the genetically-encoded Ca2+ biosensor R-GECO was imaged with a motorized fluorescence stereo microscope (SMZ-25; Nikon) equipped with P2-SHR PLAN APO 1x/0.16 objective lens (Nikon) and an sCMOS camera (ORCA-Flash4.0 V2; Hamamatsu Photonics) (13 (link), 53 ). R-GECO was excited using a mercury lamp (Intensilight Hg Illuminator; Nikon), a 561/14-nm excitation filter (FF01-561/14-25, Semrock), and a 575-nm dichroic mirror (Di02-R561-25 × 36; Semrock). The red fluorescent signal passing through a 609/54-nm filter (FF01-609/54-25; Semrock) was acquired with the sCMOS camera using NIS-Elements imaging software (Nikon).
3
Optical Modulation and Fluorescence Imaging
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Optical setup is composed of two parts; the wavefront shaping part for the excitation beam and the fluorescence imaging part for measuring R-GECO1 signals. A continuous-wave laser diode (λ = 488 nm, 60 mW, Cobolt) is used to activate optoFGFR1 which absorbs a wavelength of 488 nm. A beam from the laser diode is expended by 4f lens system to cover whole active area of the SLM before passing through a half-wave plate (WPH10E-488, Thorlabs) and a polarizer (LPVISE100-A, Thorlabs). The SLM (X10468-01, Hamamatsu Photonics Inc. Japan) is placed at a conjugate image plane of the mouse skull. A CCD camera (Lt365R, Lumenera Inc., USA) is used as a detector for both the wavefront shaping and the fluorescence signals. For measuring fluorescence images, a LED (λ = 565 nm, 880 mW, Thorlabs) is used to excite the R-GECO1 of which excitation and emission peaks are 561 nm and 600 nm, respectively. Bandpass filters are used as excitation (FF01–561/14–25, Semrock, Rochester, NY, USA) and emission (FF01–607/36–25, Semrock, Rochester, NY, USA) filters.
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