Fluorescence
microscopy was performed at a custom inverted microscope described
in detail in a previous publication.31 (link) Light
from a solid-state laser (561 nm, DPSS-System, MPB) was intensity-adjusted
using a half-wave plate and a polarizing beam splitter (WPH05M-561
and PBS101, THORLABS). The beam passed through a refractive beam-shaping
device (piShaper 6_6_VIS, AdlOptica) to create a flat illumination
profile. To achieve evanescent-field illumination, the beam excentrically
entered the oil immersion objective lens (100× NA 1.49 UAPON,
Olympus). Fluorescence emission was collected by the same objective
and filtered through suitable band-pass filters (605/64, AHF Analsentechnik)
before detection on a CMOS camera (Zyla 4.2, Andor). During acquisitions,
the temperature was stabilized at 23 °C (H101-CRYO-BL, Okolab),
and z-positioning of the sample was stabilized via
a piezo stage (Z-INSERT100, Piezoconcept and CRISP, ASI). The camera
was operated with the open source acquisition software μManager32 (link) and images were acquired with 2 × 2 pixel2 binning and field of view cropping to the central 700 ×
700 (prebinned) pixels to achieve an effective pixel width of 130
nm and a field of view matching the circular flat illumination profile
ca. 130 μm in diameter.
microscopy was performed at a custom inverted microscope described
in detail in a previous publication.31 (link) Light
from a solid-state laser (561 nm, DPSS-System, MPB) was intensity-adjusted
using a half-wave plate and a polarizing beam splitter (WPH05M-561
and PBS101, THORLABS). The beam passed through a refractive beam-shaping
device (piShaper 6_6_VIS, AdlOptica) to create a flat illumination
profile. To achieve evanescent-field illumination, the beam excentrically
entered the oil immersion objective lens (100× NA 1.49 UAPON,
Olympus). Fluorescence emission was collected by the same objective
and filtered through suitable band-pass filters (605/64, AHF Analsentechnik)
before detection on a CMOS camera (Zyla 4.2, Andor). During acquisitions,
the temperature was stabilized at 23 °C (H101-CRYO-BL, Okolab),
and z-positioning of the sample was stabilized via
a piezo stage (Z-INSERT100, Piezoconcept and CRISP, ASI). The camera
was operated with the open source acquisition software μManager32 (link) and images were acquired with 2 × 2 pixel2 binning and field of view cropping to the central 700 ×
700 (prebinned) pixels to achieve an effective pixel width of 130
nm and a field of view matching the circular flat illumination profile
ca. 130 μm in diameter.
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