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

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.

SUBMITTER: Whelan DR

PROVIDER: S-EPMC4300460 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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

Whelan Donna R DR Bell Toby D M TD

Scientific reports 20150121

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 ...[more]

PMID: 25603780

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Project description:Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput 'peptidomics' methods , an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regards to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS based peptidome analysis. We report on a novel adaptation of the standard solid phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. Expense and time requirements were comparable for both SPE and mSPE, but more interfering contaminants from the urine matrix were evident in the SPE preparations (e.g., clogging of the LC-MS columns, yellowish background coloration of prepared samples due to retained urobilin, lower peptide yields) when compared to the mSPE method. When we compared data from technical replicates of 4 runs, the mSPE method provided significantly improved efficiencies for the preparation of samples from urine (e.g., mSPE peptide identification 82% versus 18% with SPE; p = 8.92E-05). Additionally, peptide identifications, when applying the mSPE method, highlighted the biology of differential activation of urine peptidases during acute renal transplant rejection with distinct laddering of specific peptides, which was obscured for most proteins when utilizing the conventional SPE method. In conclusion, the mSPE method was found to be superior to the conventional, standard SPE method for urine peptide sample preparation when applying LC-MS peptidomics analysis due to the optimized sample clean up that provided improved experimental inference from the confidently identified peptides.

Dataset Information

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

Publications

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

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