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I am trying to measure the volume of (mostly dried) proteins I have imaged. Mathematically speaking, does anyone have a suggested method for doing so? The only methods I can think of are to ignore convolution and simply multiply individual pixel height by pixel area, or to assume a roughly symmetric sphere or ellipsoid. Both have their inherent flaws and assumptions.
Have you tried the bearing analysis function of Nanoscope Analysis? I use it pretty often for big features like cells but it should also be accurate on small features like proteins. In the past I also used the Wyco volume analysis feature but it's definitely less precise.
I agree on your other points (the pixel technique). What you can also do is use a "tip check" reference sample to get an estimation of the tip radius. Then you can use convolution/dilatation analysis programs to get the exact geometry of the features you imaged. I found this reference but can't access the full article. I think the main author developed his own labware-based program:
aLaboratoire en Mécanique Roberval, FRE CNRS-UTC 2833, Université de Technologie de Compiègne, BP 60319, 60206 Compiègne Cedex, France
bLaboratoire de Tribologie et Dynamique des Systèmes, UMR CNRS-ECL 5513, Ecole Centrale de Lyon, BP 163, 69131 Ecully Cedex, France
SFM images are the result of interactions between a sharp tip with a quasi-spherical apex and the surface of a sample. As a consequence, SFM images are highly influenced by the tip geometry especially when the surface features are sharper than the probe. For topographic images, this phenomenon is known as dilation. It can result in an image where the features reflect the tip apex characteristics rather than the sample’s surface. Mathematical algorithms are used to show the possibility of computing a curvature image from a topographic image to deduce the radius of the tip. In addition, the interpretation of a surface image obtained with modes other than topography can be achieved by comparing this image to a curvature image. As an example, the Tapping Mode AFM phase image contrast can show similarities or discrepancies with the curvature image contrast, leading to a direct relation between the phase and a topographic or a physical contrast.
Good luck,
Alex.
Permalink to AFM Deconvolution Software at MIAWiki:
http://confocal-manawatu.pbworks.com/w/page/43681258/AFM%20Deconvolution%20Software
Cheers,
Dmitry
Nuclei Volume Estimation:
http://confocal-manawatu.pbworks.com/w/page/16346886/Confocal%20Measurements%20Lab%20ImageJ
Very helpful links!
Thanks Dimitri!
Thank you both, those should be very helpful!
I should add that in doing further research in the literature, I have found that, since a Schneider, et al. 1998 paper, it appears to have become conventional to assume that an AFM-imaged protein adopts a spherical-cap shape, and to calculate the volume appropriately: V = (pi*h/6)(3r^2+h^2).
Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy. Schneider SW, Lärmer J, Henderson RM, Oberleithner H. Source Department of Physiology, University of Münster, Robert-Kochstrasse 27a, D-48149 Münster, Germany. Abstract Proteins are usually identified by their molecular weights, and atomic force microscopy (AFM) produces images of single molecules in three dimensions. We have used AFM to measure the molecular volumes of a number of proteins and to determine any correlation with their known molecular weights. We used native proteins (the TATA-binding protein Tbp, a fusion protein of glutathione-S-transferase and the renal potassium channel protein ROMK1, the immunoglobulins IgG and IgM, and the vasodilator-stimulated phosphoprotein VASP) and also denatured proteins (the red blood cell proteins actin, Band 3 and spectrin separated by SDS-gel electrophoresis and isolated from nitrocellulose). Proteins studied had molecular weights between 38 and 900 kDa and were imaged attached to a mica substrate. We found that molecular weight increased with an increasing molecular volume (correlation coefficient = 0.994). Thus, the molecular volumes measured with AFM compare well with the calculated volumes of the individual proteins. The degree of resolution achieved (lateral 5 nm, vertical 0.2 nm) depended upon the firm attachment of the proteins to the mica. This was aided by coating the mica with suitable detergent and by imaging using the AFM tapping mode which minimizes any lateral force applied to the protein. We conclude that single (native and denatured) proteins can be imaged by AFM in three dimensions and identified by their specific molecular volumes. This new approach permits detection of the number of monomers of a homomultimeric protein and study of single proteins under physiological conditions at the molecular level. Schneider SW, Larmer J, Henderson RM, Oberleithner H. Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy. Pflugers Arch.1998;435:362–367.
Department of Physiology, University of Münster, Robert-Kochstrasse 27a, D-48149 Münster, Germany.
Proteins are usually identified by their molecular weights, and atomic force microscopy (AFM) produces images of single molecules in three dimensions. We have used AFM to measure the molecular volumes of a number of proteins and to determine any correlation with their known molecular weights. We used native proteins (the TATA-binding protein Tbp, a fusion protein of glutathione-S-transferase and the renal potassium channel protein ROMK1, the immunoglobulins IgG and IgM, and the vasodilator-stimulated phosphoprotein VASP) and also denatured proteins (the red blood cell proteins actin, Band 3 and spectrin separated by SDS-gel electrophoresis and isolated from nitrocellulose). Proteins studied had molecular weights between 38 and 900 kDa and were imaged attached to a mica substrate. We found that molecular weight increased with an increasing molecular volume (correlation coefficient = 0.994). Thus, the molecular volumes measured with AFM compare well with the calculated volumes of the individual proteins. The degree of resolution achieved (lateral 5 nm, vertical 0.2 nm) depended upon the firm attachment of the proteins to the mica. This was aided by coating the mica with suitable detergent and by imaging using the AFM tapping mode which minimizes any lateral force applied to the protein. We conclude that single (native and denatured) proteins can be imaged by AFM in three dimensions and identified by their specific molecular volumes. This new approach permits detection of the number of monomers of a homomultimeric protein and study of single proteins under physiological conditions at the molecular level.