Blue-green Fluorescence Research Articles

Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy

Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, and vice versa. They can be utilized as genetically encodable probes and bear large potential for a wide array of applications, in particular for new protein tracking schemes and subdiffraction resolution microscopy. However, the currently described monomeric RSFPs emit only blue-green or green fluorescence; the spectral window for their use is thus rather limited. Using a semirational engineering approach based on the crystal structure of the monomeric nonswitchable red fluorescent protein mCherry, we generated rsCherry and rsCherryRev. These two novel red fluorescent RSFPs exhibit fluorescence emission maxima at ∼610nm. They display antagonistic switching modes, i.e., in rsCherry irradiation with yellow light induces the off-to-on transition and blue light the on-to-off transition, whereas in rsCherryRev the effects of the switching wavelengths are reversed. We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules.

Multicolor Fluorescence Imaging of Leaves—A Useful Tool for Visualizing Systemic Viral Infections in Plants†

Multicolor fluorescence induced by UV light is a sensitive and specific tool that may be used to provide information about the primary and secondary metabolism of plants by monitoring signals of the chlorophyll fluorescence (Chl-F) and blue-green fluorescence (BGF), respectively. We have followed the systemic infection of Nicotiana benthamiana plants with the Pepper mild mottle virus (PMMoV) by means of a multicolor fluorescence-imaging system, to detect differences between two strains of PMMoV during the infection process and to establish a correlation between the virulence and changes induced in the host plant. Changes in both BGF and Chl-F were monitored. BGF increased mainly in the abaxial side of the leaf during pathogenesis and the corresponding images showed a clear vein-associated pattern in leaves of infected plants. HPLC analysis of leaf extracts was carried out to identify compounds emitting BGF, and determined that chlorogenic acid was one of the main contributors. BGF imaging was able to detect viral-induced changes in asymptomatic (AS) leaves before detection of the virus itself. Chl-F images confirmed our previous results of alterations in the photosynthetic apparatus of AS leaves from infected plants that were detected with other imaging techniques. Fluorescence ratios F440/F690 and F440/F740, which increase during pathogenesis, were excellent indicators of biotic stress.

Synthesis and optical properties of 2,6-anthracene-bridged periodic mesostructured organosilicas

2,6-Bis(triethoxysilyl)anthracene was first synthesized by rhodium-catalyzed disilylation of 2,6-bis(trifluoromethanesulfonyloxy)anthracene with triethoxysilane. Using the new precursor, anthracene-bridged periodic mesostructured organosilicas were obtained without cleavage of the Si–C bondings. The anthracene-silica hybrids showed absorption at near ultraviolet region and blue–green fluorescence with a quantum yield of 0.13–0.15. The absorption and fluorescence behaviors of the 2,6-anthracene-silica hybrid film were compared with those of the previously reported 9,10-anthracene-silica hybrid film. Their absorbances suggest that the 2,6-anthracene groups were packed more densely than the 9,10-anthracene groups in the mesoporous frameworks due to smaller steric hindrance. In spite of the densely packed structure, the 2,6-anthracene-silica hybrid film showed fluorescence emission at shorter wavelengths and a higher quantum yield than those of the 9,10-anthracene-silica hybrid film, suggesting relatively weak interaction between the anthracene groups in the framework because of restriction of intramolecular rotation motion for substitution at 2,6-positions of anthracene. Dilution of the anthracene groups in the framework with trimethoxysilane promoted monomeric fluorescence and an increase of its quantum yield up to 0.18.

Base-Mediated Cyclization Reaction of 2-Alkynylphenylphosphine Oxides: Synthesis and Photophysical Properties of Benzo[b]phosphole Oxides

The base-mediated intramolecular cyclization reaction of 2-alkynylphenylphosphine oxides affords benzo[ b]phosphole oxides, which show intense blue-green fluorescence. Benzo[ b]phospholes are also prepared by the reduction of benzo[ b]phosphole oxides.

