Featuring technique of paticular proteins for brain tissue using acoustic impedance microscope

Abstract

Two-dimensional acoustic impedance imaging is useful for observation of tissue conditions without histochemical processing. Previously, we showed that direct heavy metal binding to particular channel proteins of living cerebellar tissues was useful for the featuring of an acoustic impedance, and visualized the distribution of particular molecules in vital tissues acoustically. In this report, we would prove the attractive extension of acoustic impedance imaging to feature arbitrary proteins using immunoreactivity. Quantum Dot (QD) nanoparticles are semiconductor nanocrystals of about 500 Cadmium (Cd) atoms and other elements, and used as fluorescent crystal in the immunohistochemical observation. Immunoconditioned QDs are bound to the primary immunomolecules, primary antibodies, binding to particular proteins. Chemical fixed cerebellar slices removed from postnatal 20 to 28 day rat were sliced 400 μm thickness, treated with primary antibodies to either calbindin D-28k or GABA receptor α6 subtype, and observed using acoustic microscope HMS-1000 (Honda Electronics Co. Ltd.). Calbindin D-28k is calciumbinding proteins distributed in cerebellar Purkinje cells, and GABA receptor α6 subtype is specific GABA receptor-constituent protein in cerebellar granule cells. Cerebellar slices were treated with 0.1% triton-X 100, mounted on a flat 0.7 mm thick culture dish made of hydrophilic polystyrene, and reference water. Treatment with the antibodies to calbindin D-28k and immune molecules with QD made the high acoustic impedance dots about 1.8 [106 Ns/m3] appeared in Purkinje cell layer. The intensity of impedance of these dots corresponded to the density of calbindin D-28k molecules in Purkinje cells. On the other hand, treatment with the antibodies to GABAR α6 subtype showed little enhancement in granule cell layer. GABA α6 subtype proteins would be about 10000 times fewer than calbindin D-28k, so that GABA α6-QD immunocomplex could not be featured in the acoustic image. We suggest that many kinds of proteins would be able to feature acoustically using QD immunocomplex, if cells have some amount of them.