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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

Schematic depicting optical setup for ratiometric localisation imaging.A. A single laser at 671 nm is used to provide excitation and the collected light is split into two bands using a dichroic mirror. The bands are imaged side by side on an electron multiplying CCD (EMCCD). An optional cylindrical lens allows astigmatism based 3D localisation. B. A vector field that shows the distribution of lateral chromatic shifts between the channels measured with a bead calibration sample. This vector field was used for chromatic shift compensation during fitting of the single molecule events as detailed in the Methods. The longest arrows shown correspond to a shift magnitude of ∼90 nm. C. Single molecule events are observed as flashes with an intensity component in each channel. When these intensities are plotted against each other, discreet populations emerge corresponding to each fluorochrome in the sample. One such plot, obtained from a sample in which neurons had been transfected with GFP-alpha-sap97 and subsequently labeled with antibodies against GFP (Alexa 647 secondary) and synapsin (Alexa 750 secondary) is shown. Inset: Recorded emission spectra of Alexa 647 (green) and Alexa 750 (red), the black trace is the transmission curve of the dichroic mirror in the splitter device.
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pone-0020645-g001: Schematic depicting optical setup for ratiometric localisation imaging.A. A single laser at 671 nm is used to provide excitation and the collected light is split into two bands using a dichroic mirror. The bands are imaged side by side on an electron multiplying CCD (EMCCD). An optional cylindrical lens allows astigmatism based 3D localisation. B. A vector field that shows the distribution of lateral chromatic shifts between the channels measured with a bead calibration sample. This vector field was used for chromatic shift compensation during fitting of the single molecule events as detailed in the Methods. The longest arrows shown correspond to a shift magnitude of ∼90 nm. C. Single molecule events are observed as flashes with an intensity component in each channel. When these intensities are plotted against each other, discreet populations emerge corresponding to each fluorochrome in the sample. One such plot, obtained from a sample in which neurons had been transfected with GFP-alpha-sap97 and subsequently labeled with antibodies against GFP (Alexa 647 secondary) and synapsin (Alexa 750 secondary) is shown. Inset: Recorded emission spectra of Alexa 647 (green) and Alexa 750 (red), the black trace is the transmission curve of the dichroic mirror in the splitter device.

Mentions: A single inexpensive 671 nm diode laser could induce reversible photochemical conversion [7], [17] in a number of near-infrared fluorescent dyes including Alexa 647, 680, & 750 that are commercially available as antibody conjugates. The stochastic ‘blinking’ of the fluorescent labels was enhanced by a ‘switching’ buffer containing a primary thiol [7] (for details see Materials and Methods). Single molecule events were detected in two channels by placing a dichroic mirror into the detection path. This split the image into two components which we call here ‘short’ (680–740 nm) and ‘long’ (740–8301 nm) channels. The images at these wavelength were digitized with a single electron multiplying charge-coupled device (emCCD) camera by focussing each of the two images simultaneously onto two halves of the camera (see Figure 1A). To add axial position encoding, a cylindrical lens was used to introduce astigmatism in the images [15].


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)

Schematic depicting optical setup for ratiometric localisation imaging.A. A single laser at 671 nm is used to provide excitation and the collected light is split into two bands using a dichroic mirror. The bands are imaged side by side on an electron multiplying CCD (EMCCD). An optional cylindrical lens allows astigmatism based 3D localisation. B. A vector field that shows the distribution of lateral chromatic shifts between the channels measured with a bead calibration sample. This vector field was used for chromatic shift compensation during fitting of the single molecule events as detailed in the Methods. The longest arrows shown correspond to a shift magnitude of ∼90 nm. C. Single molecule events are observed as flashes with an intensity component in each channel. When these intensities are plotted against each other, discreet populations emerge corresponding to each fluorochrome in the sample. One such plot, obtained from a sample in which neurons had been transfected with GFP-alpha-sap97 and subsequently labeled with antibodies against GFP (Alexa 647 secondary) and synapsin (Alexa 750 secondary) is shown. Inset: Recorded emission spectra of Alexa 647 (green) and Alexa 750 (red), the black trace is the transmission curve of the dichroic mirror in the splitter device.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020645-g001: Schematic depicting optical setup for ratiometric localisation imaging.A. A single laser at 671 nm is used to provide excitation and the collected light is split into two bands using a dichroic mirror. The bands are imaged side by side on an electron multiplying CCD (EMCCD). An optional cylindrical lens allows astigmatism based 3D localisation. B. A vector field that shows the distribution of lateral chromatic shifts between the channels measured with a bead calibration sample. This vector field was used for chromatic shift compensation during fitting of the single molecule events as detailed in the Methods. The longest arrows shown correspond to a shift magnitude of ∼90 nm. C. Single molecule events are observed as flashes with an intensity component in each channel. When these intensities are plotted against each other, discreet populations emerge corresponding to each fluorochrome in the sample. One such plot, obtained from a sample in which neurons had been transfected with GFP-alpha-sap97 and subsequently labeled with antibodies against GFP (Alexa 647 secondary) and synapsin (Alexa 750 secondary) is shown. Inset: Recorded emission spectra of Alexa 647 (green) and Alexa 750 (red), the black trace is the transmission curve of the dichroic mirror in the splitter device.
Mentions: A single inexpensive 671 nm diode laser could induce reversible photochemical conversion [7], [17] in a number of near-infrared fluorescent dyes including Alexa 647, 680, & 750 that are commercially available as antibody conjugates. The stochastic ‘blinking’ of the fluorescent labels was enhanced by a ‘switching’ buffer containing a primary thiol [7] (for details see Materials and Methods). Single molecule events were detected in two channels by placing a dichroic mirror into the detection path. This split the image into two components which we call here ‘short’ (680–740 nm) and ‘long’ (740–8301 nm) channels. The images at these wavelength were digitized with a single electron multiplying charge-coupled device (emCCD) camera by focussing each of the two images simultaneously onto two halves of the camera (see Figure 1A). To add axial position encoding, a cylindrical lens was used to introduce astigmatism in the images [15].

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