Automated Fiber Diameter and Porosity Measurements of Plasma Clots in Scanning Electron Microscopy Images

Abstract

Scanning Electron Microscopy (SEM) is a powerful, high-resolution imaging technique widely used to analyze the structure of fibrin networks. Currently, structural features, such as fiber diameter, length, density, and porosity, are mostly analyzed manually, which is tedious and may introduce user bias. A reliable, automated structural image analysis method would mitigate these drawbacks. We evaluated the performance of DiameterJ (an ImageJ plug-in) for analyzing fibrin fiber diameter by comparing automated DiameterJ outputs with manual diameter measurements in four SEM data sets with different imaging parameters. We also investigated correlations between biophysical fibrin clot properties and diameter, and between clot permeability and DiameterJ-determined clot porosity. Several of the 24 DiameterJ algorithms returned diameter values that highly correlated with and closely matched the values of the manual measurements. However, optimal performance was dependent on the pixel size of the images—best results were obtained for images with a pixel size of 8–10 nm (13–16 pixels/fiber). Larger or smaller pixels resulted in an over- or underestimation of diameter values, respectively. The correlation between clot permeability and DiameterJ-determined clot porosity was modest, likely because it is difficult to establish the correct image depth of field in this analysis. In conclusion, several DiameterJ algorithms (M6, M5, T3) perform well for diameter determination from SEM images, given the appropriate imaging conditions (13–16 pixels/fiber). Determining fibrin clot porosity via DiameterJ is challenging.

A guide to molecular and functional investigations of platelets to bridge basic and clinical sciences

“…Although SEM provides valuable information about clot and fibrin networks 89,90 , typical analysis is manual and tedious. Newer automated platforms are now being developed to increase accuracy and reproducibility in imaging clots 91 . Several studies have used SEM to facilitate understanding of thrombus formation, platelet adhesion and platelet properties under flow and mechanical stress 81,[92][93][94] .…”

A systematic review and comparison of automated tools for quantification of fibrous networks

“…A major drawback of the included tools is the fact that the pores are mostly measured in 2D images, while it is important to measure pores in 3D to get reliable information since pores can have different sizes in all three dimensions. Pore size and porosity as measured by DiameterJ were indeed shown to be poorly correlated with functional permeability measurements of plasma clots 8 . While pore sizes have been measured in 3D [66][67][68] , and it was shown that it is possible to work with 2D images while correcting for the missing third dimension 69 , these tools are unfortunately not publicly available.…”

“…Until recently, quantification of fiber diameters was often done manually, which is inefficient and sensitive to observer bias 8,60 . We tested eight available tools to automatically measure fiber diameter.…”

“…The first data set was a series of scanning electron microscopy (SEM) images of clots prepared from plasma from healthy controls or patients with severe postpartum hemorrhage (PPH) 8 . This study was approved by the Medical Ethics Committee of the Erasmus University Medical Center Rotterdam (MEC-2016-218) and all participants provided written informed consent.…”

“…SEM provides high resolution, but requires extensive sample preparation such as dehydration and sputter coating, which can affect network properties and introduce imaging artefacts 7 . Preparation of samples for confocal microscopy is less invasive, but this method has a limited resolution and can therefore not reliably measure the diameter of fibers, which are often below 200 nm in biological materials 8 and synthetic hydrogels 9 . Recently developed super-resolution methods such as stimulated emission depletion (STED) microscopy offer a good alternative, but are so far not exploited much for fibrous networks.…”