UV Ratiometric Imaging Of Isolated Ventricular Cardiomyocytes Using An LED Based Illuminator

Abstract

Ratiometric fluorescence microscopy methods allow researchers to obtain calibrated images of dynamic changes in the physical properties of cells and tissues independent of dye concentration.The wavelength changes required for ratiometric imaging are routinely achieved using a short-arc source in combination either with a diffraction grating or interference filters mounted in a filter wheel. Filter wheels typically switch positions in around 50ms, scanning monochromators can achieve wavelength changes within a ms. These approaches generally limit ratio imaging rates to a little over 10Hz, and have the inherent drawbacks of short lamp life and high thermal emissions. An LED based system has much higher stability than a short-arc source, and with sub-microsecond wavelength switching times allows the very highest speeds to be obtained. Here we made use of the popular Ca2+ probe Fura-2 to record images of intracellular [Ca2+] in isolated ventricular cardiomyocytes at frame rates in excess of 100Hz using a simple and inexpensive LED based system.Transient spatial gradients of Ca2+ can exist in single ventricular cardiomyocytes during spontaneous release of Ca2+ from the sarcoplasmic reticulum. This can result intracellular Ca2+ waves traveling at 100-200microns/s along the length of the cell. Rapid imaging is required to resolve the time course and pattern of intracellular Ca2+ release.Conventionally Fura-2 ratios are calculated by monitoring the fluorescence signal elicited from excitation at 340 and 380nm, however short wavelength LEDs (340nm) are not available currently. Alternatively, reliable ratiometric measurements can be made by exciting Fura-2 at its isobestic point (360nm) and 380nm. We have followed fast spatial changes in Ca2+ by switching wavelength in the microsecond time domain using commercial LEDs emitting at peak wavelengths of 365nm and 385nm.