Table 3 Important factors to consider for a successful SMLM experiment
  • Labeling considerations

Type of fluorophore
  • - Control wild-type strain for specific autofluorescence in different spectral read-out channels

  • - Evaluate the illumination intensities for different wavelengths for your specific organism. Which wavelengths and doses are tolerated, what are signs of phototoxicity? Generally, fluorophores of long wavelength and with low level of ROS production are favorable

  • - Evaluate your targets applicable orthogonal multi-targeting methods which go alongside with some fluorophore choices, e.g. FP utility depends on biologically undisturbed genetic fusions, dyes are extrinsic labels which are often not membrane-permeable and thus often need fixation protocols, etc. Signal detection at longer wavelengths typically improves S/N ratio, lowers phototoxicity and increases maximal imaging depth

  • - Check for fluorophore-pair photophysics, e.g. brightness, bleaching rate, reversible blinking rates, preferably in the environment of your organism; check dye pairs for compatible switching buffers

  • - Check FPs for compatible maturation times (e.g. faster than POI replenishment) and possible oligomerization tendencies (commonly dependent on POI abundance and density)

Functionality controls
  • - Control for biological function and localization of POIs by growth and functionality assays, western blot, tagging of POI by different fluorophore tags, C- or N-terminal or in-loop tagging variants, etc.

  • - Minor growth and functionality deficiencies might be compensated for by slower growth at lower temperatures

Sample preparation
  • - Use transparent, defined, sterile filtered medium to avoid background. If possible, avoid fluorescent supplements in medium

  • - Harvest cells in exponential growth phase, not in stationary phase

  • - The optimal temperature for growth is not necessarily the optimal temperature for imaging, growth at lower temperatures might lead to less background in cells (but needs additional assays controlling for altered functionality)

Ectopic induction of POI
  • - Prepare fresh inducer stock from powder to avoid degradation effects ensuring reproducible induction conditions

  • - Use only minimal concentrations of inducer (typically only a fraction of amounts from standard protocols) as overexpression of POI might cause high background fluorescence, inclusion bodies, aggregates or phenotype artifacts

  • - After induction, allow for sufficient FP maturation time before imaging

  • - Optimal fixation conditions are target-, fluorophore- and organism-dependent. In general, fixation with 1–4% formaldehyde (final concentration) for 15–30 min is a good start. Addition of 0.05%–1% glutaraldehyde (final concentration) can improve the fixation results. Alternatively, also ice-cold methanol fixation can be tested

  • - To quench excess formaldehyde, the first washing step with PBS should contain sodium borohydride or ammonium chloride

  • - Several washing steps are needed to remove all excess formaldehyde

  • - Carefully check if the fluorescence of the label or the spatial organization of your target is impaired by fixation

  • - Charge and size of dyes can lead to unspecific or insufficient staining

  • - Blocking with neutral or charge masking compounds and/or intense washing with buffers containing higher salt concentrations (>100 mM) and low concentrations of detergent might reduce unspecific staining

  • - Cell membrane permeabilization and cell wall digestion improves staining

  • - Prolonged staining combined with low (up to 1000-fold lower than conventional immunofluorescence) covalent dye/label concentrations can improve S/N ratio

  • - Use only minimal dye concentrations for live cell staining by electroporation/membrane-permeable dyes to avoid remaining free dyes

  • - Perform a control staining of a sample without the target epitope to evaluate the degree of non-specific staining

  • - For non-covalent labels with fixable groups, a finalizing post-fixation step following staining and washing prevents detachment of labels over time, which can be caused by the addition of thiol-containing imaging buffers

Cover glass slides
  • - Use high-precision cover glasses with defined thickness and matching the specifications of your objective

  • - Clean thoroughly, prepare fresh

  • - For agarose pads, use high purity grade low gelling agarose to minimize heat degradation effects of the media which causes background and growth impairments

  • - For multi-well cover glasses, immobilize sample firmly onto a cover glass surface using poly-l-lysine (or organism-specific substances, e.g. ConA for targeting the α-linked mannose residues of the S. cerevisiae polysaccharides)

General buffers
  • - Use high-quality chemicals with high purity grade for minimizing contaminants causing background

  • - Prepare fresh and sterile filtered buffers

  • - Use buffer with high salt concentration to wash out unwanted fluorescence (e.g. fluorescent metabolites, free fluorophores)

Fiducial markers
  • - Sonicate thoroughly prior to loading onto the sample to avoid fiducial aggregates

  • - Adjust concentration to a density of at least three fiducials in focus in a typical ROI

