Technology for Counting Macromolecules in Single Cells

Rigney, David

Abstract

The general aim of the proposed pilot project is to show the feasibility of ultra-sensitive instrumentation and methodology that will allow us to count the number of molecules of virtually any species of protein or ribonucleic acid within individual cells. If successfully implemented, the technology should permit single-cell counting of essential macromolecules that are present at the level of one to ten molecules per cell. The availability of such technology will greatly extend the types of medical diagnoses that may be made by examining as few as a hundred cells from a patient, e.g., cells from amniotic fluid, from a fine-needle tumor biopsy, or from cardiac biops through catheterization. Its availability will also permit correlation of a cell's physiology with its macromolecular content and will therefore be of considerable value to molecular, cell, and developmental biologists. The technology will make use of antibody and nucleic acid probes that bind stereo-specifically to the target molecule of interest, thus exploiting the enormous potential of commercially available hybridoma and genetic engineering products. The target-probe complex will be bound to enzyme molecules using biotin-avidin methods, and the enzyme molecules will then b used as amplifiers to generate a fluorescent signal. A novel high-sensitivi y microphotometer will be built to measure the rate of change of fluorescence surrounding individual cells, thereby providing an indirect measurement of the number of target molecules in each cell. In order to achieve ultra-sensitivity, we propose to incorporate the following principles of detection into the instrumentation design: (1) discrimination of the fluorophore on the basis of its diffusion constant, by means of spatial fluctuation analysis, using a video-rate laser scanning microscope; and (2) discrimination of the fluorophore on the basis of its fluorescence lifetime using acousto-optic laser light modulation and lock-in amplification in the 1-25 megahertz range.
Specific aims of the project axe to evaluate the sensitivity of the propose technology and to devise algorithms for estimating the number of molecules of interest in individual cells, using data acquired by means of the technology.