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SFMetrics: an analysis tool for scanning force microscopy images of biomolecules.

Sánchez H, Wyman C - BMC Bioinformatics (2015)

Bottom Line: Scanning force microscopy (SFM) allows direct, rapid and high-resolution visualization of single molecular complexes; irregular shapes and differences in sizes are immediately revealed by the scanning tip in three-dimensional images.Most existing SFM software tools are aimed at broad general use: from material-surface analysis to visualization of biomolecules.Assembled in a single user interface, SFMetrics serves as a semi-automatic analysis tool capable of measuring several geometrical properties (length, volume and angles) from DNA and protein complexes, but is also applicable to other samples with irregular shapes.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Erasmus University Medical Center, Rotterdam 3000CA, The Netherlands. h.sanchezgonzalez@erasmusmc.nl.

ABSTRACT

Background: Scanning force microscopy (SFM) allows direct, rapid and high-resolution visualization of single molecular complexes; irregular shapes and differences in sizes are immediately revealed by the scanning tip in three-dimensional images. However, high-throughput analysis of SFM data is limited by the lack of versatile software tools accessible to SFM users. Most existing SFM software tools are aimed at broad general use: from material-surface analysis to visualization of biomolecules.

Results: We present SFMetrics as a metrology toolbox for SFM, specifically aimed at biomolecules like DNA and proteins, which features (a) semi-automatic high-throughput analysis of individual molecules; (b) ease of use working within MATLAB environment or as a stand-alone application; (c) compatibility with MultiMode (Bruker), NanoWizard (JPK instruments), Asylum (Asylum research), ASCII, and TIFF files, that can be adjusted with minor modifications to other formats.

Conclusion: Assembled in a single user interface, SFMetrics serves as a semi-automatic analysis tool capable of measuring several geometrical properties (length, volume and angles) from DNA and protein complexes, but is also applicable to other samples with irregular shapes.

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Related in: MedlinePlus

Example analysing multiple features of DNA-protein complexes with SFMetrics. A) SFM image of filaments formed by human RAD51 protein on double-stranded DNA. Image size: 2×2 μm, height is represented by color in the range of −2.5-2.5 nm. B) Volume analysis of the filament zoomed in panel A. The filament is surrounded by free protein complexes, not bound to DNA. However, these do not affect volume calculations performed only for the biggest object (the filament). i.u. = intensity units. C) Skeleton length tool is used for estimating the length of the filament. After appropriately adjusting the threshold slider only the filament axis is highlighted and background is discarded. Angle tool can be used to determine kink’s openness (in red).
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Fig4: Example analysing multiple features of DNA-protein complexes with SFMetrics. A) SFM image of filaments formed by human RAD51 protein on double-stranded DNA. Image size: 2×2 μm, height is represented by color in the range of −2.5-2.5 nm. B) Volume analysis of the filament zoomed in panel A. The filament is surrounded by free protein complexes, not bound to DNA. However, these do not affect volume calculations performed only for the biggest object (the filament). i.u. = intensity units. C) Skeleton length tool is used for estimating the length of the filament. After appropriately adjusting the threshold slider only the filament axis is highlighted and background is discarded. Angle tool can be used to determine kink’s openness (in red).

