Abstract
Recently, mechanobiology has received increased attention. For investigation of biofilm and cellular tissue, measurements of the surface topography and deformation in real-time are a pre-requisite for understanding the growth mechanisms. In this paper, a novel three-dimensional (3D) fluorescent microscopic method for surface profilometry and deformation measurements is developed. In this technique a pair of cameras are connected to a binocular fluorescent microscope to acquire micrographs from two different viewing angles of a sample surface doped or sprayed with fluorescent microparticles. Digital image correlation technique is used to search for matching points in the pairing fluorescence micrographs. After calibration of the system, the 3D surface topography is reconstructed from the pair of planar images. When the deformed surface topography is compared with undeformed topography using fluorescent microparticles for movement tracking of individual material points, the full field deformation of the surface is determined. The technique is demonstrated on topography measurement of a biofilm, and also on surface deformation measurement of the biofilm during growth. The use of 3D imaging of the fluorescent microparticles eliminates the formation of bright parts in an image caused by specular reflections. The technique is appropriate for non-contact, full-field and real-time 3D surface profilometry and deformation measurements of materials and structures at the microscale.
Highlights
Three-dimensional (3D) measurements of topography and displacement at the microscale can shed light in the investigations in many disciplines such as biology, medicine, biomedical engineering, materials science and engineering, and mechanical engineering
The typical characteristic in-plane dimensions of biological tissues are on the order of 1~20 mm, and the out-of-plane feature dimensions are on the order of submicron to tens of μm; these relatively small dimensions make it difficult for real-time, non-contact, full field profilometry and deformation measurements for purpose of tracking 3D deformations in-vivo or in-vitro
We demonstrate the fluorescent stereo microscopy (FSM) technique for 3D surface topography measurement and deformation measurement of a Bacillus subtilis biofilm
Summary
Three-dimensional (3D) measurements of topography and displacement at the microscale can shed light in the investigations in many disciplines such as biology, medicine, biomedical engineering, materials science and engineering, and mechanical engineering. In this paper we utilize stereo-imaging of fluorescent particles on a biomaterial sample to determine the surface topography and to measure simultaneously the 3D surface deformations for situations where these particles follow the movements of material points on the material surface [25] Both fluorescent nanoparticles and microparticles can be used for observations at different scales. A sample can be stained with multi-fluorescent probes for multi-purpose observations; and the fluorescent particles serve as random texture or markers for surface profilometry and for tracking surface deformations at different scales; (2) Fluorescence imaging avoids unwanted specular reflections, to allow pairing the corresponding points observed on the pair of micrographs through digital image correlation; this characteristic is especially helpful for observation of biosamples under in-vivo or in-vitro conditions where moist surface usually induces reflection when white light is used; (3) The fluorescent light will not induce as much heating as white light on a sample surface to alter the environmental condition for a biomaterial under investigation; and (4) Fluorescent particles have excellent biocompatibility with biomaterials, and enabling investigations of living biological samples such as biofilms and tissues. We demonstrate the FSM technique for 3D surface topography measurement and deformation measurement of a Bacillus subtilis biofilm
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