2.2 Mounting Media:
Composition
and Choice
Gloxy [6]: Cysteamine hydrochloride 50 mM, glucose 10% w/v,
glucose oxidase 0.5 mg/ml, catalase 40 μg/ml in TN buffer
(50 mM Tris (pH 8.0) and 10 mM NaCl).
We use the following stock solutions:
1. Cysteamine hydrochloride (Sigma-Aldrich M6500, 1 M in
H 2 O, stored at À20
C).
2. Glucose 400 g/l, stored at À20
C.
3. Glucose Oxidase (Sigma-Aldrich G2133, 5 mg/ml, stored at
4
C).
4. Catalase (Sigma-Aldrich C3515, 4 mg/ml, stored at 4
C).
Vectashield/TDE [14]: Vectashield 20% v/v, TDE 70% v/v in
PBS.
1. Vectashield
®
Antifade Mounting Medium (Vectorlabs
H-1000, stored at +4
C).
2. 2,2
0 -Thiodiethanol (TDE, Sigma-Aldrich 166782).
Mix 20% v/v Vectashield with 70% v/v TDE and 10% v/v
PBS 10Â. For mounting, incubate in solutions with increasing
concentrations of TDE in PBS: 10%, 25%, 50%, 10 min each.
Then replace with the final solution, Vectashield/TDE.
OxEA [15]: Cysteamine hydrochloride 50 mM, OxyFluor™ 3% v/v, sodium DL-lactate 20% v/v in PBS adjusted
to pH 8–8.5 with 1 M aqueous NaOH.
We use the following stock solutions:
1. Cysteamine hydrochloride (Sigma-Aldrich M6500, 1 M in
PBS, stored at À20
C).
2. OxyFluor™ (Sigma-Aldrich SAE0059, stored at À20
C) [16].
3. Sodium DL-lactate (Sigma-Aldrich L1375, 60% w/w syrup,
stored at +4
C).
2.3 Instrumentation
The super-resolution experiments were performed on a Leica SR
GSD system built on the base of the DMI6000 B inverted widefield microscope, consisting of the HCX PL APO 100Â/1.47 Oil
CORR TIRF PIFOC objective and 1.6Â magnification lens that
provide an equivalent pixel size of 100 nm on an EMCCD camera
(Andor iXon3 DU-897U-CS0-#BV); continuous wave fiber lasers
(MPBC Inc., 488 nm 300 mW, 532 nm 1000 mW, 642 nm
500 mW), a diode laser (405 nm 30 mW) and the suppressed
motion (SuMo) sample stage.
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Composition
and Choice
Gloxy [6]: Cysteamine hydrochloride 50 mM, glucose 10% w/v,
glucose oxidase 0.5 mg/ml, catalase 40 μg/ml in TN buffer
(50 mM Tris (pH 8.0) and 10 mM NaCl).
We use the following stock solutions:
1. Cysteamine hydrochloride (Sigma-Aldrich M6500, 1 M in
H 2 O, stored at À20
C).
2. Glucose 400 g/l, stored at À20
C.
3. Glucose Oxidase (Sigma-Aldrich G2133, 5 mg/ml, stored at
4
C).
4. Catalase (Sigma-Aldrich C3515, 4 mg/ml, stored at 4
C).
Vectashield/TDE [14]: Vectashield 20% v/v, TDE 70% v/v in
PBS.
1. Vectashield
®
Antifade Mounting Medium (Vectorlabs
H-1000, stored at +4
C).
2. 2,2
0 -Thiodiethanol (TDE, Sigma-Aldrich 166782).
Mix 20% v/v Vectashield with 70% v/v TDE and 10% v/v
PBS 10Â. For mounting, incubate in solutions with increasing
concentrations of TDE in PBS: 10%, 25%, 50%, 10 min each.
Then replace with the final solution, Vectashield/TDE.
OxEA [15]: Cysteamine hydrochloride 50 mM, OxyFluor™ 3% v/v, sodium DL-lactate 20% v/v in PBS adjusted
to pH 8–8.5 with 1 M aqueous NaOH.
We use the following stock solutions:
1. Cysteamine hydrochloride (Sigma-Aldrich M6500, 1 M in
PBS, stored at À20
C).
2. OxyFluor™ (Sigma-Aldrich SAE0059, stored at À20
C) [16].
3. Sodium DL-lactate (Sigma-Aldrich L1375, 60% w/w syrup,
stored at +4
C).
2.3 Instrumentation
The super-resolution experiments were performed on a Leica SR
GSD system built on the base of the DMI6000 B inverted widefield microscope, consisting of the HCX PL APO 100Â/1.47 Oil
CORR TIRF PIFOC objective and 1.6Â magnification lens that
provide an equivalent pixel size of 100 nm on an EMCCD camera
(Andor iXon3 DU-897U-CS0-#BV); continuous wave fiber lasers
(MPBC Inc., 488 nm 300 mW, 532 nm 1000 mW, 642 nm
500 mW), a diode laser (405 nm 30 mW) and the suppressed
motion (SuMo) sample stage.
Practical Aspects of Super-Resolution Imaging and Segmentation of. . .
273