LED Excitation and Photomultiplier Tube Biasing and Gating Circuitry for Fluorescence Instrumentation

Abstract

Fluorescence technologies have only begun exploiting the transient recording of lifetime-based signals and images for low nanosecond lifetimes, but the method has tremendous potential for scientific and medical applications. Low nanosecond lifetime recording in real-time can enable the tracking of metabolite concentrations in cells and tissues (e.g. cancerous tissues) without introducing foreign substances. It will also enable the tracking of reactive species (e.g. ozone) and intermediate/short-lived states in chemical reactions in the atmosphere. Current techniques all employ laser excitation, but LEDs can also be used which cause considerably less damage to live tissue. We have developed a high speed fluorescence prototype using high intensity LED pulses and novel PMT gating technology. Precision timing circuitry generates tunable width pulse signals which are driven through the LED using a comparator-based push-pull architecture. The timing circuitry also generates PMT gating pulses which are applied to the dynode chain via high voltage operational amplifiers. LED pulses with fall times (99%) as short as 2ns and PMT gating times (10% to 90%) of 3.6ns have been achieved. The prototype has been used to successfully measure the fluorescent lifetimes of Alexa Fluor 610X dye (1.7ns and 4.7ns) and riboflavin (4.5ns). Lifetimes of acridine orange were measured as follows: alone (2ns), in solution with ssDNA (3.7ns), in solution with dsDNA (5.8ns), and in solution with dsRNA (5.9ns). Finally, dsRNA was heated and allowed to cool revealing lifetimes that started at 3.7ns when hot and increased to nearly 5ns when cool.