The setup was a single-molecule-sensitive,
wide-field microscope equipped with a single MEMS micromirror, which
has been previously described in detail.22 (link) Use of a NA 1.49, 60× oil immersion objective (APON60XOTIRF,
Olympus) and ∼1.8× postmagnification (OptoSplit II, Cairn)
led to an effective camera pixel size of 122 nm. The fluorescence
light was filtered with a zt532/640rpc dichroic mirror (Chroma) and
multibandpass filter ZET532/640 (Chroma), and imaged on a EMCCD camera
(iXon Life 888, Andor). For all measurements, the central area of
the EMCCD camera with 512 × 512 pixels was selected, thus resulting
in a total FOV of (62.5 μm)2; the camera was recording
30,000 frames at 20 Hz frame rate, except for the experiments on buffer
acidification with 15,000 frames at 10 Hz. Buffer acidification and
cell measurements were performed with a refractive beam shaping device
to generate a flat-field illumination (piShaper 6_6_VIS, AdlOptica),
whereas all other experiments were performed with an active MEMS micromirror.
The excitation intensity for the full FOV was measured to 0.48 kW
cm–2 for single-molecule photoswitching using the
MEMS illumination and 0.72 kW cm–2 for cell measurements
using the piShaper.
wide-field microscope equipped with a single MEMS micromirror, which
has been previously described in detail.22 (link) Use of a NA 1.49, 60× oil immersion objective (APON60XOTIRF,
Olympus) and ∼1.8× postmagnification (OptoSplit II, Cairn)
led to an effective camera pixel size of 122 nm. The fluorescence
light was filtered with a zt532/640rpc dichroic mirror (Chroma) and
multibandpass filter ZET532/640 (Chroma), and imaged on a EMCCD camera
(iXon Life 888, Andor). For all measurements, the central area of
the EMCCD camera with 512 × 512 pixels was selected, thus resulting
in a total FOV of (62.5 μm)2; the camera was recording
30,000 frames at 20 Hz frame rate, except for the experiments on buffer
acidification with 15,000 frames at 10 Hz. Buffer acidification and
cell measurements were performed with a refractive beam shaping device
to generate a flat-field illumination (piShaper 6_6_VIS, AdlOptica),
whereas all other experiments were performed with an active MEMS micromirror.
The excitation intensity for the full FOV was measured to 0.48 kW
cm–2 for single-molecule photoswitching using the
MEMS illumination and 0.72 kW cm–2 for cell measurements
using the piShaper.
Free full text: Click here
