Examples of Chl a fluorescence imaging
A wide range of examples of Chl a fluorescence imaging are provided within Oxborough (2004). Other examples can be found in Omasa et al. (1987), Daley et al. (1989), Fenton and Crofts (1990), Genty and Meyer (1995), Leipner et al. (2001), Siebke and Weis (1995), Scholes and Rolfe (1996), Bennoun and Beal (1997), Niyogi et al. (1997, 1998), Oxborough and Baker (1997a, b), Osmond et al. (1999), Küpper et al. (2000), Nedbal et al. (2000), Meng et al. (2001), Rolfe and Scholes (2002), Zangerl et al. (2002), and Barbagallo et al. (2003). The example in Fig. 3 illustrates a recent development in the field of Chl a fluorescence imaging; that of using an imaging system as a multi-channel fluorometer, with the ability to record multiple continuous fluorescence traces. In this instance, 10 traces were recorded simultaneously, in real time (only one trace is shown in the figure). There is no practical reason why this number should not be increased to several hundred, or even several thousand, should a particular application benefit from this facility.
The system used for these measurements utilizes 16 panels of 100 orange LEDs to provide measuring pulses, actinic illumination and super-saturating pulses for measurement of Fm and F'm. These LED panels are mounted on ball and socket joints and arranged in a slightly elongated dome (to take account of the 4:3 aspect ratio of the camera field of view). This arrangement provides a low level of self-shading and simplifies the generation of a uniform light field. Output from the LEDs is regulated through pulse width modulation using an ultra-fast switching circuit (Bartington Associates, Essex, UK). This allows for the incident photon irradiance to be varied from less than 5 µmol m–2 s–1 up to the maximum output, without changing the forward voltage. This approach avoids the output instability and spectral variation that are characteristic of voltage regulation.
Images of Fm, F'm and F' were generated by synchronizing the camera shutter to a measuring pulse of between 250 µs and 1 ms at a photon irradiance of 4500 µmol m–2 s–1 (the shorter pulse lengths were used when the continuous photon irradiance was low, to minimize the actinic effect). To generate images of Fm and F'm, a sequence of images was taken at 20 Hz over the last 600 ms of an 800 ms saturating pulse with a photon irradiance of 4500 µmol m–2 s–1. The image with the highest mean value was taken to be the Fm or F'm value. Images of Fo were generated by applying 2 µs pulses, at a photon irradiance of 4500 µmol m–2 s–1, at 400 µs intervals, over a 25 ms integration period.