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4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.

Baddeley D, Crossman D, Rossberger S, Cheyne JE, Montgomery JM, Jayasinghe ID, Cremer C, Cannell MB, Soeller C - PLoS ONE (2011)

Bottom Line: Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%.The number of marker species that can be distinguished depends on the mean photon number of single molecule events.Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine and Health Sciences, University of Auckland, Auckland, New Zealand.

ABSTRACT

Background: Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample.

Methodology/principal findings: We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk.

Conclusions/significance: Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.

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Related in: MedlinePlus

Two-dimensional super-resolution imaging of the distribution of Ryanodine receptors (red) and Caveolin (green), using Alexa 680 and Alexa 750 secondaries, in the periphery of isolated rat cardiac myocytes and overview of dye properties.Panel A shows the sample at conventional resolution, panel B the super-resolved image. Comparison of enlarged detail (C & D) shows that apparent overlap in the diffraction-limited images is not seen in the corresponding super-resolution image. E. Histogram of mean photon number per event of a dataset of ∼400 ratiometric super-resolution images. The mean photon numbers were calculated for each image in the dataset, the histogram of actual photon numbers per single molecule event are shown in panel F. Scale bars B: 1 µm, D: 200 nm.
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pone-0020645-g002: Two-dimensional super-resolution imaging of the distribution of Ryanodine receptors (red) and Caveolin (green), using Alexa 680 and Alexa 750 secondaries, in the periphery of isolated rat cardiac myocytes and overview of dye properties.Panel A shows the sample at conventional resolution, panel B the super-resolved image. Comparison of enlarged detail (C & D) shows that apparent overlap in the diffraction-limited images is not seen in the corresponding super-resolution image. E. Histogram of mean photon number per event of a dataset of ∼400 ratiometric super-resolution images. The mean photon numbers were calculated for each image in the dataset, the histogram of actual photon numbers per single molecule event are shown in panel F. Scale bars B: 1 µm, D: 200 nm.

Mentions: This method of almost cross-talk free dual-colour super-resolution imaging was used to investigate the spatial relationship between two types of protein, the membrane protein caveolin-3 (CAV3) and the ryranodine receptor (RyR), a protein located on the sarcoplasmic reticulum, near the surface membrane of cardiac ventricular myocytes [18]. Using indirect immunofluorescence labelling and the dye pair Alexa 680 and Alexa 750 to label RyR and CAV3, respectively, we obtained dual colour super-resolution images (Figure 2A,B). Both proteins form clusters or aggregates and many areas that appear to contain co-localizing structures at conventional resolution (Figure 2A) often showed little overlap in the corresponding region of the super-resolution image. The increased information in the super-resolution data was used to calculate improved estimates of co-localizing protein fractions. Based on the diffraction limited data (Figure 2A) 28.6% of CAV3 were co-localized with RyRs while the co-localizing fraction in the super-resolution data (Figure 2B) was only 4.9%, indicating that the apparent partial co-localization between the proteins at diffraction-limited resolution is almost entirely due to optical blurring.


4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.

Baddeley D, Crossman D, Rossberger S, Cheyne JE, Montgomery JM, Jayasinghe ID, Cremer C, Cannell MB, Soeller C - PLoS ONE (2011)

Two-dimensional super-resolution imaging of the distribution of Ryanodine receptors (red) and Caveolin (green), using Alexa 680 and Alexa 750 secondaries, in the periphery of isolated rat cardiac myocytes and overview of dye properties.Panel A shows the sample at conventional resolution, panel B the super-resolved image. Comparison of enlarged detail (C & D) shows that apparent overlap in the diffraction-limited images is not seen in the corresponding super-resolution image. E. Histogram of mean photon number per event of a dataset of ∼400 ratiometric super-resolution images. The mean photon numbers were calculated for each image in the dataset, the histogram of actual photon numbers per single molecule event are shown in panel F. Scale bars B: 1 µm, D: 200 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105105&req=5

pone-0020645-g002: Two-dimensional super-resolution imaging of the distribution of Ryanodine receptors (red) and Caveolin (green), using Alexa 680 and Alexa 750 secondaries, in the periphery of isolated rat cardiac myocytes and overview of dye properties.Panel A shows the sample at conventional resolution, panel B the super-resolved image. Comparison of enlarged detail (C & D) shows that apparent overlap in the diffraction-limited images is not seen in the corresponding super-resolution image. E. Histogram of mean photon number per event of a dataset of ∼400 ratiometric super-resolution images. The mean photon numbers were calculated for each image in the dataset, the histogram of actual photon numbers per single molecule event are shown in panel F. Scale bars B: 1 µm, D: 200 nm.
Mentions: This method of almost cross-talk free dual-colour super-resolution imaging was used to investigate the spatial relationship between two types of protein, the membrane protein caveolin-3 (CAV3) and the ryranodine receptor (RyR), a protein located on the sarcoplasmic reticulum, near the surface membrane of cardiac ventricular myocytes [18]. Using indirect immunofluorescence labelling and the dye pair Alexa 680 and Alexa 750 to label RyR and CAV3, respectively, we obtained dual colour super-resolution images (Figure 2A,B). Both proteins form clusters or aggregates and many areas that appear to contain co-localizing structures at conventional resolution (Figure 2A) often showed little overlap in the corresponding region of the super-resolution image. The increased information in the super-resolution data was used to calculate improved estimates of co-localizing protein fractions. Based on the diffraction limited data (Figure 2A) 28.6% of CAV3 were co-localized with RyRs while the co-localizing fraction in the super-resolution data (Figure 2B) was only 4.9%, indicating that the apparent partial co-localization between the proteins at diffraction-limited resolution is almost entirely due to optical blurring.

Bottom Line: Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%.The number of marker species that can be distinguished depends on the mean photon number of single molecule events.Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine and Health Sciences, University of Auckland, Auckland, New Zealand.

ABSTRACT

Background: Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample.

Methodology/principal findings: We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk.

Conclusions/significance: Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.

Show MeSH
Related in: MedlinePlus