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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

SMLM images of microtubules prepared using standard protocols but with careful initial preparation and application of the fixative solutions show some sub-diffraction artifacts but preserve much of the filamentous architecture.(A–D) COS-7 cells stained for tubulin using Alexa Fluor 647 after fixation with (A) -20°C methanol following a PBS wash, (B) −20°C methanol allowed to equilibrate to room temperature during fixation, (C) room temperature 4% paraformaldehyde, and (D) 3% glutaraldehyde following pre-extraction using 0.3% Triton X-100. White arrows indicate discontinuousness of filaments, blue arrows indicate abnormal curvature of the filaments, red arrows indicate structured but non-filamentous localizations. Scale bar: 1 μm.
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f3: SMLM images of microtubules prepared using standard protocols but with careful initial preparation and application of the fixative solutions show some sub-diffraction artifacts but preserve much of the filamentous architecture.(A–D) COS-7 cells stained for tubulin using Alexa Fluor 647 after fixation with (A) -20°C methanol following a PBS wash, (B) −20°C methanol allowed to equilibrate to room temperature during fixation, (C) room temperature 4% paraformaldehyde, and (D) 3% glutaraldehyde following pre-extraction using 0.3% Triton X-100. White arrows indicate discontinuousness of filaments, blue arrows indicate abnormal curvature of the filaments, red arrows indicate structured but non-filamentous localizations. Scale bar: 1 μm.

Mentions: Figure 3 further investigates sub-diffraction artifacts by directly comparing four common protocols used for preparing cells for MT staining and confocal imaging (Full protocols in SI Methods 3). Figure 3a shows a cell fixed using methanol kept at −20°C throughout the fixation. Some epitope damage is observed since there are discontinuous filaments present, (white arrow) despite the use of increased antibody concentration for methanol fixed stains. Minimal background/nonspecific stain is observed without the use of pre-extraction or blocking steps. Figure 3b shows a cell fixed in methanol at −20°C but allowed to sit in ambient conditions for the 20 minutes of fixation, thus raising the temperature of the methanol. There is some indication of non-native curvature in these MTs (blue arrow) as well as structure and clustering in the non-specific dyes localized near the filaments (red arrow). This clustering of the non-specific stain is not observed under any of the other trialled or optimized fixation methods. Figure 3c shows MTs fixed using room temperature, 4% PFA: while MT filaments can be observed in this image and the average cross section of these single MTs is comparable with optimized protocols (~60–65 nm), the image suffers from some discontinuousness in the MT structures as well as increased background/non-specific stain. Finally, Figure 3d shows MTs in a cell fixed using a pre-extraction step and 3% GA in CSB, demonstrating well-conserved architecture and a low level of background/non-specific stain. While methanol is an often-used fixative for MT structure preservation, these results show that GA is preferable and invite further investigation into the biochemical causes for the differences between methanol and GA fixed cells. They are also an excellent illustration of a previously sufficient protocol yielding unexpected artifacts at a sub-diffraction level.


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

Whelan DR, Bell TD - Sci Rep (2015)

SMLM images of microtubules prepared using standard protocols but with careful initial preparation and application of the fixative solutions show some sub-diffraction artifacts but preserve much of the filamentous architecture.(A–D) COS-7 cells stained for tubulin using Alexa Fluor 647 after fixation with (A) -20°C methanol following a PBS wash, (B) −20°C methanol allowed to equilibrate to room temperature during fixation, (C) room temperature 4% paraformaldehyde, and (D) 3% glutaraldehyde following pre-extraction using 0.3% Triton X-100. White arrows indicate discontinuousness of filaments, blue arrows indicate abnormal curvature of the filaments, red arrows indicate structured but non-filamentous localizations. Scale bar: 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: SMLM images of microtubules prepared using standard protocols but with careful initial preparation and application of the fixative solutions show some sub-diffraction artifacts but preserve much of the filamentous architecture.(A–D) COS-7 cells stained for tubulin using Alexa Fluor 647 after fixation with (A) -20°C methanol following a PBS wash, (B) −20°C methanol allowed to equilibrate to room temperature during fixation, (C) room temperature 4% paraformaldehyde, and (D) 3% glutaraldehyde following pre-extraction using 0.3% Triton X-100. White arrows indicate discontinuousness of filaments, blue arrows indicate abnormal curvature of the filaments, red arrows indicate structured but non-filamentous localizations. Scale bar: 1 μm.
Mentions: Figure 3 further investigates sub-diffraction artifacts by directly comparing four common protocols used for preparing cells for MT staining and confocal imaging (Full protocols in SI Methods 3). Figure 3a shows a cell fixed using methanol kept at −20°C throughout the fixation. Some epitope damage is observed since there are discontinuous filaments present, (white arrow) despite the use of increased antibody concentration for methanol fixed stains. Minimal background/nonspecific stain is observed without the use of pre-extraction or blocking steps. Figure 3b shows a cell fixed in methanol at −20°C but allowed to sit in ambient conditions for the 20 minutes of fixation, thus raising the temperature of the methanol. There is some indication of non-native curvature in these MTs (blue arrow) as well as structure and clustering in the non-specific dyes localized near the filaments (red arrow). This clustering of the non-specific stain is not observed under any of the other trialled or optimized fixation methods. Figure 3c shows MTs fixed using room temperature, 4% PFA: while MT filaments can be observed in this image and the average cross section of these single MTs is comparable with optimized protocols (~60–65 nm), the image suffers from some discontinuousness in the MT structures as well as increased background/non-specific stain. Finally, Figure 3d shows MTs in a cell fixed using a pre-extraction step and 3% GA in CSB, demonstrating well-conserved architecture and a low level of background/non-specific stain. While methanol is an often-used fixative for MT structure preservation, these results show that GA is preferable and invite further investigation into the biochemical causes for the differences between methanol and GA fixed cells. They are also an excellent illustration of a previously sufficient protocol yielding unexpected artifacts at a sub-diffraction level.

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