Use of a highly sensitive two-dimensional luminescence imaging system to monitor endogenous bioluminescence in plant leaves
© Flor-Henry et al; licensee BioMed Central Ltd. 2004
Received: 07 July 2004
Accepted: 18 November 2004
Published: 18 November 2004
All living organisms emit spontaneous low-level bioluminescence, which can be increased in response to stress. Methods for imaging this ultra-weak luminescence have previously been limited by the sensitivity of the detection systems used.
We developed a novel configuration of a cooled charge-coupled device (CCD) for 2-dimensional imaging of light emission from biological material. In this study, we imaged photon emission from plant leaves. The equipment allowed short integration times for image acquisition, providing high resolution spatial and temporal information on bioluminescence. We were able to carry out time course imaging of both delayed chlorophyll fluorescence from whole leaves, and of low level wound-induced luminescence that we showed to be localised to sites of tissue damage. We found that wound-induced luminescence was chlorophyll-dependent and was enhanced at higher temperatures.
The data gathered on plant bioluminescence illustrate that the equipment described here represents an improvement in 2-dimensional luminescence imaging technology. Using this system, we identify chlorophyll as the origin of wound-induced luminescence from leaves.
It is well documented that essentially all living systems spontaneously generate and emit very low levels of light (reviewed in ). This ultra-weak bioluminescence is generally characterized by emission of photons (sometimes termed "biophotons") at an intensity less than 10-14 W.cm-2 (< 1000 photons.sec-1.cm-2). This is in contrast to the more widely known bioluminescence from animals that visibly glow, such as certain species of jellyfish, fireflies and beetles, which employ fluorescent proteins and/or luciferase enzymes to catalyse reactions that result in chemiluminescence. Ultra-weak bioluminescence is generally considered to result from oxidative chemistry occurring within cells [1, 2], though in most cases, the source of observed emissions has not been identified. Nevertheless, changes in emission levels in response to stress – particularly oxidative stress – have been observed in many systems, including bacteria , plants (e.g. [2, 4–6]) and animals [7, 8]. For this reason, measurement of ultra-weak bioluminescence may be a useful non-invasive technique to monitor rapid perturbations to cellular activity and for early detection of diseased and damaged cells.
In plants, increases in spontaneous low-level luminescence have been observed in response to pathogen infection , salt stress , osmotic stress  and mechanical damage or wounding [2, 4, 6, 11]. It is suggested that luminescence is produced by singlet oxygen and excited carbonyl species generated as a result of lipid peroxidation reactions [2, 11]. Lipid peroxidation in wounded and pathogen-infected plant tissues is a common consequence of the generation of ROS, which also act as signals to induce plant defence responses .
Quantitative measurements of such ultra-weak photon emissions are normally obtained using sensitive photomultiplier tubes as photon counting devices. However, more recently, 2-dimensional photomultiplier tubes and cooled charge-coupled device (CCD) cameras have also been employed as a means of imaging the spatial distribution of light emission from diseased and damaged plant tissues [2–6, 9]. The clearest images were obtained by Chen et al., , using a micro-channel plate coupled to a cooled CCD to image light captured through a lens. However, the acquisition time required for what is a relatively weak signal, was 1 hour, preventing a detailed temporal investigation of stress-induced bioluminescence.
Here, we present a novel configuration of a cooled CCD that enables high sensitivity, high resolution 2-dimensional imaging with short integration times. We demonstrate the utility of the system to image and quantify delayed chlorophyll fluorescence and wound-induced luminescence from plant leaves.
Results and Discussion
Construction of a luminescence imager
Imaging delayed chlorophyll fluorescence
Spatial and temporal resolution of ultra-weak luminescence in wounded leaves
Origin of wound-induced luminescence
Temperature-dependence of wound-induced luminescence
The novel configuration of cooled CCD that we have used in this study to capture 2-dimensional images of plant leaves provides excellent temporal and spatial resolution of bioluminescence/chemiluminescence processes in biological materials, which are important markers of various forms of stress and disease. Using this system, we are able to define the origin of wound-induced luminescence from plant leaves as chlorophyll, and suggest that this arises via the temperature-dependent release of energy from excited triplet carbonyls produced by oxidative stress.
Plants of Arabidopsis thaliana, Columbia ecotype, were grown in soil in a glasshouse at 22°C with a 16-hour photoperiod. Wounding was performed by crushing leaves with a haemostat. To generate leaves lacking chlorophyll, Arabidopsis plants grown for 3 weeks under 16 h light/8 h dark, were watered with 0.1 mM norflurazon (Sigma Aldrich PS1044) and grown under constant illumination at 120 μmol/m2/sec. Under these conditions, newly emerging leaf tissue was white. Leaves with white regions were taken 15 days after the start of norflurazon treatment, and used for luminescence imaging.
Two dimensional luminescence imaging apparatus
The imager used is a two-dimensional imaging system based on a slow-scan 'scientific grade' Silicon Photo Area-Detector [SPAD] (Integrated SPAD Imaging System – PiXx*ell 1A, Biolumonics Ltd), which consists of a cooled ultra-sensitive area photo-detector within a stainless steel vacuum vessel and associated image acquisition electronics. The input image is coupled to the detector with optical fibres (numerical aperture = 1). The detector contains 222,336 pixels, and in the experiments presented here, ran at an operating temperature of -58°C. The combination of the proprietary low read-out noise electronics, cooling of the detector and the optical fibre coupling results in a sensitive imaging system capable of recording the faintest luminescences at high resolution, (40 μm to 200 μm depending on geometry of sample and integration time). The spectral absorption response of the SPAD ranges from the near UV to IR, thus including the entire visible spectrum up to the band-gap energy barrier of Silicon (1.1 eV) i.e. at approx 1100 nm; however, for ultimate sensitivity the detector output is monochrome, i.e. there is no wavelength selectivity in the present instrument. Further information is available on request from Michel Flor-Henry, Biolumonics Ltd.
Coloured filters were obtained from Lee Filters (Andover, UK). We used lighting effect filters LEE 182 – "light red," LEE 118 – "light blue," LEE 183 – "moonlight blue" and LEE 735 – "velvet green." Spectral characteristics of the filters can be viewed at http://www.leefilters.com, though readers should note that the transmittance spectra provided with the filters covers the range 300–800 nm, rather than the more limited 400–700 nm spectra shown on the web site. Filters were placed between the leaf and the sample stage prior to imaging.
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