allows to image deeper in the sample as compared to TIRF
microscopy but allows to keep low background intensity
[17]. HiLo illumination can be used with any mounting
medium.
3. Labeling method. Cysteamine- or β-mercaptoethanol-containing buffers may cleave antibodies which can decrease the specific labeling and increase background, while TDE-containing
media destabilize phalloidin labeling [18].
4. Possibility to refresh the buffer. Gloxy buffer needs to be
replaced each 2–4 h, OxEA—each 6–12 h, while Vectashieldbased buffers do not need to be replaced.
3.2 Basic SMLM
Experiment
Workflow of acquisition processing:
A typical acquisition session is composed of the following steps.
1. Preview. It consists of the search of the ROI and focusing using
conventional epifluorescence or TIRF/HiLo illumination with
low laser power (1–5% of maximal power). In the case of the
TIRF/HiLo mode the best direction (azimuth) of laser illumination to a sample has to be chosen as well. The step is completed by capturing a wide-field image (Fig. 1a). It is important
that the dSTORM experiment will be carried out under the
same conditions (x, y, z position, epi/TIRF, direction of laser
illumination if TIRF/HiLo) to have a direct comparison with
the classical (epifluorescence) microscopy image and avoid artifacts in the super-resolution image.
2. Pumping. Illumination of the sample with high intensity light
in order to bring most fluorescent molecules into the dark
state. At the very beginning, when all the molecules are in the
bright state, due to very strong excitation, the fluorescence is
very intense because all the dyes emit at the same time. After a
little time (in the order of seconds, depending on kinetics of the
fluorophore and excitation intensity), with occupation of the
dark states and depletion of the on state, the intensity of
fluorescence decreases because progressively less molecules
stay in the on state until the number of shining molecules
becomes less than one per diffraction-limited region. At this
time one begins to see the light from each “on” molecule
separately, and it is the time to start the acquisition, the next
step of the experiment.
3. Acquisition. With constant laser intensity, which may be as high
as during pumping or lower, one gets a number of shining
single-molecule events (“blinks”) per frame with certain exposure time (Fig. 1b). This number is progressively dropping with
photobleaching of the molecules, but it can be increased using
“backpumping” (e.g., with a 405-nm laser). With gradual
increase of backpumping laser intensity, it is possible to keep
Practical Aspects of Super-Resolution Imaging and Segmentation of. . .
275
microscopy but allows to keep low background intensity
[17]. HiLo illumination can be used with any mounting
medium.
3. Labeling method. Cysteamine- or β-mercaptoethanol-containing buffers may cleave antibodies which can decrease the specific labeling and increase background, while TDE-containing
media destabilize phalloidin labeling [18].
4. Possibility to refresh the buffer. Gloxy buffer needs to be
replaced each 2–4 h, OxEA—each 6–12 h, while Vectashieldbased buffers do not need to be replaced.
3.2 Basic SMLM
Experiment
Workflow of acquisition processing:
A typical acquisition session is composed of the following steps.
1. Preview. It consists of the search of the ROI and focusing using
conventional epifluorescence or TIRF/HiLo illumination with
low laser power (1–5% of maximal power). In the case of the
TIRF/HiLo mode the best direction (azimuth) of laser illumination to a sample has to be chosen as well. The step is completed by capturing a wide-field image (Fig. 1a). It is important
that the dSTORM experiment will be carried out under the
same conditions (x, y, z position, epi/TIRF, direction of laser
illumination if TIRF/HiLo) to have a direct comparison with
the classical (epifluorescence) microscopy image and avoid artifacts in the super-resolution image.
2. Pumping. Illumination of the sample with high intensity light
in order to bring most fluorescent molecules into the dark
state. At the very beginning, when all the molecules are in the
bright state, due to very strong excitation, the fluorescence is
very intense because all the dyes emit at the same time. After a
little time (in the order of seconds, depending on kinetics of the
fluorophore and excitation intensity), with occupation of the
dark states and depletion of the on state, the intensity of
fluorescence decreases because progressively less molecules
stay in the on state until the number of shining molecules
becomes less than one per diffraction-limited region. At this
time one begins to see the light from each “on” molecule
separately, and it is the time to start the acquisition, the next
step of the experiment.
3. Acquisition. With constant laser intensity, which may be as high
as during pumping or lower, one gets a number of shining
single-molecule events (“blinks”) per frame with certain exposure time (Fig. 1b). This number is progressively dropping with
photobleaching of the molecules, but it can be increased using
“backpumping” (e.g., with a 405-nm laser). With gradual
increase of backpumping laser intensity, it is possible to keep
Practical Aspects of Super-Resolution Imaging and Segmentation of. . .
275