Multiple-wavelength Imaging Research Articles

Diagnostic morphometric applicability of confocal laser scanning microscopy in Osteoarchaeology

AbstractThe confocal laser scanning microscope (CLSM) has become an essential tool for a wide range of biological and clinical studies and imaging applications. The major imaging modes of the CLSM include single and serial optical sections from thick specimens (100 µm), multiple wavelength images and three‐dimensional reconstruction. Such images provide measurement of length, surface or volume of objects. Surprisingly, this advanced microscopic method has been very rarely used in anthropological research, although numerous clinical and basic science research projects showed its value to study, bone growth, bone micro‐architecture and 3D bone morphometry. We present the basic principles and advantages of CLSM and outline practical aspects of specimen preparation, image collection and digital image processing specifically appropriate for historical bone and hair material. Copyright © 2009 John Wiley & Sons, Ltd.

Site-specific Modification of AAV Vector Particles With Biophysical Probes and Targeting Ligands Using Biotin Ligase

We have developed a highly specific and robust new method for labeling adeno-associated virus (AAV) vector particles with either biophysical probes or targeting ligands. Our approach uses the Escherichia coli enzyme biotin ligase (BirA), which ligates biotin to a 15-amino-acid biotin acceptor peptide (BAP) in a sequence-specific manner. In this study we demonstrate that by using a ketone isotere of biotin as a cofactor we can ligate this probe to BAP-modified AAV capsids. Because ketones are absent from AAV, BAP-modified AAV particles can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage modification methodology in the context of a mammalian cell lysate for the labeling of AAV vector particles with various fluorophores, and for the attachment of a synthetic cyclic arginine-glycine-aspartate (RGD) peptide (c(RGDfC)) to target integrin receptors that are present on neovasculature. Fluorophore labeling allowed the straightforward determination of intracellular particle distribution. Ligand conjugation mediated a significant increase in the transduction of endothelial cells in vitro, and permitted the intravascular targeting of AAV vectors to tumor-associated vasculature in vivo. These results suggest that this approach holds significant promise for future studies aimed at understanding and modifying AAV vector-cellular interactions.

Oximetry with a multiple wavelength SLO.

Oximetry may be useful for understanding pathologies of the eye. We use a prototype scanning laser ophthalmoscope capable of simultaneous multiple wavelength imaging to record fundus images. The system is run under non-confocal conditions in using slit apertures with a width of up to 600 microm. The laser lines launched into the SLO were the 633 nm line of a HeNe-laser, and the 815 nm line from a tunable (700 to 900 nm) cw Ti:Sapphire-laser. These wavelengths were selected because of their availability and absorption characteristics. The difference in absorption at these two wavelengths is used to assess blood oxygen content. Images were averaged to improve the signal to noise ratio. This simultaneous method of measuring oxygen content may be preferred to other techniques such as sequential SLO imaging at different wavelengths, or spot analysis using a modified fundus camera. The advantages of this technique are that image registration is not required, and a large area of the retina can be assessed concurrently.

Principles and practices of laser scanning confocal microscopy.

The laser scanning confocal microscope (LSCM) is an essential tool for many biomedical imaging applications at the level of the light microscope. The basic principles of confocal microscopy and the evolution of the LSCM into today's sophisticated instruments are outlined. The major imaging modes of the LSCM are introduced including single optical sections, multiple wavelength images, three-dimensional reconstructions, and living cell and tissue sequences. Practical aspects of specimen preparation, image collection, and image presentation are included along with a primer on troubleshooting the LSCM for the novice.