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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

Correlative confocal and super-resolution imaging of a human cardiac tissue section.The section was ∼10 µm thick and was labeled with phalloidin for f-Actin (Alexa 488), WGA for the cell membrane and extracellular matrix (Alexa 594), along with antibodies for the ryanodine receptor (RyR, Alexa 680) and calsequestrin (CSQ, Alexa 750). In addition to the applied labelling, a strong endogenous fluorescence signal from lipofuscin was recorded. The shorter wavelength labels (Actin, WGA, and RyR) were imaged on a confocal microscope, and the sample then taken to the localisation microscope where super-resolution imaging of the longer wavelength labels (RyR, lipofuscin, CSQ) was performed. Panel A shows an overview of the cellular structure across a large tissue area that is indicated by the actin labeling (largely muscle cell contractile protein). Scale bar 100 µm. Panel B is a projection of a confocal stack taken of the region indicated in A. Scale bar 10 µm. Panel C shows a confocal stack of a small detail area from B and panel D shows an optically sectioned super-resolution stack, within the region covered by the confocal stack in C. Panels E & F compare corresponding confocal (F) and super-resolution (E) images both using the RyR-Alexa 647 signal. Note the good correlation between the data and the improvement in resolution in E. Scale bar 1 µm. G: 3-colour super-resolution image of a small area in the tissue sample, note the improved resolution as compared to the conventional resolution image section. Since the ratios of Alexa 647 and lipofuscein were relatively close some crosstalk did occur. Scale bar 500 nm.
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pone-0020645-g003: Correlative confocal and super-resolution imaging of a human cardiac tissue section.The section was ∼10 µm thick and was labeled with phalloidin for f-Actin (Alexa 488), WGA for the cell membrane and extracellular matrix (Alexa 594), along with antibodies for the ryanodine receptor (RyR, Alexa 680) and calsequestrin (CSQ, Alexa 750). In addition to the applied labelling, a strong endogenous fluorescence signal from lipofuscin was recorded. The shorter wavelength labels (Actin, WGA, and RyR) were imaged on a confocal microscope, and the sample then taken to the localisation microscope where super-resolution imaging of the longer wavelength labels (RyR, lipofuscin, CSQ) was performed. Panel A shows an overview of the cellular structure across a large tissue area that is indicated by the actin labeling (largely muscle cell contractile protein). Scale bar 100 µm. Panel B is a projection of a confocal stack taken of the region indicated in A. Scale bar 10 µm. Panel C shows a confocal stack of a small detail area from B and panel D shows an optically sectioned super-resolution stack, within the region covered by the confocal stack in C. Panels E & F compare corresponding confocal (F) and super-resolution (E) images both using the RyR-Alexa 647 signal. Note the good correlation between the data and the improvement in resolution in E. Scale bar 1 µm. G: 3-colour super-resolution image of a small area in the tissue sample, note the improved resolution as compared to the conventional resolution image section. Since the ratios of Alexa 647 and lipofuscein were relatively close some crosstalk did occur. Scale bar 500 nm.

Mentions: The ability to use localisation microscopy in fixed tissue and spectrally distinguish several markers was investigated using thick (∼10 µm) tissue sections from human heart. The sections were labelled with Alexa 488 phalloidin (for actin filaments) and Alexa 594 wheat germ agglutinin (WGA) (for extracellular matrix and cell membranes). In addition, we labelled intracellular proteins with indirect immunofluorescence with Alexa 647 and Alexa 750 for cardiac ryanodine receptor (RyRs) and calsequestrin (CSQ) respectively. With confocal microscopy, low magnification overview images of large tissue areas (Figure 3A) as well as smaller regions at diffraction-limited resolution (Figure 3B,C) were used to select several cells for multicolor super-resolution imaging. After transfer of the sample to a super-resolution microscope the signal from the two near infrared stains (labelling RyR and CSQ) and an intrinsic signal from lipofuscin (pigment granules that are present in the aging heart [19] which was also simultaneously excited at 671 nm) generated sufficient contrast for good single molecule detection of the three markers (Figure 3D–G). Lipofuscin blinking was supported by the dSTORM mountant, since samples mounted in pure glycerol exhibited an ∼10-fold reduced lipofuscin event rate during illumination at 671 nm. These data also illustrate the benefit of using near IR dyes as fixed tissue autofluorescence was reduced by almost two orders of magnitude in the regime >680 nm as compared to the visible range at ∼500 nm (see Supplementary Figure S4, Supplementary Text S2).