Archaeoglobus infectus sp. nov., a novel thermophilic, chemolithoheterotrophic archaeon isolated from a deep-sea rock collected at Suiyo Seamount, Izu-Bonin Arc, western Pacific Ocean

A novel thermophilic, strictly anaerobic archaeon, designated strain Arc51T, was isolated from a rock sample collected from a deep-sea hydrothermal field in Suiyo Seamount, Izu-Bonin Arc, western Pacific Ocean. Cells of the isolate were irregular cocci with single flagella and exhibited blue-green fluorescence at 436 nm. The optimum temperature, pH and NaCl concentration for growth were 70 degrees C, pH 6.5 and 3 % (w/v), respectively. Strain Arc51T could grow on thiosulfate or sulfite as an electron acceptor in the presence of hydrogen. This strain required acetate as a carbon source for its growth, suggesting that the reductive acetyl CoA pathway for CO2 fixation was incomplete. In addition, coenzyme M (2-mercaptoethanesulfonic acid), which is a known methyl carrier in methanogenesis, was also a requirement for growth of the strain. Analysis of the 16S rRNA gene sequence revealed that the isolate was similar to members of the genus Archaeoglobus, with sequence similarities of 93.6-97.2 %; the closest relative was Archaeoglobus veneficus. Phylogenetic analyses of the dsrAB and apsA genes, encoding the alpha and beta subunits of dissimilatory sulfite reductase and the alpha subunit of adenosine-5'-phosphosulfate reductase, respectively, produced results similar to those inferred from comparisons based on the 16S rRNA gene sequence. On the basis of phenotypic and phylogenetic data, strain Arc51T represents a novel species of the genus Archaeoglobus, for which the name Archaeoglobus infectus sp. nov. is proposed. The type strain is Arc51T (=NBRC 100649T=DSM 18877T).

Laser-Induced Fluorescence Ratios of Cajanus Cajan L. under the Stress of Cadmium and its Correlation with Pigment Content and Pigment Ratios

Laser-induced fluorescence spectra were used to characterize the effect of cadmium on the pigment status of the leaves of Cajanus cajan L. Laser-induced fluorescence spectra of untreated as well as cadmium treated (0.01 mM, 0.10 mM, and 1.00 mM) Cajanus cajan L. were recorded using the 355 nm line of a Nd:YAG laser as the excitation source and a monochromator with an intensified charge-coupled device as a detector in the region 400-800 nm. The fluorescence intensity ratios (FIR) of control as well as treated Cajanus cajan L. have been calculated by evaluating curve fitted parameters using a Gaussian spectral function. In addition, some growth parameters, such as photosynthetic pigment content, were also measured. The 355 nm line of the laser-light-excited leaves not only showed a fluorescence emission in the red spectral region (650-800 nm), but also in the blue-green region (400-570 nm). The chlorophyll FIR F690/F740 strongly correlated with the photosynthetic pigment content (total chlorophyll and carotenoids) and its ratio. Consequently, a correlation was also seen between the ratio of the blue-green fluorescence F470/F540 and the photosynthetic pigment content. The results indicated that the plants treated with 0.01 mM of cadmium exhibited better growth, while higher concentrations of cadmium were hazardous for Cajanus cajan L.

Use of fluorescence information for automated phytoplankton investigation by image analysis

Automated identification and quantification of algae in microscopic images is a tool that allows high taxonomic resolution with reasonable technical efforts. However, in samples containing various non-algal objects, this is still not a satisfactorily solved problem. We show that autofluorescence information improves discrimination of algae from non-algal objects as well as phycoerythrin (PE) containing algae from others. We analyse the stability of the autofluorescence to estimate its constraints. Cold and dark storage of glutaraldehyde fixed samples maintains autofluorescence sufficiently for 3 weeks. Under repeated excitations, chlorophyll a (Chl a) or PE autofluorescence show an exponential decrease followed by an intermediate maximum. A peak also occurs in emission wavelength ranges without chlorophyll and PE fluorescence. The unspecific autofluorescence causing the peaks is at least partly identical with the blue-green fluorescence (BGF) in plant cells. BGF interferes with identification of algae, thus correction of pigment autofluorescence with such unspecific fluorescence allows a more reliable algal discrimination procedure. A classification scheme for discrimination of Chl a and PE-containing algae shows a high performance in a test with natural samples. Integration of fluorescence and bright-field image information provides a powerful tool for phytoplankton analysis in complex samples.

