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Image artifacts in single molecule localization microscopy: why optimization of sample preparation protocols matters.

Whelan DR, Bell TD - Sci Rep (2015)

Bottom Line: As a result of the up to an order-of-magnitude improvement in spatial resolution, substantially more detail is observed, including changes in distribution and ultrastructure caused by the many steps required to fix, permeabilize, and stain a sample.We present three well-optimized fixation protocols for staining microtubules, mitochondria and actin in a mammalian cell line and then discuss various artifacts in relation to images obtained from samples prepared using the protocols.The potential for such errors to go undetected in SMLM images and the complications in defining a 'good' image using previous parameters applied to confocal microscopy are also discussed.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, Monash University, Clayton, Victoria 3800, Australia.

ABSTRACT
Single molecule localization microscopy (SMLM) techniques allow for sub-diffraction imaging with spatial resolutions better than 10 nm reported. Much has been discussed relating to different variations of SMLM and all-inclusive microscopes can now be purchased, removing the need for in-house software or hardware development. However, little discussion has occurred examining the reliability and quality of the images being produced, as well as the potential for overlooked preparative artifacts. As a result of the up to an order-of-magnitude improvement in spatial resolution, substantially more detail is observed, including changes in distribution and ultrastructure caused by the many steps required to fix, permeabilize, and stain a sample. Here we systematically investigate many of these steps including different fixatives, fixative concentration, permeabilization concentration and timing, antibody concentration, and buffering. We present three well-optimized fixation protocols for staining microtubules, mitochondria and actin in a mammalian cell line and then discuss various artifacts in relation to images obtained from samples prepared using the protocols. The potential for such errors to go undetected in SMLM images and the complications in defining a 'good' image using previous parameters applied to confocal microscopy are also discussed.

No MeSH data available.


Related in: MedlinePlus

Optimized protocols using paraformaldehyde, glutaraldehyde and methanol preserve ultrastructure for SMLM imaging of mitochondria, actin and microtubules.(A–C) COS-7 cells fixed using the optimized 3.7% paraformaldehyde protocol and stained using Alexa Fluor 647 in conjunction with (A) anti-Tom20, a protein component of the import receptor on the outer membrane of mitochondria, (B) phalloidin which strongly associates with filamentous actin, and (C) anti-α/β-tubulin, the unit protein that polymerizes into microtubules. (D–F) Similarly stained mitochondria (D), actin (E) and microtubules (F) in cells fixed using the optimized 3% glutaraldehyde protocol. (G–I) Similarly stained mitochondria (G), actin (H) and microtubules (I) in cells fixed using the optimized −20°C methanol protocol. All images show sub-diffraction structure with the exception of G–H that show substantial damage. Scale bar: 1 μm.
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f1: Optimized protocols using paraformaldehyde, glutaraldehyde and methanol preserve ultrastructure for SMLM imaging of mitochondria, actin and microtubules.(A–C) COS-7 cells fixed using the optimized 3.7% paraformaldehyde protocol and stained using Alexa Fluor 647 in conjunction with (A) anti-Tom20, a protein component of the import receptor on the outer membrane of mitochondria, (B) phalloidin which strongly associates with filamentous actin, and (C) anti-α/β-tubulin, the unit protein that polymerizes into microtubules. (D–F) Similarly stained mitochondria (D), actin (E) and microtubules (F) in cells fixed using the optimized 3% glutaraldehyde protocol. (G–I) Similarly stained mitochondria (G), actin (H) and microtubules (I) in cells fixed using the optimized −20°C methanol protocol. All images show sub-diffraction structure with the exception of G–H that show substantial damage. Scale bar: 1 μm.

Mentions: As such, the three optimized protocols detailed above are a result of careful, systematic consideration of many of the different facets and steps of sample preparation and were initially developed specifically for MT fixation. Conclusively, all three fixatives—paraformaldehyde (PFA), glutaraldehyde (GA) and methanol—when used in accordance with the protocols outlined, give exemplary SMLM images of MTs (Figure 1). The PFA fixation protocol also worked well for MC networks but actin microstructure was lost; conversely the GA fixation protocol worked well for actin but MC appeared susceptible to shrinkage as has been observed before24. Overall, methanol gave unsatisfactory results for both MC and actin stains as expected based on previous literature and the precipitory action of methanol on the MC membrane252627.


