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Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.

Wu Y, Wu X, Lu R, Zhang J, Toro L, Stefani E - Sci Rep (2015)

Bottom Line: Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing.The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy.We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.

ABSTRACT
Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy. With ≥6 GW∙cm(-2) depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 8-fold wider field of view. We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data agree with a theoretical model. Our results encourage further increasing the linear scanning speed for photobleaching reduction in STED microscopy.

No MeSH data available.


Related in: MedlinePlus

Photobleaching rates of piezo-stage and resonant scanning in STED microscopy.(a) Normalized image intensity of the 2nd image in the time series for Sample #2. Under the equal-irradiance condition (pixel size 110 × 100 nm), piezo-state caused about the same photobleaching as resonant scanning at Zoom 8, despite its exposure time-span is 100-fold longer. Under the equal-lines condition (pixel size 15 × 15 nm), piezo-stage resulted in much more severe photobleaching. (b–d) show fluorescence gain ratio  with respect to piezo-stage scanning as a function of exposure divisor n, under the equal-irradiance condition for Sample #2, Sample #1 and Sample #3, respectively. Data points (columns) were fitted to  expressed in Eq. (4) (solid lines). Fitted parameters are listed in Table 2.
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f7: Photobleaching rates of piezo-stage and resonant scanning in STED microscopy.(a) Normalized image intensity of the 2nd image in the time series for Sample #2. Under the equal-irradiance condition (pixel size 110 × 100 nm), piezo-state caused about the same photobleaching as resonant scanning at Zoom 8, despite its exposure time-span is 100-fold longer. Under the equal-lines condition (pixel size 15 × 15 nm), piezo-stage resulted in much more severe photobleaching. (b–d) show fluorescence gain ratio with respect to piezo-stage scanning as a function of exposure divisor n, under the equal-irradiance condition for Sample #2, Sample #1 and Sample #3, respectively. Data points (columns) were fitted to expressed in Eq. (4) (solid lines). Fitted parameters are listed in Table 2.

Mentions: Scanning speed of a piezo-stage is much slower than a resonant scanner (see Table 1). Under the equal-irradiance condition, Eq. (1) dictates that the pixel size of the piezo-stage ought to be 110 × 110 nm. This size would be too large for a practical STED imaging experiment. The equal-lines condition is what a practical STED imaging experiment would use, under which the pixel size was chosen to be the same as in resonant scanning (15 × 15 nm), the excitation laser power was reduced to 3.9 μW, and the depletion power was kept constant to preserve the optical resolution. In Fig. 7, we show the photobleaching comparison of piezo-stage and resonant scanning. Due to the slowness of piezo-stage, we only took two images for each time series to measure photobleaching (each image with 3 minutes of normalized imaging time). Figure 7a shows the normalized image intensity of the second image to quantify photobleaching in Sample #2. Under the equal-irradiance condition, though the exposure time of the piezo-stage is 100-fold longer than resonant scanning at Zoom 8, their photobleaching rates are about the same. Piezo-stage scanning under the more practical equal-lines condition caused much more severe photobleaching due to the excessive depletion illumination dose. For the four equal-irradiance cases, using the piezo-stage scanning case as the reference, the fluorescence gain as a function of exposure divisor n was plotted in Fig. 7b. Its profile is consistent with the function profile depicted in Fig. 3a. Piezo-stage scanning and Zoom 8 together show the slow entry, while Zoom 4, Zoom 2 and Zoom 1 roughly belong to the quasi-linearly growth phase of the profile. Similar results were obtained for Sample #1 and Sample #3, as illustrated in Fig. 7c,d, respectively. The experimental data points were fitted to the theoretical model expressed by Eq. (4), and the fitted parameter (k and δ) values are summarized in Table 2.


Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.

Wu Y, Wu X, Lu R, Zhang J, Toro L, Stefani E - Sci Rep (2015)

Photobleaching rates of piezo-stage and resonant scanning in STED microscopy.(a) Normalized image intensity of the 2nd image in the time series for Sample #2. Under the equal-irradiance condition (pixel size 110 × 100 nm), piezo-state caused about the same photobleaching as resonant scanning at Zoom 8, despite its exposure time-span is 100-fold longer. Under the equal-lines condition (pixel size 15 × 15 nm), piezo-stage resulted in much more severe photobleaching. (b–d) show fluorescence gain ratio  with respect to piezo-stage scanning as a function of exposure divisor n, under the equal-irradiance condition for Sample #2, Sample #1 and Sample #3, respectively. Data points (columns) were fitted to  expressed in Eq. (4) (solid lines). Fitted parameters are listed in Table 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589784&req=5

f7: Photobleaching rates of piezo-stage and resonant scanning in STED microscopy.(a) Normalized image intensity of the 2nd image in the time series for Sample #2. Under the equal-irradiance condition (pixel size 110 × 100 nm), piezo-state caused about the same photobleaching as resonant scanning at Zoom 8, despite its exposure time-span is 100-fold longer. Under the equal-lines condition (pixel size 15 × 15 nm), piezo-stage resulted in much more severe photobleaching. (b–d) show fluorescence gain ratio with respect to piezo-stage scanning as a function of exposure divisor n, under the equal-irradiance condition for Sample #2, Sample #1 and Sample #3, respectively. Data points (columns) were fitted to expressed in Eq. (4) (solid lines). Fitted parameters are listed in Table 2.
Mentions: Scanning speed of a piezo-stage is much slower than a resonant scanner (see Table 1). Under the equal-irradiance condition, Eq. (1) dictates that the pixel size of the piezo-stage ought to be 110 × 110 nm. This size would be too large for a practical STED imaging experiment. The equal-lines condition is what a practical STED imaging experiment would use, under which the pixel size was chosen to be the same as in resonant scanning (15 × 15 nm), the excitation laser power was reduced to 3.9 μW, and the depletion power was kept constant to preserve the optical resolution. In Fig. 7, we show the photobleaching comparison of piezo-stage and resonant scanning. Due to the slowness of piezo-stage, we only took two images for each time series to measure photobleaching (each image with 3 minutes of normalized imaging time). Figure 7a shows the normalized image intensity of the second image to quantify photobleaching in Sample #2. Under the equal-irradiance condition, though the exposure time of the piezo-stage is 100-fold longer than resonant scanning at Zoom 8, their photobleaching rates are about the same. Piezo-stage scanning under the more practical equal-lines condition caused much more severe photobleaching due to the excessive depletion illumination dose. For the four equal-irradiance cases, using the piezo-stage scanning case as the reference, the fluorescence gain as a function of exposure divisor n was plotted in Fig. 7b. Its profile is consistent with the function profile depicted in Fig. 3a. Piezo-stage scanning and Zoom 8 together show the slow entry, while Zoom 4, Zoom 2 and Zoom 1 roughly belong to the quasi-linearly growth phase of the profile. Similar results were obtained for Sample #1 and Sample #3, as illustrated in Fig. 7c,d, respectively. The experimental data points were fitted to the theoretical model expressed by Eq. (4), and the fitted parameter (k and δ) values are summarized in Table 2.

Bottom Line: Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing.The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy.We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.

ABSTRACT
Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy. With ≥6 GW∙cm(-2) depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 8-fold wider field of view. We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data agree with a theoretical model. Our results encourage further increasing the linear scanning speed for photobleaching reduction in STED microscopy.

No MeSH data available.


Related in: MedlinePlus