Mentions: SFMetrics function was tested using SFM image data of biomolecules typically generated in our laboratory. DNA fragments of 2 kbp were incubated with RAD51 protein, a strand exchange protein that binds DNA and forms stable defined nucleoprotein filaments in the conditions used [7]. Circular dsDNA (pUC19 plasmid), or the nucleoprotein filaments were deposited on freshly cleaved mica, rinsed with water and dried with filtered air [1]. Molecules were imaged in air using a Nanoscope III or IV (Bruker) operating in Tapping Mode™. SFM images were flattened to remove background slope by subtracting a 1st order plane fit with ‘NanoScope Analysis’ software (Bruker) and saved in the original format before opening in SFMetrics. Figure 1 shows a screen-shot of the SFMetrics starting window. After selecting the type of image (JPK, Multimode, Asylum, TIFF/JPEG/PNG, or ASCII) a new window for selecting specific parts of the image is opened (Figure 2). This is useful for large images filled with many objects to analyse. The selected area can be called by typing a number previously assigned by the user in the main window. Specific DNA (Figures 1 and 3) or DNA-protein complexes (Figure 4) are tagged with a number and selected with a square selection tool and the cropped region is displayed in the adjacent window. Volume of the object, in this case DNA or a RAD51 nucleoprotein filament, is calculated after detecting the edges (red contour-line in Figure 4B) of the complex using the sliding threshold tool. The ‘Plot line’ tool allows measurements of distances along a path defined by freehand tracing (red line in Figure 3A) and can be used for determining protein positions on DNA, for example. Height (Figure 3B) and width (Figure 3D) profiles from the line can be acquired by defining the desired width of a line perpendicular to the user-defined path (yellow line in Figure 3A). In order to illustrate this feature, we measured length, width and height of circular DNA molecules using two probes with different radius of curvature. Forty-five (from 15 images) and thirty-six DNA molecules (from 13 images) were measured with AppNano ACT-W probe (nominal radius of curvature 5 nm) and Nanosensors SSS-NCH probe (nominal radius of curvature 2 nm), respectively and hereafter probe A and probe B. Length measured by freehand tracing was 1000 ± 64 (STD) nm (probe A) and 991 ± 46 (STD) nm (probe B). These measurements were significantly (p < 0.05) longer than the skeleton length: 835 ± 64 (STD) nm (probe A) and 847 ± 68 (STD) nm (probe B) but similar between the probes. DNA molecules were 2686 bp in length corresponding to ~892 nm of B-form dsDNA. The different length measured accounted for an overestimation of about 10% of the expected length due to pixel oversampling and user inaccuracy when freehand tracing that did not happen in skeleton length determination. Radius of curvature of the probe, however, is expected to affect significantly the width of the scanned molecules. We measured the FWHM of the DNA molecules in every point along the freehand-traced line (at least 200 data points per DNA molecule) as described above. Figure 5 shows the results obtained with each probe. Probe A produced DNA images with a median FWHM of 6.9 nm (with 6.4 and 7.6 nm as 25th and 75th percentiles respectively), while probe B produced DNA images with a median FWHM of 4.4 nm (with 3.9 and 5.9 nm as 25th and 75th percentiles respectively), significantly thinner (p < 0.05). The average height of the DNA was 0.5 ± 0.08 (STD) nm and 0.3 ± 0.1 (STD), for probe A and B respectively.Figure 1


SFMetrics: an analysis tool for scanning force microscopy images of biomolecules.

Sánchez H, Wyman C - BMC Bioinformatics (2015)

Example analysing multiple features of DNA-protein complexes with SFMetrics. A) SFM image of filaments formed by human RAD51 protein on double-stranded DNA. Image size: 2×2 μm, height is represented by color in the range of −2.5-2.5 nm. B) Volume analysis of the filament zoomed in panel A. The filament is surrounded by free protein complexes, not bound to DNA. However, these do not affect volume calculations performed only for the biggest object (the filament). i.u. = intensity units. C) Skeleton length tool is used for estimating the length of the filament. After appropriately adjusting the threshold slider only the filament axis is highlighted and background is discarded. Angle tool can be used to determine kink’s openness (in red).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4314933&req=5