  • - Match fiducial brightness with sample brightness to prevent superposing the single-molecule signals during imaging

  • - For imaging in two parallel channels: calibration slide (e.g. a fine spatial grid) of multi-colored fiducials for channel overlay

  • - Check if sample quality (sample appearance, S/N and photoswitching efficiencies) is preserved after (long-term) storage at 4°C

  • - Addition of sodium azide to fixed samples prevents the growth of contaminants

Imaging conditions
Laser power
  • - Control laser power post-objective before each experiment

  • - Adjust laser power (e.g. activation illuminations) for constant fluorophore blinking at sufficiently low density

  • - Check for fluorophore bleaching

Illumination mode
  • - Laser power and background in the target plane change with applied imaging mode (epifluorescence, light sheet, HILO, TIRF)

  • - Pulsing of the photoactivating lasers can reduce possible phototoxicity and offers temporal control for fluorophore activation

Switching buffers
  • - Quality demands as for general buffers above

  • - Switching buffers have to be adjusted to both dyes of the selected dye combination

  • - pH or redox components influence fluorophore switching behavior

  • - Apply oxygen removing buffers directly before imaging and tightly seal the sample to prevent uptake of new atmospheric oxygen

  • - Replace buffer regularly as enzymatic buffer exhaust themselves over time and might cause pH changes (e.g. GLOX buffer drops pH)

Imaging conditions and parameters
  • - Use appropriate immersion oil for your objective and avoid air bubbles in the oil

  • - If implemented, use a focus-stabilizing system to avoid z-drift

  • - Image fluorophores with excitation maxima at longer wavelengths first to avoid photobleaching and cross-talk

  • - Camera frame rate should be fast enough to temporally resolve the molecule of interest kinetics

  • - For structural studies, single fluorophore blinks should be recorded in only a few camera frames for maximal S/N

  • - For 3D read-out: match the spatial resolution needed to answer your biological question with a compatible 3D technique. Read-out range and sensitivity is different for each 3D method

Imaging controls
  • - Negative: to check autofluorescence/background in all spectral channels used, a wild-type strain, a without correct epitope stained sample, for drug studies check non-treated strains

  • - Positive: easy-to-image “standard strain” to control for stable setup configuration and thus constant read-out quality (S/N, photoswitching efficiencies) and to check for proper sample preparations.

  • - Check for phototoxicity effects in live cell studies

  • - When imaging dynamics: prepare controls for (i) the freely diffusive cytosolic fluorophore(s) used as labels to benchmark the purely diffusive signal distribution (e.g. for confinement effects of small microbial volumes, possible inclusion bodies for overexpression) and (ii) a fixed control to access the immobile signal distribution (where the apparent movement is only determined by the acquired localization precision)

Post-processing prior data analysis
Localization routine
  • - Check if the chosen localization algorithm fits fluorescent spots reliably

  • - Determine the experimentally achieved localization precision

  • - Check for fluorophore recall rates and false positives

Drift correction
  • - Use fiducial markers to correct for x–y-drift (by bead traces or cross-correlation) or apply cross-correlation on the target directly if possible (needs high fluorophore densities per frame, typically only possible for large ROIs and samples with highly abundant target molecule)

Channel alignment
  • - Parallel read-out: use a dense, ideally fine spatial grid as calibration sample for channel alignment before your experiment

  • - For sequential read-out in the same spectral channel, fiducial markers are sufficient to overlay the image sequences

  • - Choose a visualization reflecting your achieved resolution to avoid interpretation errors (in co-localization, clustering analysis, etc.)

  • - Choose a visualization with well-adjusted intensity scaling to mimic real fluorescence images one is used to

Data analysis
  • - Characterize and quantify for over- and undercounting bias/error in your measurements

  • - Optimize clustering algorithm thresholds/parameters to identify clusters properly while at the same time avoiding merging clusters into one cluster and omitting sparse molecules

  • - Check and correct for self-clustering artifacts of blinking probes

  • - For live cell samples measured for long observation times (e.g. in sequential imaging modes or for long parallel read-out), control for possible target movements during the read-out time

  • - Consider filtering trajectories for sufficient length (e.g. >6 steps) to provide enough statistics to extract robust diffusion characteristics

The table gives an overview of common tips and tricks and discusses the pitfalls of an SMLM experiment sorted by the different stages from study design over sample preparation to data analysis. Factors explicitly relevant for microbial samples are marked in bold; factors relevant for multi-color imaging in italics and bold.