by optimizing the processing parameters. For example, to
reduce the noise level, it is necessary to increase the threshold
of single-molecule detection, if it was quite low during the
experiment. Vice versa, if the threshold was pretty high, it is
desirable to reduce it in order to get more events and obtain a
better reconstructed image. The frames with undesirable artifacts (non-blinking areas, especially in the very first frames)
should be truncated, thus also improving the results.
3.3 Experimental
Parameters of dSTORM
Data Collection
and Processing
Numerous experimental parameters that can be changed by the
user are summarized here.
1. Laser power, can be chosen differently for pumping, acquisition, and backpumping. Strong excitation intensity during
acquisition increases the speed of “blinking,” reduces the number of “on” molecules, and ensures faster completion of the
acquisition. However, too strong excitation can also lead to
increase of the background brightness and reduction of the
number of the localized molecules. This parameter has to be
adjusted for each combination of fluorophore-imaging buffer.
Backpumping intensity is normally set to zero for pumping and
for the beginning of acquisition and is turned on with gradually
increasing intensity when the number of events per frame
drops.
2. Frame exposure time. Ideally, it has to be equal to the average
on-time of the fluorophores. If the exposure time is too short,
molecules will appear on many consecutive frames, their signal
to noise ratio (SNR) will be reduced. On the other side, if the
exposure time is too long, the density of localizations per frame
will be higher and the SNR can also be reduced because the
background is acquired during the whole frame exposure time
while the signal from the fluorophores is detected only during
their on-time.
3. Illumination mode: epi-/HiLo/TIRF, azimuth of laser illumination (when available), affects the penetration depth and the
axial resolution. It is advised to make pumping in
epi-illumination mode to send higher energy to the specimen
and move all the volume of the sample to the dark state; this
provides lower background during the acquisition.
4. EM camera gain for EMCCD detectors. Normally it is set up to
maximum, but to be diminished in case of saturation due to
strong fluorescence and/or high exposure times (i.e., adjust
and calibrate the dynamic range of the camera).
5. Threshold of single-molecular detection in photons/pixel.
Affects the real-time reconstructed super-resolution image
(if available); it can be changed in the post-processing step.
Usually lower threshold increases the noise level, a high
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277
reduce the noise level, it is necessary to increase the threshold
of single-molecule detection, if it was quite low during the
experiment. Vice versa, if the threshold was pretty high, it is
desirable to reduce it in order to get more events and obtain a
better reconstructed image. The frames with undesirable artifacts (non-blinking areas, especially in the very first frames)
should be truncated, thus also improving the results.
3.3 Experimental
Parameters of dSTORM
Data Collection
and Processing
Numerous experimental parameters that can be changed by the
user are summarized here.
1. Laser power, can be chosen differently for pumping, acquisition, and backpumping. Strong excitation intensity during
acquisition increases the speed of “blinking,” reduces the number of “on” molecules, and ensures faster completion of the
acquisition. However, too strong excitation can also lead to
increase of the background brightness and reduction of the
number of the localized molecules. This parameter has to be
adjusted for each combination of fluorophore-imaging buffer.
Backpumping intensity is normally set to zero for pumping and
for the beginning of acquisition and is turned on with gradually
increasing intensity when the number of events per frame
drops.
2. Frame exposure time. Ideally, it has to be equal to the average
on-time of the fluorophores. If the exposure time is too short,
molecules will appear on many consecutive frames, their signal
to noise ratio (SNR) will be reduced. On the other side, if the
exposure time is too long, the density of localizations per frame
will be higher and the SNR can also be reduced because the
background is acquired during the whole frame exposure time
while the signal from the fluorophores is detected only during
their on-time.
3. Illumination mode: epi-/HiLo/TIRF, azimuth of laser illumination (when available), affects the penetration depth and the
axial resolution. It is advised to make pumping in
epi-illumination mode to send higher energy to the specimen
and move all the volume of the sample to the dark state; this
provides lower background during the acquisition.
4. EM camera gain for EMCCD detectors. Normally it is set up to
maximum, but to be diminished in case of saturation due to
strong fluorescence and/or high exposure times (i.e., adjust
and calibrate the dynamic range of the camera).
5. Threshold of single-molecular detection in photons/pixel.
Affects the real-time reconstructed super-resolution image
(if available); it can be changed in the post-processing step.
Usually lower threshold increases the noise level, a high
Practical Aspects of Super-Resolution Imaging and Segmentation of. . .
277