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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

3D super-resolution imaging of GFP-alpha-SAP97 (antibody labelled with an Alexa 647 secondary, green) and Synapsin (with an Alexa 750 secondary, red) in a primary hippocampal culture.4D imaging was performed using dual-colour 3D localisation based on astigmatism in conjunction with the ratiometric multi-colour approach. A. Comparison between detail in a super-resolution image (left) with the corresponding conventional diffraction limited image (right) of the two proteins. B & C. 3D rendering of the region indicated in A using super-resolution (B) and conventional resolution (C). Note that in the high resolution data (B) ‘lateral’ as well as ‘axial’ synapses can be distinguished. The 2D images shown on the axes are average projections along the respective directions. Scale bars A: 1 µm, B: 500 nm.
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pone-0020645-g004: 3D super-resolution imaging of GFP-alpha-SAP97 (antibody labelled with an Alexa 647 secondary, green) and Synapsin (with an Alexa 750 secondary, red) in a primary hippocampal culture.4D imaging was performed using dual-colour 3D localisation based on astigmatism in conjunction with the ratiometric multi-colour approach. A. Comparison between detail in a super-resolution image (left) with the corresponding conventional diffraction limited image (right) of the two proteins. B & C. 3D rendering of the region indicated in A using super-resolution (B) and conventional resolution (C). Note that in the high resolution data (B) ‘lateral’ as well as ‘axial’ synapses can be distinguished. The 2D images shown on the axes are average projections along the respective directions. Scale bars A: 1 µm, B: 500 nm.

Mentions: Ratiometric multi-colour localisation microscopy can be extended to full 3D localisation by introducing a cylindrical lens, analogous to single colour imaging [15]. With this setup (see also Figure 1A) we imaged neuronal cells in a primary hippocampal culture in which the pre-synaptic protein synapsin and the postsynaptic protein SAP97 were labelled with Alexa 750 and Alexa 647 (respectively). Figure 4 shows dual-colour 3D localisation images where synapses between axially adjacent processes are clearly resolved in the 3D localisation data, while difficult to discern at conventional resolution (Figure 4H). In tissue samples, an axial resolution of better than 65 nm (std. dev.) could be routinely achieved with a lateral resolution of ∼15 nm (std. dev.), corresponding to a PSF volume of <0.1 aL, a volume ∼300 times smaller than in diffraction-limited optical microscopy.


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)

3D super-resolution imaging of GFP-alpha-SAP97 (antibody labelled with an Alexa 647 secondary, green) and Synapsin (with an Alexa 750 secondary, red) in a primary hippocampal culture.4D imaging was performed using dual-colour 3D localisation based on astigmatism in conjunction with the ratiometric multi-colour approach. A. Comparison between detail in a super-resolution image (left) with the corresponding conventional diffraction limited image (right) of the two proteins. B & C. 3D rendering of the region indicated in A using super-resolution (B) and conventional resolution (C). Note that in the high resolution data (B) ‘lateral’ as well as ‘axial’ synapses can be distinguished. The 2D images shown on the axes are average projections along the respective directions. Scale bars A: 1 µm, B: 500 nm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020645-g004: 3D super-resolution imaging of GFP-alpha-SAP97 (antibody labelled with an Alexa 647 secondary, green) and Synapsin (with an Alexa 750 secondary, red) in a primary hippocampal culture.4D imaging was performed using dual-colour 3D localisation based on astigmatism in conjunction with the ratiometric multi-colour approach. A. Comparison between detail in a super-resolution image (left) with the corresponding conventional diffraction limited image (right) of the two proteins. B & C. 3D rendering of the region indicated in A using super-resolution (B) and conventional resolution (C). Note that in the high resolution data (B) ‘lateral’ as well as ‘axial’ synapses can be distinguished. The 2D images shown on the axes are average projections along the respective directions. Scale bars A: 1 µm, B: 500 nm.
Mentions: Ratiometric multi-colour localisation microscopy can be extended to full 3D localisation by introducing a cylindrical lens, analogous to single colour imaging [15]. With this setup (see also Figure 1A) we imaged neuronal cells in a primary hippocampal culture in which the pre-synaptic protein synapsin and the postsynaptic protein SAP97 were labelled with Alexa 750 and Alexa 647 (respectively). Figure 4 shows dual-colour 3D localisation images where synapses between axially adjacent processes are clearly resolved in the 3D localisation data, while difficult to discern at conventional resolution (Figure 4H). In tissue samples, an axial resolution of better than 65 nm (std. dev.) could be routinely achieved with a lateral resolution of ∼15 nm (std. dev.), corresponding to a PSF volume of <0.1 aL, a volume ∼300 times smaller than in diffraction-limited optical microscopy.

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