Sample Preparation for GSDIM Localization Microscopy – Protocols and Tips

The preservation of a specific cellular structure strongly depends on the fixation method. The exact fixation protocol should be optimized for the structure of interest. For the majority of structures a Paraformaldehyde / Formaldehyde fixation is sufficient. Small amounts of Glutaraldehyde (0.05 % to 0.2 % (v/v)) in addition to Paraformaldehyde / Formaldehyde as a fixant can strongly improve structure preservation. Glutaraldehyde can induce a hazy fluorescent background therefore quenching with ammonium, NaBH₄ or other suitable reagents is recommended. Methanol fixation is common for microtubule preservation, but might not be sufficient for other structures.

Super-resolution imaging requires excellent optical performance throughout the whole optical system – not only within the microscope and objective. The coverslip is a major parameter with high impact on imaging results. To obtain optimal super-resolution image quality a flat coverslip is required.

Unfortunately, while being uncritical for standard widefield applications, not all chambered coverslip products can guarantee the degree of flatness optimal for GSD. Furthermore, chambered coverslip products may not provide the necessary mechanical stability for high-resolution imaging. The respective products should be tested by the researcher for their suitability. A better flatness and high stability can be generally achieved by stress free mounting of a glass coverslip, e.g. in specially designed coverslip holders.

In our case, we recommend an alternative method for easy and flat coverslip mounting specifically tailored for GSD imaging utilizing high precision coverslips and a silicone glue to fasten and seal the specimen (see below).
 

Reagents

• β-Mercaptoethylamine (MEA) (e.g. Sigma-Aldrich, # 30070, also known as Cysteamine)
• Phosphate Buffered Saline (PBS)
• HCl / NaOH for pH adjustment
  
  

Buffer

PBS containing 10 mM β-Mercaptoethylamine (MEA), adjusted to pH 7.4. Dissolve the β-Mercaptoethylamine in PBS and adjust to pH 7.4. The β-Mercaptoethylamine concentration can be varied over a wide range to optimize the GSD image – the usable range is in general between 10 and 100 mM (recommended concentrations for testing 10 / 30 / 100 mM).

β-Mercaptoethylamine solutions must be freshly prepared before use. Alternatively, stock solutions can be prepared and stored at –20°C and freshly thawed before use. The frozen aliquots can be stored for several weeks.

Illumination

EPI: yes
TIRF: yes

Reagents

• Glucose Oxidase (e.g. Sigma-Aldrich G2133)
• Catalase (e.g. Sigma-Aldrich C1345)
• Glucose
• Phosphate Buffered Saline (PBS)
• HCl / NaOH for pH adjustment
  
  

Buffer

PBS containing 10 % (w/v) glucose, 0.5 mg/mL glucoseoxidase and 40 μg/mL catalase adjusted to pH 7.4.
Note: It is recommended to prepare a glucose stock solution and mix the reagents freshly before mounting the coverslip.

Illumination

EPI: yes
TIRF: yes

Reagents

• Polyvinyl alcohol (PVA) with a molecular weight of 25,000, 88 mol% hydrolyzed (e.g. Polysciences, #02975-500)
• Phosphate Buffered Saline (PBS)
• HCl / NaOH for pH adjustment
  
  

Hardware

Spincoater

Buffer

Prepare a solution of 1 % PVA in PBS. Adjust the pH to 7.4.

Spincoating

Place the coverslip on the spincoater and dry the coverslip with brief 5–10 sec spin at approx. 3,000 rpm. Drop 50 μl of the PVA-solution onto the sample and spin the sample at approx. 3,000 rpm for approximately 30 s. Let the PVA-film dry for a few minutes and mount the sample. The sample can be stored at room temperature and used for GSD imaging for up to three months.

No additional mounting medium is required. The PVA-film is protecting the sample.

Illumination

EPI: yes
TIRF: not possible

Reagents

• Glycerol (analytical grade)
• Mowiol 4-88 (e.g. Calbiochem #475904)
• Distilled water
• TRIS
• HCl / NaOH for pH adjustment
  
  

Embedding medium

• Start with 6 g Glycerol
• Add 2.4 g Mowiol 4-88
• Add 6 ml Aqua dest.
• Add 12 ml 0.2 M TRIS buffer pH 8
• Stir for 4 hours (magnetic stirrer)
• Let suspension rest for 2 hours
• Incubate for 10 min at 50 °C (water bath)
• Centrifuge at 5,000 x g for 15 min
• Take the supernatant and freeze it in aliquots at –20 °C
  
  

Illumination

EPI: yes
TIRF: not possible

Reagents

• Vectashield^® (e.g. Vector Laboratories, # H-1000)
• 50 mM Tris in Glycerol
• HCl / NaOH for pH adjustment
  
  

Buffer

• 50 mM Tris/Glycerol buffer containing 10 % Vectashield^® adjusted to pH 7.4.
• Dissolve Tris in Glycerol to prepare a 50 mM stock solution. Mix 10 % Vectashield^® with 90 % Tris/Glycerol buffer. Adjust to pH 7.4. Samples can be stored over several weeks at 4 °C.
  
  

Illumination

EPI: yes
TIRF: not possible

GSDIM is suitable to create super-resolution images using fluorescent proteins. E.g. the fluorescent protein YFP / Venus displayed astonishing results. Furthermore, other fluorescent proteins should work with this technology (including GFP). To combine the power of organic fluorophores with the advantages of genetically encoded fusion proteins (e.g. if no suitable antibody is available), the use of tags like SNAP-, Halo- or CLIP-Tag is recommended. These tags can be genetically linked to the protein of interest and later on labeled with organic fluorophores (e.g. after fixation and permeabilization).

The following aqueous embedding was successfully used to image YFP in fixed and permeabilized mammalian cells:

• Catalase SIGMA, C1345 40 ug/mL
• Glucose Oxidase SIGMA G2133 0.5 mg/mL
• Glucose 10 mg/mL
• β-Mercaptoethylamine (Cysteamine) 1 mM
• Diluted in PBS, adjusted to pH 7.4
  
  

Fig. 3: PtK2 cells with AlexaFluor 647 stained microtubules in red and AlexaFluor 488 stained clathrin in green. Courtesy of Prof. Dr. Stefan Hell, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.

The Leica SR GSD 3D is equipped with a 405 nm laser. The lifetime of fluorophores in the off state can be shortened by illuminating the sample additionally with UV-light. The shorter lifetime leads to an increase of fluorophores in the on state and therefore can be used to increase the number of detected events per frame. By keeping the number of events per frame in an optimal range, it is possible to a) efficiently detect well-separated single fluorophores and b) keep the time to acquire a super-resolution image short, because it is possible to accumulate the required number of events within fewer images.

When acquiring the longer wavelength channel of dual-color images, it is important to use the 405 nm laser carefully! The 405 nm wavelength can excite green dyes and therefore bleach the shorter wavelength fluorophores while acquiring the longer wavelength super-resolution image. The optimal settings should be determined for each sample type separately.

Protocol Guide for the Leica SR GSD 3D