Image artifacts in single molecule localization microscopy: why optimization of sample preparation protocols matters.

Whelan DR, Bell TD - Sci Rep (2015)

Optimized protocols using paraformaldehyde, glutaraldehyde and methanol preserve ultrastructure for SMLM imaging of mitochondria, actin and microtubules.(A–C) COS-7 cells fixed using the optimized 3.7% paraformaldehyde protocol and stained using Alexa Fluor 647 in conjunction with (A) anti-Tom20, a protein component of the import receptor on the outer membrane of mitochondria, (B) phalloidin which strongly associates with filamentous actin, and (C) anti-α/β-tubulin, the unit protein that polymerizes into microtubules. (D–F) Similarly stained mitochondria (D), actin (E) and microtubules (F) in cells fixed using the optimized 3% glutaraldehyde protocol. (G–I) Similarly stained mitochondria (G), actin (H) and microtubules (I) in cells fixed using the optimized −20°C methanol protocol. All images show sub-diffraction structure with the exception of G–H that show substantial damage. Scale bar: 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300460&req=5

f1: Optimized protocols using paraformaldehyde, glutaraldehyde and methanol preserve ultrastructure for SMLM imaging of mitochondria, actin and microtubules.(A–C) COS-7 cells fixed using the optimized 3.7% paraformaldehyde protocol and stained using Alexa Fluor 647 in conjunction with (A) anti-Tom20, a protein component of the import receptor on the outer membrane of mitochondria, (B) phalloidin which strongly associates with filamentous actin, and (C) anti-α/β-tubulin, the unit protein that polymerizes into microtubules. (D–F) Similarly stained mitochondria (D), actin (E) and microtubules (F) in cells fixed using the optimized 3% glutaraldehyde protocol. (G–I) Similarly stained mitochondria (G), actin (H) and microtubules (I) in cells fixed using the optimized −20°C methanol protocol. All images show sub-diffraction structure with the exception of G–H that show substantial damage. Scale bar: 1 μm.
Mentions: As such, the three optimized protocols detailed above are a result of careful, systematic consideration of many of the different facets and steps of sample preparation and were initially developed specifically for MT fixation. Conclusively, all three fixatives—paraformaldehyde (PFA), glutaraldehyde (GA) and methanol—when used in accordance with the protocols outlined, give exemplary SMLM images of MTs (Figure 1). The PFA fixation protocol also worked well for MC networks but actin microstructure was lost; conversely the GA fixation protocol worked well for actin but MC appeared susceptible to shrinkage as has been observed before24. Overall, methanol gave unsatisfactory results for both MC and actin stains as expected based on previous literature and the precipitory action of methanol on the MC membrane252627.

Bottom Line: As a result of the up to an order-of-magnitude improvement in spatial resolution, substantially more detail is observed, including changes in distribution and ultrastructure caused by the many steps required to fix, permeabilize, and stain a sample.We present three well-optimized fixation protocols for staining microtubules, mitochondria and actin in a mammalian cell line and then discuss various artifacts in relation to images obtained from samples prepared using the protocols.The potential for such errors to go undetected in SMLM images and the complications in defining a 'good' image using previous parameters applied to confocal microscopy are also discussed.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, Monash University, Clayton, Victoria 3800, Australia.

ABSTRACT
Single molecule localization microscopy (SMLM) techniques allow for sub-diffraction imaging with spatial resolutions better than 10 nm reported. Much has been discussed relating to different variations of SMLM and all-inclusive microscopes can now be purchased, removing the need for in-house software or hardware development. However, little discussion has occurred examining the reliability and quality of the images being produced, as well as the potential for overlooked preparative artifacts. As a result of the up to an order-of-magnitude improvement in spatial resolution, substantially more detail is observed, including changes in distribution and ultrastructure caused by the many steps required to fix, permeabilize, and stain a sample. Here we systematically investigate many of these steps including different fixatives, fixative concentration, permeabilization concentration and timing, antibody concentration, and buffering. We present three well-optimized fixation protocols for staining microtubules, mitochondria and actin in a mammalian cell line and then discuss various artifacts in relation to images obtained from samples prepared using the protocols. The potential for such errors to go undetected in SMLM images and the complications in defining a 'good' image using previous parameters applied to confocal microscopy are also discussed.

No MeSH data available.


Related in: MedlinePlus