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.


STORM imaging of microtubules (see section 2.2 for more details) in Vectashield. (A): Widefield image (B): Single frame, (C1): Reconstructed STORM image, with blow-up on the ROI in (C2). scale-bar = 5 μm for (A),(B),(C1) and 1 μm for C2.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3675867&req=5

g001: STORM imaging of microtubules (see section 2.2 for more details) in Vectashield. (A): Widefield image (B): Single frame, (C1): Reconstructed STORM image, with blow-up on the ROI in (C2). scale-bar = 5 μm for (A),(B),(C1) and 1 μm for C2.

Mentions: In eukaryotic cells, microtubules form extended polymer networks from dimers of α- and β- tubulin. Each microtubule is a polymer tube of 25 nm in outer diameter [19], well below the diffraction limit. To test Vectashield as a STORM buffer for Alexa-647, we used as a test-sample fixed cells in which microtubules were immunostained with primary antibodies against α-tubulin and with secondary antibody fragments labeled with Alexa-647 (see Section 2.2). We then simply added Vectashield (see Section 2.2) turned on our excitation laser to full power (≈ 1.5 kW.cm−2) and recorded 10.000 frames. Figure 1 shows a typical dataset, with Fig. 1(A) showing an image reconstructed by projecting the mean frame intensity, and subtracting from it the projected minimum pixel intensity, yielding an image which is similar to a widefield image. After an initial bleaching step lasting a few frames (typically 10–20), single molecule blinking could be observed, as shown in Fig. 1(B) where we display the raw data captured in frame 2500. A sparse population of dyes can be seen in the fluorescent state, as is required to localize them individually. We reconstructed a STORM image (Fig. 1(C1)) from all localized molecules (according to Section 2.3) with a higher magnification view given in Fig. 1(C2) Since there is high temporal variability in the fluorescence signal, this data could also be processed with a SOFI [12] algorithm, and would yield an image of resolution intermediate between the STORM and the widefield images.


Simple buffers for 3D STORM microscopy.

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

STORM imaging of microtubules (see section 2.2 for more details) in Vectashield. (A): Widefield image (B): Single frame, (C1): Reconstructed STORM image, with blow-up on the ROI in (C2). scale-bar = 5 μm for (A),(B),(C1) and 1 μm for C2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g001: STORM imaging of microtubules (see section 2.2 for more details) in Vectashield. (A): Widefield image (B): Single frame, (C1): Reconstructed STORM image, with blow-up on the ROI in (C2). scale-bar = 5 μm for (A),(B),(C1) and 1 μm for C2.
Mentions: In eukaryotic cells, microtubules form extended polymer networks from dimers of α- and β- tubulin. Each microtubule is a polymer tube of 25 nm in outer diameter [19], well below the diffraction limit. To test Vectashield as a STORM buffer for Alexa-647, we used as a test-sample fixed cells in which microtubules were immunostained with primary antibodies against α-tubulin and with secondary antibody fragments labeled with Alexa-647 (see Section 2.2). We then simply added Vectashield (see Section 2.2) turned on our excitation laser to full power (≈ 1.5 kW.cm−2) and recorded 10.000 frames. Figure 1 shows a typical dataset, with Fig. 1(A) showing an image reconstructed by projecting the mean frame intensity, and subtracting from it the projected minimum pixel intensity, yielding an image which is similar to a widefield image. After an initial bleaching step lasting a few frames (typically 10–20), single molecule blinking could be observed, as shown in Fig. 1(B) where we display the raw data captured in frame 2500. A sparse population of dyes can be seen in the fluorescent state, as is required to localize them individually. We reconstructed a STORM image (Fig. 1(C1)) from all localized molecules (according to Section 2.3) with a higher magnification view given in Fig. 1(C2) Since there is high temporal variability in the fluorescence signal, this data could also be processed with a SOFI [12] algorithm, and would yield an image of resolution intermediate between the STORM and the widefield images.

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.