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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

The timing of permeabilization of cells significantly affects the distribution of fluorophores within the cell, resulting in differences in the ‘cleanliness’ of the image as well as the degree to which the image is representative of the biologically native structure and distribution.(A–D) COS-7 cells stained for tubulin using Alexa Fluor 647 after (A) fixation with −20°C methanol, (B) pre-extraction with 0.3% Triton X-100 in HEPES buffer before 3% glutaraldehyde fixation, (C) simultaneous permeabilization and fixation (0.3% glutaraldehyde with 0.3% Triton X-100), and (D) fixation with 3% glutaraldehyde prior to permeabilization with 0.3% Triton X-100. Scale bar: 1 μm.
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f4: The timing of permeabilization of cells significantly affects the distribution of fluorophores within the cell, resulting in differences in the ‘cleanliness’ of the image as well as the degree to which the image is representative of the biologically native structure and distribution.(A–D) COS-7 cells stained for tubulin using Alexa Fluor 647 after (A) fixation with −20°C methanol, (B) pre-extraction with 0.3% Triton X-100 in HEPES buffer before 3% glutaraldehyde fixation, (C) simultaneous permeabilization and fixation (0.3% glutaraldehyde with 0.3% Triton X-100), and (D) fixation with 3% glutaraldehyde prior to permeabilization with 0.3% Triton X-100. Scale bar: 1 μm.

Mentions: Like the MTs in Fig 1i, Fig 4a shows a cell fixed in −20°C methanol. In this cell almost no non-specific stain is detected despite a high antibody concentration used. Similarly, cells pre-extracted with 0.3% Triton X-100 in growth medium have a very low amount of non-specific stain (Fig 4b) (SI Methods 4). Permeabilization of the cells in a pre-fixation step with a low concentration of GA (0.3% with 0.3% Triton X-100) was observed to leave some cytosolic non-specific stain (Fig 4c) while permeabilizing after fixation (2% GA, followed by 0.3% Triton X-100) yielded the highest amount of non-specific localizations (Fig 4d). In all images the MTs themselves have been stained well with continuous filaments intact and yielding average cross-sections of 55–65 nm. These observations led us to hypothesize that the localized fluorophores not associated with polymeric tubulin were associated with cytosolic dimeric tubulin and were not non-specific in the traditional sense. If they were truly non-specific we would expect to see non-polymeric localizations even with the cytosol removed.


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

Whelan DR, Bell TD - Sci Rep (2015)

The timing of permeabilization of cells significantly affects the distribution of fluorophores within the cell, resulting in differences in the ‘cleanliness’ of the image as well as the degree to which the image is representative of the biologically native structure and distribution.(A–D) COS-7 cells stained for tubulin using Alexa Fluor 647 after (A) fixation with −20°C methanol, (B) pre-extraction with 0.3% Triton X-100 in HEPES buffer before 3% glutaraldehyde fixation, (C) simultaneous permeabilization and fixation (0.3% glutaraldehyde with 0.3% Triton X-100), and (D) fixation with 3% glutaraldehyde prior to permeabilization with 0.3% Triton X-100. Scale bar: 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: The timing of permeabilization of cells significantly affects the distribution of fluorophores within the cell, resulting in differences in the ‘cleanliness’ of the image as well as the degree to which the image is representative of the biologically native structure and distribution.(A–D) COS-7 cells stained for tubulin using Alexa Fluor 647 after (A) fixation with −20°C methanol, (B) pre-extraction with 0.3% Triton X-100 in HEPES buffer before 3% glutaraldehyde fixation, (C) simultaneous permeabilization and fixation (0.3% glutaraldehyde with 0.3% Triton X-100), and (D) fixation with 3% glutaraldehyde prior to permeabilization with 0.3% Triton X-100. Scale bar: 1 μm.
Mentions: Like the MTs in Fig 1i, Fig 4a shows a cell fixed in −20°C methanol. In this cell almost no non-specific stain is detected despite a high antibody concentration used. Similarly, cells pre-extracted with 0.3% Triton X-100 in growth medium have a very low amount of non-specific stain (Fig 4b) (SI Methods 4). Permeabilization of the cells in a pre-fixation step with a low concentration of GA (0.3% with 0.3% Triton X-100) was observed to leave some cytosolic non-specific stain (Fig 4c) while permeabilizing after fixation (2% GA, followed by 0.3% Triton X-100) yielded the highest amount of non-specific localizations (Fig 4d). In all images the MTs themselves have been stained well with continuous filaments intact and yielding average cross-sections of 55–65 nm. These observations led us to hypothesize that the localized fluorophores not associated with polymeric tubulin were associated with cytosolic dimeric tubulin and were not non-specific in the traditional sense. If they were truly non-specific we would expect to see non-polymeric localizations even with the cytosol removed.

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