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)

Correlative confocal and super-resolution imaging of a human cardiac tissue section.The section was ∼10 µm thick and was labeled with phalloidin for f-Actin (Alexa 488), WGA for the cell membrane and extracellular matrix (Alexa 594), along with antibodies for the ryanodine receptor (RyR, Alexa 680) and calsequestrin (CSQ, Alexa 750). In addition to the applied labelling, a strong endogenous fluorescence signal from lipofuscin was recorded. The shorter wavelength labels (Actin, WGA, and RyR) were imaged on a confocal microscope, and the sample then taken to the localisation microscope where super-resolution imaging of the longer wavelength labels (RyR, lipofuscin, CSQ) was performed. Panel A shows an overview of the cellular structure across a large tissue area that is indicated by the actin labeling (largely muscle cell contractile protein). Scale bar 100 µm. Panel B is a projection of a confocal stack taken of the region indicated in A. Scale bar 10 µm. Panel C shows a confocal stack of a small detail area from B and panel D shows an optically sectioned super-resolution stack, within the region covered by the confocal stack in C. Panels E & F compare corresponding confocal (F) and super-resolution (E) images both using the RyR-Alexa 647 signal. Note the good correlation between the data and the improvement in resolution in E. Scale bar 1 µm. G: 3-colour super-resolution image of a small area in the tissue sample, note the improved resolution as compared to the conventional resolution image section. Since the ratios of Alexa 647 and lipofuscein were relatively close some crosstalk did occur. Scale bar 500 nm.
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getmorefigures.php?uid=PMC3105105&req=5

pone-0020645-g003: Correlative confocal and super-resolution imaging of a human cardiac tissue section.The section was ∼10 µm thick and was labeled with phalloidin for f-Actin (Alexa 488), WGA for the cell membrane and extracellular matrix (Alexa 594), along with antibodies for the ryanodine receptor (RyR, Alexa 680) and calsequestrin (CSQ, Alexa 750). In addition to the applied labelling, a strong endogenous fluorescence signal from lipofuscin was recorded. The shorter wavelength labels (Actin, WGA, and RyR) were imaged on a confocal microscope, and the sample then taken to the localisation microscope where super-resolution imaging of the longer wavelength labels (RyR, lipofuscin, CSQ) was performed. Panel A shows an overview of the cellular structure across a large tissue area that is indicated by the actin labeling (largely muscle cell contractile protein). Scale bar 100 µm. Panel B is a projection of a confocal stack taken of the region indicated in A. Scale bar 10 µm. Panel C shows a confocal stack of a small detail area from B and panel D shows an optically sectioned super-resolution stack, within the region covered by the confocal stack in C. Panels E & F compare corresponding confocal (F) and super-resolution (E) images both using the RyR-Alexa 647 signal. Note the good correlation between the data and the improvement in resolution in E. Scale bar 1 µm. G: 3-colour super-resolution image of a small area in the tissue sample, note the improved resolution as compared to the conventional resolution image section. Since the ratios of Alexa 647 and lipofuscein were relatively close some crosstalk did occur. Scale bar 500 nm.
Mentions: The ability to use localisation microscopy in fixed tissue and spectrally distinguish several markers was investigated using thick (∼10 µm) tissue sections from human heart. The sections were labelled with Alexa 488 phalloidin (for actin filaments) and Alexa 594 wheat germ agglutinin (WGA) (for extracellular matrix and cell membranes). In addition, we labelled intracellular proteins with indirect immunofluorescence with Alexa 647 and Alexa 750 for cardiac ryanodine receptor (RyRs) and calsequestrin (CSQ) respectively. With confocal microscopy, low magnification overview images of large tissue areas (Figure 3A) as well as smaller regions at diffraction-limited resolution (Figure 3B,C) were used to select several cells for multicolor super-resolution imaging. After transfer of the sample to a super-resolution microscope the signal from the two near infrared stains (labelling RyR and CSQ) and an intrinsic signal from lipofuscin (pigment granules that are present in the aging heart [19] which was also simultaneously excited at 671 nm) generated sufficient contrast for good single molecule detection of the three markers (Figure 3D–G). Lipofuscin blinking was supported by the dSTORM mountant, since samples mounted in pure glycerol exhibited an ∼10-fold reduced lipofuscin event rate during illumination at 671 nm. These data also illustrate the benefit of using near IR dyes as fixed tissue autofluorescence was reduced by almost two orders of magnitude in the regime >680 nm as compared to the visible range at ∼500 nm (see Supplementary Figure S4, Supplementary Text S2).

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