While discussing the problem of studying photosynthesis in intact leaves, Fabrice Rappaport, a research director at the Paris-based CNRS Institut de Biologie Physico-Chimique, invoked a challenge from math history. He likened the measurement task to that of constructing a square with the same area as a given circle and using only a compass and straightedge. Squaring the circle cannot be done, a consequence of the transcendental nature of pi. Likewise, time-resolving absorption changes when studying photosynthesis seems unachievable because increasing the light intensity to improve sensitivity decreases time resolution. However, solving this problem is easier than solving the pi problem, because the measurement of photosynthesis can be improved by changing the nature of the probing light. “Short and weak light pulses may be used,” Rappaport said. “Their duration determines the time resolution of the measurement, and their intensity may be kept low enough to avoid an actinic effect.” Applying that concept promises to be easier than ever, thanks to the use of new LEDs. For the study of photosynthesis, pulsed LEDs offer significant advantages over light sources such as xenon flashlamps and lasers. LEDs cost less, sustain a high repetition rate and have low pulse-to-pulse variation in terms of intensity and spatial distribution.Institute researchers Daniel Béal and Pierre Joliot have developed a prototype spectrophotometer to measure absorption changes as a way to study photosynthesis in vivo. Their device has two arms, each containing a photodiode and associated electronics. One arm is for reference, while the other contains the sample being measured. The sample is illuminated by an actinic excitation beam that initiates photosynthesis. The pulsed measuring beam is driven by visible LEDs at wavelengths such as 485, 518 and 810 nm and is carried via optical fibers to the sample’s surface. The photodiode measures absorption on the back of the sample. The two devised the device to get the total picture of photosynthesis, a process that relies on an electron-transfer chain. Because photosynthesis is triggered by light, the process can be started as needed. Both thermodynamic and kinetic parameters of the basic steps of photosynthesis are fairly well known as a result of extensive spectroscopic studies of the biochemically purified complexes.But the entire chain is more than the simple sum of its parts. For example, the various complexes in the chain are embedded in membranes, which can restrict the diffusion of soluble electron carriers. Other factors also act to shift reactions from what isolated process components would seem to dictate. “In many instances, understanding the elementary steps of the isolated complexes is not sufficient to understand the function of the overall chain,” Rappaport said.Short and weakFor researchers, the solution is to do time-resolved studies of intact samples — a challenge, given the fact that such specimens often strongly absorb or scatter light. A typical change resulting from photosynthesis might adjust the total absorption of a sample only 1/10 to 1 percent. Reliably detecting such a slight change puts a premium on a high signal-to-noise ratio, something difficult to achieve with a continuous source because the light detecting the change also will induce it.That fact drives the need for short and weak light pulses, the latter because exposure must be kept low enough to avoid an actinic effect. As for duration, shorter is better because pulse width determines time resolution. In addition, a high and sustainable repetition rate is important so that the light remains consistent throughout all experiments.“In order to accurately time-resolve a process which develops during the time interval between two successive flashes, several experiments are needed,” Rappaport said.Another advantage of this approach, he noted, is that the sensitivity of the measurement is not affected by the time resolution. That is particularly important in studying biological processes, which can span several orders of magnitude in time.A laboratory version of the spectrophotometer has been used to investigate the cyclic electron flow of spinach leaves, where researchers concluded that two flows were not physically isolated and were in dynamic competition. Having first been described and deployed several years ago, the device now has a time resolution of about 10 ms, a repetition rate of 100 μs, and a signal-to-noise ratio of 100,000:1.A commercial version of the device, the JTS-10, or Joliot Type Spectrometer 10, has been developed by Bio-Logic SAS of Claix, France, under a license from CNRS. The first prototypes became available in July. Zohra Mana, a Bio-Logic applications engineer, said that the device has removable lights and a large choice of probe and actinic sources. “JTS-10 is a unique and highly sensitive instrument, dedicated for absorbance and fluorescence changes in visible and near-infrared-wavelength ranges. An ultraviolet upgrade will be developed in the near future,” Mana said.The absorbance of a leaf (left) is shown by oxidation in intense red light, followed by reduction in the dark, with probing done with a source at 705 nm. Fluorescence is on the right, with continuous light at 520 nm and a short green probe light for a source. The curves were taken with one instrument. Courtesy of Bio-Logic SAS.As with its research predecessor, the commercial instrument has a reference and measurement arm but differs in that it has only a leaf or other sample in the measurement arm. It also uses a dichroic mirror and not optical fibers to create the two arms. Because it has an LED array at the entrance of the lightguide, it has an alternate light source suitable for fluorescence measurement.In the laboratory, researchers are looking into extending the device into the near-ultraviolet, from 280 to 380 nm, as well as at using shorter pulse widths, which is possible for samples that absorb less than 90 percent of incoming light. They also are attempting to design procedures to activate the mitochondrial respiratory chain via light. “This would enable us to apply the same techniques to the study of the mitochondrial processes, the dysfunction of which is commonly associated with myopathy or neural degeneracy,” Rappaport said.Contact: Fabrice Rappaport, CNRS Institut de Biologie Physico-Chimique, Paris; e-mail: fabrice.rappaport@ibpc.fr; Zohra Mana, Bio-Logic SAS, Claix, France; e-mail: photosynthesis@bio-logic.info.