Fig4: Example analysing multiple features of DNA-protein complexes with SFMetrics. A) SFM image of filaments formed by human RAD51 protein on double-stranded DNA. Image size: 2×2 μm, height is represented by color in the range of −2.5-2.5 nm. B) Volume analysis of the filament zoomed in panel A. The filament is surrounded by free protein complexes, not bound to DNA. However, these do not affect volume calculations performed only for the biggest object (the filament). i.u. = intensity units. C) Skeleton length tool is used for estimating the length of the filament. After appropriately adjusting the threshold slider only the filament axis is highlighted and background is discarded. Angle tool can be used to determine kink’s openness (in red).
Mentions: SFMetrics function was tested using SFM image data of biomolecules typically generated in our laboratory. DNA fragments of 2 kbp were incubated with RAD51 protein, a strand exchange protein that binds DNA and forms stable defined nucleoprotein filaments in the conditions used [7]. Circular dsDNA (pUC19 plasmid), or the nucleoprotein filaments were deposited on freshly cleaved mica, rinsed with water and dried with filtered air [1]. Molecules were imaged in air using a Nanoscope III or IV (Bruker) operating in Tapping Mode™. SFM images were flattened to remove background slope by subtracting a 1st order plane fit with ‘NanoScope Analysis’ software (Bruker) and saved in the original format before opening in SFMetrics. Figure 1 shows a screen-shot of the SFMetrics starting window. After selecting the type of image (JPK, Multimode, Asylum, TIFF/JPEG/PNG, or ASCII) a new window for selecting specific parts of the image is opened (Figure 2). This is useful for large images filled with many objects to analyse. The selected area can be called by typing a number previously assigned by the user in the main window. Specific DNA (Figures 1 and 3) or DNA-protein complexes (Figure 4) are tagged with a number and selected with a square selection tool and the cropped region is displayed in the adjacent window. Volume of the object, in this case DNA or a RAD51 nucleoprotein filament, is calculated after detecting the edges (red contour-line in Figure 4B) of the complex using the sliding threshold tool. The ‘Plot line’ tool allows measurements of distances along a path defined by freehand tracing (red line in Figure 3A) and can be used for determining protein positions on DNA, for example. Height (Figure 3B) and width (Figure 3D) profiles from the line can be acquired by defining the desired width of a line perpendicular to the user-defined path (yellow line in Figure 3A). In order to illustrate this feature, we measured length, width and height of circular DNA molecules using two probes with different radius of curvature. Forty-five (from 15 images) and thirty-six DNA molecules (from 13 images) were measured with AppNano ACT-W probe (nominal radius of curvature 5 nm) and Nanosensors SSS-NCH probe (nominal radius of curvature 2 nm), respectively and hereafter probe A and probe B. Length measured by freehand tracing was 1000 ± 64 (STD) nm (probe A) and 991 ± 46 (STD) nm (probe B). These measurements were significantly (p < 0.05) longer than the skeleton length: 835 ± 64 (STD) nm (probe A) and 847 ± 68 (STD) nm (probe B) but similar between the probes. DNA molecules were 2686 bp in length corresponding to ~892 nm of B-form dsDNA. The different length measured accounted for an overestimation of about 10% of the expected length due to pixel oversampling and user inaccuracy when freehand tracing that did not happen in skeleton length determination. Radius of curvature of the probe, however, is expected to affect significantly the width of the scanned molecules. We measured the FWHM of the DNA molecules in every point along the freehand-traced line (at least 200 data points per DNA molecule) as described above. Figure 5 shows the results obtained with each probe. Probe A produced DNA images with a median FWHM of 6.9 nm (with 6.4 and 7.6 nm as 25th and 75th percentiles respectively), while probe B produced DNA images with a median FWHM of 4.4 nm (with 3.9 and 5.9 nm as 25th and 75th percentiles respectively), significantly thinner (p < 0.05). The average height of the DNA was 0.5 ± 0.08 (STD) nm and 0.3 ± 0.1 (STD), for probe A and B respectively.Figure 1

Bottom Line: Scanning force microscopy (SFM) allows direct, rapid and high-resolution visualization of single molecular complexes; irregular shapes and differences in sizes are immediately revealed by the scanning tip in three-dimensional images.Most existing SFM software tools are aimed at broad general use: from material-surface analysis to visualization of biomolecules.Assembled in a single user interface, SFMetrics serves as a semi-automatic analysis tool capable of measuring several geometrical properties (length, volume and angles) from DNA and protein complexes, but is also applicable to other samples with irregular shapes.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Erasmus University Medical Center, Rotterdam 3000CA, The Netherlands. h.sanchezgonzalez@erasmusmc.nl.

ABSTRACT

Background: Scanning force microscopy (SFM) allows direct, rapid and high-resolution visualization of single molecular complexes; irregular shapes and differences in sizes are immediately revealed by the scanning tip in three-dimensional images. However, high-throughput analysis of SFM data is limited by the lack of versatile software tools accessible to SFM users. Most existing SFM software tools are aimed at broad general use: from material-surface analysis to visualization of biomolecules.

Results: We present SFMetrics as a metrology toolbox for SFM, specifically aimed at biomolecules like DNA and proteins, which features (a) semi-automatic high-throughput analysis of individual molecules; (b) ease of use working within MATLAB environment or as a stand-alone application; (c) compatibility with MultiMode (Bruker), NanoWizard (JPK instruments), Asylum (Asylum research), ASCII, and TIFF files, that can be adjusted with minor modifications to other formats.

Conclusion: Assembled in a single user interface, SFMetrics serves as a semi-automatic analysis tool capable of measuring several geometrical properties (length, volume and angles) from DNA and protein complexes, but is also applicable to other samples with irregular shapes.

Show MeSH
Related in: MedlinePlus