Limits...
Simple buffers for 3D STORM microscopy.

Olivier N, Keller D, Rajan VS, Gönczy P, Manley S - Biomed Opt Express (2013)

Bottom Line: This usually relies on the utilization of complex buffers, containing different chemicals and sensitive enzymatic systems, limiting the reproducibility of the method.We report here that the commercial mounting medium Vectashield can be used for STORM of Alexa-647, and yields images comparable or superior to those obtained with more complex buffers, especially for 3D imaging.We expect that this advance will promote the versatile utilization of 3D STORM by removing one of its entry barriers, as well as provide a more reproducible way to compare optical setups and data processing algorithms.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Experimental Biophysics, School of Basic Sciences, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland.

ABSTRACT
3D STORM is one of the leading methods for super-resolution imaging, with resolution down to 10 nm in the lateral direction, and 30-50 nm in the axial direction. However, there is one important requirement to perform this type of imaging: making dye molecules blink. This usually relies on the utilization of complex buffers, containing different chemicals and sensitive enzymatic systems, limiting the reproducibility of the method. We report here that the commercial mounting medium Vectashield can be used for STORM of Alexa-647, and yields images comparable or superior to those obtained with more complex buffers, especially for 3D imaging. We expect that this advance will promote the versatile utilization of 3D STORM by removing one of its entry barriers, as well as provide a more reproducible way to compare optical setups and data processing algorithms.

No MeSH data available.


3D STORM of Alexa-647-labeled microtubules in Vectashield: (A) Imaging performed in 25% Vectashield-75 % TRIS-Glycerol, scale-bar = 5 μm. (B1&2): axial profile taken from the two regions delimited in A (yellow for (B1), showing a single microtubule; red for (B2) showing two well-resolved microtubules crossing at a distance of ≈ 160 nm).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3675867&req=5

g006: 3D STORM of Alexa-647-labeled microtubules in Vectashield: (A) Imaging performed in 25% Vectashield-75 % TRIS-Glycerol, scale-bar = 5 μm. (B1&2): axial profile taken from the two regions delimited in A (yellow for (B1), showing a single microtubule; red for (B2) showing two well-resolved microtubules crossing at a distance of ≈ 160 nm).

Mentions: We performed 3D-STORM imaging by adding an astigmatic lens in our detection path, and used the ellipticity of the measured spots to determine the z-position of each detected molecule [17, 18] (see Section 2.3 for more details). Figure 6 shows a 3D-STORM image of microtubules immunostained with Alexa-647 imaged in a mixture of 25% Vectashield-75% TRIS-Glycerol (which also has an index of refraction of ≈1.45), where the depth information is color-coded. We quantified the axial resolution in Fig. 6(B1) by fitting the axial profile of a straight section of microtubule over 200 nm with a Gaussian function, and obtained a full-width at half maximum (FWHM) of 74 nm. Since the nano-structure of microtubules is known, we can deconvolve from this value the size of microtubules, and estimate our axial resolution as ≈ 40–50 nm. Moreover, we can see from the criss-crossing microtubules shown in Fig. 6(B2) that aberrations due to the index mismatch do not significantly degrade the resolution over several hundreds of nanometers in depth, as the size of the furthest microtubule from the coverslip is not so different from the one closest. Vectashield is therefore well suited for 3D STORM imaging; importantly this is not limited to 3D using astigmatism, and other 3D methods such as biplane imaging [24] or interferometric imaging [25] can benefit from using this buffer.


Simple buffers for 3D STORM microscopy.

Olivier N, Keller D, Rajan VS, Gönczy P, Manley S - Biomed Opt Express (2013)

3D STORM of Alexa-647-labeled microtubules in Vectashield: (A) Imaging performed in 25% Vectashield-75 % TRIS-Glycerol, scale-bar = 5 μm. (B1&2): axial profile taken from the two regions delimited in A (yellow for (B1), showing a single microtubule; red for (B2) showing two well-resolved microtubules crossing at a distance of ≈ 160 nm).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675867&req=5

g006: 3D STORM of Alexa-647-labeled microtubules in Vectashield: (A) Imaging performed in 25% Vectashield-75 % TRIS-Glycerol, scale-bar = 5 μm. (B1&2): axial profile taken from the two regions delimited in A (yellow for (B1), showing a single microtubule; red for (B2) showing two well-resolved microtubules crossing at a distance of ≈ 160 nm).
Mentions: We performed 3D-STORM imaging by adding an astigmatic lens in our detection path, and used the ellipticity of the measured spots to determine the z-position of each detected molecule [17, 18] (see Section 2.3 for more details). Figure 6 shows a 3D-STORM image of microtubules immunostained with Alexa-647 imaged in a mixture of 25% Vectashield-75% TRIS-Glycerol (which also has an index of refraction of ≈1.45), where the depth information is color-coded. We quantified the axial resolution in Fig. 6(B1) by fitting the axial profile of a straight section of microtubule over 200 nm with a Gaussian function, and obtained a full-width at half maximum (FWHM) of 74 nm. Since the nano-structure of microtubules is known, we can deconvolve from this value the size of microtubules, and estimate our axial resolution as ≈ 40–50 nm. Moreover, we can see from the criss-crossing microtubules shown in Fig. 6(B2) that aberrations due to the index mismatch do not significantly degrade the resolution over several hundreds of nanometers in depth, as the size of the furthest microtubule from the coverslip is not so different from the one closest. Vectashield is therefore well suited for 3D STORM imaging; importantly this is not limited to 3D using astigmatism, and other 3D methods such as biplane imaging [24] or interferometric imaging [25] can benefit from using this buffer.

Bottom Line: This usually relies on the utilization of complex buffers, containing different chemicals and sensitive enzymatic systems, limiting the reproducibility of the method.We report here that the commercial mounting medium Vectashield can be used for STORM of Alexa-647, and yields images comparable or superior to those obtained with more complex buffers, especially for 3D imaging.We expect that this advance will promote the versatile utilization of 3D STORM by removing one of its entry barriers, as well as provide a more reproducible way to compare optical setups and data processing algorithms.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Experimental Biophysics, School of Basic Sciences, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland.

ABSTRACT
3D STORM is one of the leading methods for super-resolution imaging, with resolution down to 10 nm in the lateral direction, and 30-50 nm in the axial direction. However, there is one important requirement to perform this type of imaging: making dye molecules blink. This usually relies on the utilization of complex buffers, containing different chemicals and sensitive enzymatic systems, limiting the reproducibility of the method. We report here that the commercial mounting medium Vectashield can be used for STORM of Alexa-647, and yields images comparable or superior to those obtained with more complex buffers, especially for 3D imaging. We expect that this advance will promote the versatile utilization of 3D STORM by removing one of its entry barriers, as well as provide a more reproducible way to compare optical setups and data processing algorithms.

No MeSH data available.