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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

Varying antibody concentration affects apparent microtubule width, non-filamentous stain, and filament continuity—all of which can affect spatial resolution.(A–C) COS-7 cells fixed using the optimized glutaraldehyde protocol and then stained for tubulin using mouse anti-β-tubulin and Alexa Fluor 647 conjugated rabbit-anti-mouse. (A) Both primary and secondary antibodies were diluted 1:50, (B) 1:500, and (C) 1:2000. Scale bar: 1 μm.
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f5: Varying antibody concentration affects apparent microtubule width, non-filamentous stain, and filament continuity—all of which can affect spatial resolution.(A–C) COS-7 cells fixed using the optimized glutaraldehyde protocol and then stained for tubulin using mouse anti-β-tubulin and Alexa Fluor 647 conjugated rabbit-anti-mouse. (A) Both primary and secondary antibodies were diluted 1:50, (B) 1:500, and (C) 1:2000. Scale bar: 1 μm.

Mentions: Images supporting this hypothesis are shown in Figure 5 which depicts changes observed in response to different antibody concentrations applied to MTs. As mentioned above, homogeneous background fluorescence does not result in localizations therefore this represents only the Alexa Fluor dyes. These cells were all fixed according to the optimized GA protocol and then stained over the dilution range of 1:50 to 1:2000 primary and secondary antibodies. In all cases the antibody solutions were administered in 200 μl aliquots with a primary incubation time of three hours and a secondary incubation time of one hour. Figure 5a shows heavily overstained MTs with an increased average MT width (91 nm) as well as a very high level of background/non-specific stain. A magnitude less antibody results in the MTs depicted in Fig 5b. These MTs have average widths of 58 nm and a significantly lowered amount of non-specific stain and depict the optimal staining density for SMLM. An image of stained MTs resulting from further dilution of the antibodies is shown in Fig 5c depicting an even lower number of non-specific localizations but also suffering from very discontinuous MT filaments. A control stain in which the primary antibody was not used showed almost no localizations demonstrating that the non-specific stain observed in Fig 5 is not simply non-specific secondary antibody. This suggests that these are not ‘traditional’ non-specific fluorophore attachment but are something specific, most likely dimeric tubulin. Previous SMLM publications featuring MTs have not identified the non-polymeric localizations seen in their published images as dimeric tubulin. Nor has it been discussed that by pre- or simultaneously extracting the cytosol from cells the resulting images appear ‘cleaner’ with less non-filamentous localizations and that this effect is achieved with significant alteration of the biology of the sample, i.e. by removing the cytosol and with it, many ‘real’ tubulin localizations. Diffraction limited confocal or epifluorescence images of MTs in a cell would also detect the dimeric tubulin but as a low-intensity homogeneous background that is easily considered noise below the signal of the MTs themselves.


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

Whelan DR, Bell TD - Sci Rep (2015)

Varying antibody concentration affects apparent microtubule width, non-filamentous stain, and filament continuity—all of which can affect spatial resolution.(A–C) COS-7 cells fixed using the optimized glutaraldehyde protocol and then stained for tubulin using mouse anti-β-tubulin and Alexa Fluor 647 conjugated rabbit-anti-mouse. (A) Both primary and secondary antibodies were diluted 1:50, (B) 1:500, and (C) 1:2000. Scale bar: 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Varying antibody concentration affects apparent microtubule width, non-filamentous stain, and filament continuity—all of which can affect spatial resolution.(A–C) COS-7 cells fixed using the optimized glutaraldehyde protocol and then stained for tubulin using mouse anti-β-tubulin and Alexa Fluor 647 conjugated rabbit-anti-mouse. (A) Both primary and secondary antibodies were diluted 1:50, (B) 1:500, and (C) 1:2000. Scale bar: 1 μm.
Mentions: Images supporting this hypothesis are shown in Figure 5 which depicts changes observed in response to different antibody concentrations applied to MTs. As mentioned above, homogeneous background fluorescence does not result in localizations therefore this represents only the Alexa Fluor dyes. These cells were all fixed according to the optimized GA protocol and then stained over the dilution range of 1:50 to 1:2000 primary and secondary antibodies. In all cases the antibody solutions were administered in 200 μl aliquots with a primary incubation time of three hours and a secondary incubation time of one hour. Figure 5a shows heavily overstained MTs with an increased average MT width (91 nm) as well as a very high level of background/non-specific stain. A magnitude less antibody results in the MTs depicted in Fig 5b. These MTs have average widths of 58 nm and a significantly lowered amount of non-specific stain and depict the optimal staining density for SMLM. An image of stained MTs resulting from further dilution of the antibodies is shown in Fig 5c depicting an even lower number of non-specific localizations but also suffering from very discontinuous MT filaments. A control stain in which the primary antibody was not used showed almost no localizations demonstrating that the non-specific stain observed in Fig 5 is not simply non-specific secondary antibody. This suggests that these are not ‘traditional’ non-specific fluorophore attachment but are something specific, most likely dimeric tubulin. Previous SMLM publications featuring MTs have not identified the non-polymeric localizations seen in their published images as dimeric tubulin. Nor has it been discussed that by pre- or simultaneously extracting the cytosol from cells the resulting images appear ‘cleaner’ with less non-filamentous localizations and that this effect is achieved with significant alteration of the biology of the sample, i.e. by removing the cytosol and with it, many ‘real’ tubulin localizations. Diffraction limited confocal or epifluorescence images of MTs in a cell would also detect the dimeric tubulin but as a low-intensity homogeneous background that is easily considered noise below the signal of the MTs themselves.

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