Development of a GFP-Expressing Aspergillus flavus Strain to Study Fungal Invasion, Colonization, and Resistance in Cottonseed

Cotton bolls were inoculated with a green fluorescent protein (GFP)-expressing Aspergillus flavus (strain 70) to monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins. The GFP strain and the wild-type did not differ significantly in pathogen aggressiveness as indicated by similar reductions in inoculated locule weight. GFP fluorescence was at least 10 times higher than the blue green yellow fluorescence (BGYF) produced in response to infection by A. flavus. The GFP produced by the strain made it possible to identify and monitor specific plant tissues colonized by the fungus. For example, the inner seed coat and cotyledon were colonized by the fungus within 72 h of inoculation and the mode of entry was invariably through the porous chalazal cap in intact seeds. The amount of GFP fluorescence was shown to be an indicator of fungal growth, colonization and, to some extent, aflatoxin production. The A. flavus strain expressing GFP should be very useful for rapidly identifying cotton lines with enhanced resistance to A. flavus colonization developed through genetic engineering or traditional plant breeding. In addition, development of GFP expressing A. flavus strain provides an easy and rapid assay procedure for studying the ecology, etiology, and epidemiology of cotton boll rot caused by A. flavus resulting in aflatoxin contamination.

Two-Photon Frequency-Upconversion Fluorescence of Multibranch Chromophores with Nitrogen-Coupling Center

Two-photon absorption (TPA) properties of multibranch chromophores with nitrogen coupling center, N-[4-{2-(3,5-di-{5-[4-(tert-butyl)phenyl]-1,3,4-oxadiazol-2-yl}phenyl)-1-ethenyl}phenyl]-N,N-diphenylamine (BPODPA), N,N-bis[4-{2-(3,5-di-{5-[4-(tert-butyl)phenyl]-1,3,4-oxadiazol-2-yl}phenyl)-1-ethenyl}phenyl]-N-phenylamine (BBPOPA) and N,N,N-tris[4-{2-(3,5-di-{5-[4-(tert-butyl)phenyl]-1,3,4-oxadiazol-2-yl}phenyl)-1-ethenyl}phenyl] amine (TBPOA),were measured by nonlinear transmission method. Single photon excited fluorescence and two-photon excited fluorescence (TPEF) of the chromophores were studied. The effect of dendronization on the TPA and TPEF characters of triphenylamine-based chromophores was investigated. When pumped with 800 nm laser irradiation, chromophores BPODPA, BBPOPA, and TBPOA showed strong two-photon excited blue green fluorescence at 502, 515, and 518 nm, respectively. These multibranched chromophores showed large TPA cross-section. The enhanced two-photon absorption found in these chromophores was attributed to the fact that the multibranched chromophores had both an extended -conjugated system and an increased intramolecular cooperative effect compared to the one-branched chromophore.

Poly(fluorenevinylene) Copolymers Containing Bis(phenyl)oxadiazole and Triphenylamine Moieties: Synthesis, Photophysics, and Redox and Electroluminescent Properties

New fluorescent fluorenevinylene copolymers incorporating bis(phenyl)oxadiazole, triphenylamine, or both moieties were synthesized, characterized, and used as the emissive materials in efficient electroluminescent diodes. In dilute solutions, the copolymers had blue-green fluorescence with high quantum yields of 0.76−0.87. All the copolymers had high glass transition temperatures (Tg = 147−160 °C) and outstanding thermal stability. Light emitting diodes based on the neat copolymer thin films showed bright greenish-yellow electroluminescence, with luminances of up to 1360 cd/m2, originating mostly from intramolecular charge-transfer excited states due to the presence of the donor/acceptor moieties in the copolymers. However, dilute (approximately 5 wt %) binary blends of the copolymers with poly(vinyl carbazole) gave stable blue electroluminescence (CIE = 0.17, 0.16) with higher luminances (1770−3400 cd/m2) and luminous efficiencies of up to 1.34 cd/A.

