Generating 3D and 3D-like animations of strongly uneven surface microareas of bloodstains from small series of partially out-of-focus digital SEM micrographs

Abstract

When dealing with microscopic still images of some kinds of samples, the out-of-focus problem represents a particularly serious limiting factor for the subsequent generation of fully sharp 3D animations. In order to produce fully-focused 3D animations of strongly uneven surface microareas, a vertical stack of six digital secondary-electron SEM micrographs of a human bloodstain microarea was acquired. Afterwards, single combined images were generated using a macrophotography and light microscope image post-processing software. Subsequently, 3D animations of texture and topography were obtained in different formats using a combination of software tools. Finally, a 3D-like animation of a texture–topography composite was obtained in different formats using another combination of software tools. By one hand, results indicate that the use of image post-processing software not concerned primarily with electron micrographs allows to obtain, in an easy way, fully-focused images of strongly uneven surface microareas of bloodstains from small series of partially out-of-focus digital SEM micrographs. On the other hand, results also indicate that such small series of electron micrographs can be utilized for generating 3D and 3D-like animations that can subsequently be converted into different formats, by using certain user-friendly software facilities not originally designed for use in SEM, that are easily available from Internet. Although the focus of this study was on bloodstains, the methods used in it well probably are also of relevance for studying the surface microstructures of other organic or inorganic materials whose sharp displaying is difficult of obtaining from a single SEM micrograph.

Introduction

Animations are nowadays a widespread tool for circulating scientific research and education (e.g. Fisk, 2008, Kano et al., 2008, Kelly and Jones, 2008, Koutmos et al., 2008, Le Marechal and El Bilani, 2008, McGrath et al., 2008, Power, 2008, Salmela et al., 2008). However, when dealing with microscopic still images (micrographs) of some kinds of samples, the out-of-focus problem represents a particularly serious limiting factor for the subsequent generation of fully sharp animations in three-dimensional mode (3D animations). Several software solutions have been previously used in order to focus light microscope images (Piper, 2008). The basis of this focusing is the superimposition of several images of the same view focused at slightly different planes. This superimposition produces a fully sharp, in-focus image from the vertical stack of images that were partially out of focus.

On the other hand, although, in contrast with light microscopes, scanning electron microscopes (SEMs) have a large depth of field, completely focused images of strongly uneven surface microareas are occasionally difficult to obtain even in this type of microscopes. In the case of bloodstains, the outermost erythrocyte layer of a thin bloodstain can be much more uneven than that previously suggested by customary SEM micrographs (e.g. Hortolà, 1992, Hortolà, 2002). Therefore, such erythrocyte layer is worth of being modelised in 3D in order to improve our knowledge of the microstructure of blood smears.

In a previous paper, I have reported the use of digital anaglyphy to improve the relief effect of SEM micrographs of bloodstains as 3D still images (Hortolà, 2009). In this paper, I report the application of some software tools to the production of 3D and 3D-like animations of strongly uneven surface microareas of bloodstains, starting from small series of partially out-of-focus digital SEM micrographs.