threshold reduces the number of events, so a compromise value
should be found. It is convenient to follow which spots on the
images are detected and adjust the threshold in a way that only
the single-molecule spots are detected (Fig. 1c).
6. Pixel size for real-time reconstruction (Fig. 1d), if available.
Commonly set to around 20 nm, and can be reduced for postprocessing (e.g., 5 or 10 nm sampling).
3.4 3D SMLM
Experiment
One of the easiest and most common ways for 3D SMLM imaging
is realized by the modification of the point spread function (PSF) of
the microscope with an astigmatic aberration.
Calibration. The astigmatic PSF deformation as a function of
the axial position of fluorophores should be calibrated by imaging
fluorescent beads using several known axial positions of the
objective.
1. Install a sample with fluorescent beads with subdiffraction size
in the microscope. For example, multi-color TetraSpeck™
Microspheres, 0.1 or 0.2 μm, can be used.
2. Put in place the cylindrical lens or activate astigmatism in the
adaptive optics module.
3. Focus the microscope in a way that the images of the beads are
closest to circular symmetry.
4. Adjust the imaging parameters (excitation intensity, exposure
time, and electron-multiplying gain of the camera) to get good
SNR. It is preferable to increase first the excitation intensity in
order to keep the exposure time minimal.
5. Image the beads as a Z-stack with a step of 50 nm around
Æ0.6–1 μm of the focus point determined in step 3.
6. Repeat the procedure (steps 1–5) for each spectral channel.
7. Focus the microscope on the beads in one of the spectral
channels. Image the beads through all the channels in this
Z-position. It is necessary to take into account axial chromatic
aberration.
8. Fit all the acquired images with a software for single-molecule
detection, which will be used for the following experiments.
Refer to the manual of the software.
Once the calibration is done, 3D SMLM experiments can
be performed with the conventional SMLM procedure, with
the difference that the maximal density of switched-on molecules on a frame should be reduced to avoid overlap of
stretched PSF spots.
3.5 SMLM Data
Processing
The first step in SMLM data processing is the determination of the
localization of each individual fluorophore to provide a full event
list from which a super-resolution image can be reconstructed. The
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should be found. It is convenient to follow which spots on the
images are detected and adjust the threshold in a way that only
the single-molecule spots are detected (Fig. 1c).
6. Pixel size for real-time reconstruction (Fig. 1d), if available.
Commonly set to around 20 nm, and can be reduced for postprocessing (e.g., 5 or 10 nm sampling).
3.4 3D SMLM
Experiment
One of the easiest and most common ways for 3D SMLM imaging
is realized by the modification of the point spread function (PSF) of
the microscope with an astigmatic aberration.
Calibration. The astigmatic PSF deformation as a function of
the axial position of fluorophores should be calibrated by imaging
fluorescent beads using several known axial positions of the
objective.
1. Install a sample with fluorescent beads with subdiffraction size
in the microscope. For example, multi-color TetraSpeck™
Microspheres, 0.1 or 0.2 μm, can be used.
2. Put in place the cylindrical lens or activate astigmatism in the
adaptive optics module.
3. Focus the microscope in a way that the images of the beads are
closest to circular symmetry.
4. Adjust the imaging parameters (excitation intensity, exposure
time, and electron-multiplying gain of the camera) to get good
SNR. It is preferable to increase first the excitation intensity in
order to keep the exposure time minimal.
5. Image the beads as a Z-stack with a step of 50 nm around
Æ0.6–1 μm of the focus point determined in step 3.
6. Repeat the procedure (steps 1–5) for each spectral channel.
7. Focus the microscope on the beads in one of the spectral
channels. Image the beads through all the channels in this
Z-position. It is necessary to take into account axial chromatic
aberration.
8. Fit all the acquired images with a software for single-molecule
detection, which will be used for the following experiments.
Refer to the manual of the software.
Once the calibration is done, 3D SMLM experiments can
be performed with the conventional SMLM procedure, with
the difference that the maximal density of switched-on molecules on a frame should be reduced to avoid overlap of
stretched PSF spots.
3.5 SMLM Data
Processing
The first step in SMLM data processing is the determination of the
localization of each individual fluorophore to provide a full event
list from which a super-resolution image can be reconstructed. The
278
Leonid Andronov et al.