Charge transport and intrinsic fluorescence in amyloid-like fibrils

The self-assembly of polypeptides into stable, conductive, and intrinsically fluorescent biomolecular nanowires is reported. We have studied the morphology and electrical conduction of fibrils made of an elastin-related polypeptide, poly(ValGlyGlyLeuGly). These amyloid-like nanofibrils, with a diameter ranging from 20 to 250 nm, result from self-assembly in aqueous solution at neutral pH. Their morphological properties and conductivity have been investigated by atomic force microscopy, scanning tunneling microscopy, and two-terminal transport experiments at the micro- and nanoscales. We demonstrate that the nanofibrils can sustain significant electrical conduction in the solid state at ambient conditions and have remarkable stability. We also show intrinsic blue-green fluorescence of the nanofibrils by confocal microscopy analyses. These results indicate that direct (label-free) excitation can be used to investigate the aggregation state or the polymorphism of amyloid-like fibrils (and possibly of other proteinaceous material) and open up interesting perspectives for the use of peptide-based nanowire structures, with tunable physical and chemical properties, for a wide range of nanobiotechnological and bioelectronic applications.

Polymorphism-dependent fluorescence of 9,10-bis(pentafluorobenzoyloxy)anthracene

Anthracene derivatives possessing pentafluorobenzoyloxy moieties at 9- and 10-positions showed polymorphism affording two types of fluorescent crystals with blue and bluish green fluorescence in their crystalline state, respectively. Their single crystal X-ray structures showed that the degree of overlap of anthracene moieties was responsible for the difference in fluorescence. Fluorescence in the crystalline state originated in the dimer emission deduced from their excitation spectra.

The drought response of Theobroma cacao (cacao) and the regulation of genes involved in polyamine biosynthesis by drought and other stresses

Drought can negatively impact pod production despite the fact that cacao production usually occurs in tropical areas having high rainfall. Polyamines (PAs) have been associated with the response of plants to drought in addition to their roles in responses to many other stresses. The constitutive and drought inducible expression patterns of genes encoding enzymes involved in PA biosynthesis were determined: an ornithine decarboxylase ( TcODC), an arginine decarboxylase ( TcADC), an S-adenosylmethionine decarboxylase ( TcSAMDC), a spermidine synthase ( TcSPDS), and a spermine synthase ( TcSPMS). Expression analysis using quantitative real-time reverse transcription-PCR (QPCR) results showed that the PA biosynthesis genes were expressed in all plant tissues examined. Constitutive expression of PA biosynthesis genes was generally highest in mature leaves and open flowers. Expression of TcODC, TcADC, and TcSAMDC was induced with the onset of drought and correlated with changes in stomatal conductance, photosynthesis, photosystem II efficiency, leaf water potential and altered emission of blue-green fluorescence from cacao leaves. Induction of TcSAMDC in leaves was most closely correlated with changes in water potential. The earliest measured responses to drought were enhanced expression of TcADC and TcSAMDC in roots along with decreases in stomatal conductance, photosynthesis, and photosystem II efficiency. Elevated levels of putrescine, spermidine, and spermine were detected in cacao leaves 13 days after the onset of drought. Expression of all five PA associated transcripts was enhanced (1.5–3-fold) in response to treatment with abscisic acid. TcODC and TcADC, were also responsive to mechanical wounding, infection by Phytophthora megakarya (a causal agent of black pod disease in cacao), the necrosis- and ethylene-inducing protein (Nep1) of Fusarium oxysporum, and flower abscission. TcSAMDC expression was responsive to all stresses except flower abscission. TcODC, although constitutively expressed at much lower levels than TcADC, TcSAMDC, TcSPDS, and TcSPMS, was highly inducible by the fungal protein Nep1 (135-fold) and the cacao pathogen Phytophthora megakarya (671-fold). The full length cDNA for ODC was cloned and characterized. Among the genes studied, TcODC, TcADC, and TcSAMDC were most sensitive to induction by drought in addition to other abiotic and biotic stresses. TcODC, TcADC, and TcSAMDC may share signal transduction pathways and/or the stress induced signal induction pathways may converge at these three genes leading to similar although not identical patterns of expression. It is possible altering PA levels in cacao will result in enhanced tolerance to multiple stresses including drought and disease as has been demonstrated in other crops.

