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Optodes visualize ammonium distribution within plant root system

BioPhotonics
Mar 2008
Gwynne D. Koch

Nitrogen compounds such as ammonium are important nutrients for plant growth. Scientists at the University of Gothenburg in Sweden have created a ratiometric imaging optode that, when coupled with a recently developed calibration method, can be used to quantify ammonium and to visualize its distribution over time close to an intact plant root system. Ammonium turnover in root systems typically is studied using a technique that employs fast-scanning microelectrodes and microscopy; however, this provides limited coverage and can be used only on roots grown in solution.

In comparison, the researchers’ technique can be used directly in soil and offers good coverage and spatial and temporal resolution, important features for examining the function, mechanisms and location of complex and chemically important processes such as nutrient uptake, nitrogen fixation and root-microbial symbiosis.

According to researcher Niklas Strömberg, the general detection principle of the imaging optode is based on changes in the luminescence properties of solute-specific indicators immobilized within thin-layered plastic films, which are induced by exposure to an analyte. The sensor film, consisting of the ammonium ionophore nonactin and the fluorescent ammonium indicator dye merocyanine-540, is placed in contact with the sample and illuminated. Solvent shifts induced by fluctuations in ammonium concentrations change the excitation-emission maxima of the fluorescent dye.

To test the technique, a large 6-month-old fruit-bearing tomato plant was transplanted into a pot with removable glass sides for easy insertion and removal of the sensor assembly. The assembly consisted of the following: a transparency film that isolated a single root structure from the rest of the plant, a 2-mm-thick sheet of paper that acted as a reservoir for the ammonium solution, an optical isolation film and the sensor film. Optical isolation minimized the scattering effects and depressed autofluorescence from the sample.

BNammonium_Fig-1_Setup.jpg

Figure 1. The optical system and sensor assembly consisted of a xenon light source (A), a dual bandpass filter changer (B), a liquid lightguide (C), focusing lenses (D), the sensor assembly in the pot (E), a macro lens (F), bandpass filters for wavelength selection (G), a CCD camera (H), a dual-filter control unit (I) and a computer (J). The close-up of the sensor assembly shows the transparency film that isolated the studied root structure (I), the ammonium solution reservoir (II), the optical isolation (III), the ammonium sensor film (IV), and the removable glass front (V) facing the illumination and the macro lens. Images reprinted with permission of Environmental Science & Technology.

Excitation radiation from a 300-W xenon UV/VIS arc lamp equipped with a dual-filter changer with 511:572-nm interference bandpass filters was delivered to the sample through a liquid lightguide (Figure 1).

Because an infinity-corrected macro imaging system was not commercially available, light emitted by the sensing membrane of the imaging optode was collected through a Nikon macro lens and an infinity-corrected achromatic lens package custom-configured by the team. For collimated and maximum throughput, Strömberg mounted 572:592-nm emission bandpass filters in the infinity region perpendicular to the optical axis between the achromatic lenses.

The filtered fluorescence signal was detected with a 60-dB, 12-bit cooled monochrome Diagnostic Instruments CCD camera used in 3 × 3 binning mode to increase the dynamic range. The use of narrow, 2-nm bandpass filters necessitated the rather long exposure time of 84 s. The time could be reduced by using a more suitable bandpass for the 511-nm excitation.

The optode film was calibrated in a flow cell before and after image capture. The responses in each pixel from the two time-separated calibrations were linked using a linear time-dependent response function, and the fluorescence ratio in each pixel was determined individually at the time of image capture, a procedure Strömberg refers to as time-correlated pixel-by-pixel calibration. This method removes drift in response over time and enables statistical measures such as analytical sensitivity, limit of detection and relative signal variation to be collected with high precision and accuracy. Ammonium was quantified through a dual-excitation, dual-emission image ratio of 511:572 nm/572:592 nm.

To visualize ammonium flow patterns, the team generated quiver plots that display velocity vectors as arrows showing the direction and magnitude of the flow (Figure 2). The results indicate that ammonium uptake for tomato plants occurs over the entire root structure, but that transverse thin peripheral roots are about twice as efficient as the main root.

BNAmmonium_Fig2_2x3-inches.jpg
Figure 2. A quiver plot shows the ammonium flow patterns close to roots toward the end of the light period. The circled regions in the upper image indicate local areas of high ammonium supply (A) or consumption (B, C and D). Region B was directly associated with a fresh root structure directed toward the sensor film, indicating that ammonium acquisition is highest at apical regions of roots.

The experiment demonstrated the technique’s ability to be used in situ to study ammonium turnover in complex systems over time. Scientists at the Swedish University of Agricultural Sciences are using the sensor to examine release rates and distribution patterns from fertilizers in various soils to study their effectiveness under specific climatic conditions. Strömberg also is collaborating with researchers at the University of Gothenburg to develop imaging optodes for oxygen and chemical compounds known as catecholamines for use with fluorescence lifetime imaging for studying neurotransmission in neuronal networks.

Environmental Science & Technology, published online Jan. 31, 2008.


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