Biomedical Engineering Reference
In-Depth Information
reconstructions of cells and surfaces can be achieved with this technique. To achieve this, the sample
is scanned such that one 2D slice is recorded, the focal plane of the sample is then moved a pre-
scribed amount where the next 2D slice is recorded, and this sequence is repeated until the required
volume is scanned. Image processing software is then used to process the collected data to reconstruct
the three-dimensional object. Confocal microscopes are most often used to image biological systems
and semiconductor surfaces
[18-20]
.
Three variations of scanning are available in confocal microscope systems. In the conventional
confocal laser scanning microscope, the sample is raster scanned which results in a scanning rate
of about three frames per second. Such systems provide the highest spatial resolution, however for
higher temporal resolution, the spinning disk (Nipkow) and the programmable array microscope
(PAM) systems can provide rates of 30 frames per second
[21]
. In a Nipkow disk system, a thin
disk with hundreds of spirally patterned pinholes is spun in the light path to the objective lens. The
pinholes only allow perpendicularly oriented rays of light to penetrate, which allows high scanning
speeds independent of the laser scanning speed. PAM is a variation on this whereby an acousto- or
electro-optical filter can be patterned to automatically produce the pinhole pattern required. Such a
system can allow, e.g., up to 1000 beams to simultaneously scan the entire field at millisecond scan
speeds. High frequency scanning has the added advantage of reducing photon exposure to sensitive
samples which may be damaged due to photobleaching or phototoxicity.
Fluorescent dyes are often added to a surface or flurophores to cellular systems to enable
enhanced imaging with confocal fluorescent microscopy. Various excitation laser wavelengths
are available for these systems ranging from 442 to 647 nm depending on the flurophore excitation
and emission wavelengths used. Reflected and fluoresced light waves are emitted from the sample.
A beam splitter can be selected to reflect only the fluoresced light to the detector, which provides
enhanced signal-to-noise ratio. As described earlier, the pinhole is also used in this setup to eliminate
the out-of-focus signal and record only the light from the region of interest.
Confocal Microscopy Case Studies
In a recent study, Manara et al.
[22]
examined an electrochemically assisted process for the depo-
sition of hydroxyapatite/collagen coatings on titanium plate. The produced coatings are biomimetic
bone-like composites made of self-assembled collagen fibrils and carbonate hydroxyapatite nanoc-
rystals. As well as studying these structures chemically, structurally, and morphologically, these
workers also evaluated their ability to bind fibronectin (FN) in order to test their bioactivity. FN (a
high-molecular weight glycoprotein) aids the development of the cytoskeleton by providing binding
sites for cell surface receptors and other extracellular matrix (ECM) components. FN has been iden-
tified as a potential communication network directing bone growth and is also involved in wound
healing. Manara et al.
[22]
used laser scanning confocal microscopy to characterize the degree of FN
adsorption on the various composite coatings that they produced. Confocal images showed collagen-
FN fiber bundles more than 50 μm in size and 20 μm deep after 5 min of collagen deposition (
Figure
18.3
). It was seen from this work that when the apatite phase was inter-grown with collagen fibrils,
higher FN adsorption was recorded. The authors pointed out that this can be accounted for as FN has
at least three receptors for collagen located close to the amino terminus of the molecule. It was fur-
ther noted that FN adsorption was irregular following the specific topography of the sample.
Wang et al.
[23]
presented work evaluating the mechanical properties and corrosion behaviors of
oriented Ca(PO
3
)
2
glass. In order to produce these structures, polycrystalline Al
2
O
3
plate was inserted