Fluorescent chemosensor for metal ions based on optically active polybinaphthyls and 1,3,4-oxadiazole

Linear chiral polymers P-1 and P-2 were synthesized by the polymerization of (R)-5,5′-dibromo-6,6′-di(4-methylphenyl)-2,2′-bisoctoxy-1,1′-binaphthyl (R-M-1) with 2,5-di(4-vinylphenyl)-1,3,4-oxadiazole (M-2) and 2,5-di(4-tributylstannylphenyl)-1,3,4-oxadiazole (M-3) via Heck and Stille cross-coupling reaction, respectively. The chiral conjugated polymer P-1 can show strong green-blue fluorescence, and the chiral polymer P-2 shows strong blue fluorescence. While the conjugated polymers P-1 and P-2 were used as fluorescent chemosensor for metal ions, their fluorescence can be efficiently quenched on the addition of different metal ions. The obvious quenching effect of the polymers P-1 and P-2 indicates that the intramolecular photoinduced electron transfer (PET) or photoinduced charge transfer (PCT) between the polymer backbone and receptor-ions in the main chain of fluorescent chemosensor can lead to the pronounced fluorescence quenching. The results also show that the chiral polymers P-1 and P-2 incorporating 1,3,4-oxadiazole moiety as the recognition site can act as a special fluorescent chemosensor for the appropriate detection of the sensitive and selective sense of metal ions.

Synthesis and properties of carbazole-based hyperbranched conjugated polymers

Carbazole-based novel hyperbranched conjugated polymers linked with triphenylamine and benzene moieties were synthesized by Sonogashira coupling polycondensation of N-octadecyl- and N-octyl-3,6-diethynylcarbazoles with tris(4-iodophenyl)amine and 1,3,5-tribromobenzene. Solvent-soluble polymers with number-average molecular weights in the range of 3500–21,000 were obtained in 48–66% yields. The UV–vis absorption bands of the polymers were red-shifted compared to that of carbazole, indicating the extension of conjugation length. The polymers emitted blue–green fluorescence with high quantum yields up to 67% in CHCl3. Poly(1/3) containing triphenylamine units emits visible light and shows unique solvatochromism. The polymers were electrochemically redox-active.

Synthesis of fluorescent hyperbranched poly(aryl ether ketones) containing biphenyl units

Hyperbranched poly(aryl ether ketones) with M n = (8–10) × 103 and terminal fluorine atoms have been synthesized through the polycondensation of 3,3′,5,5′-tetramethyl-4,4′-dihydroxydiphenyl(A2) and 1,3,5-tris[4-(4-fluorobenzoyl)phenoxy]benzene (B3) taken at a molar ratio of (0.720–0.675): 1. The replacement of the terminal fluorine atoms by residues of m-N,N-dimethylaminophenol yields polymers demonstrating green-blue fluorescence both in solution and in the solid state. Fluorescence quenching is observed at concentrations of the said polymers in solutions above 3 g/l.

Fluorescent Chemosensory Conjugated Polymers Based on Optically Active Polybinaphthyls and 2,2‘-Bipyridyl Units

Chiral conjugated polymers P-1 and P-2 were synthesized by the polymerization of (S)-6,6‘-di(4-methylphenyl)-3,3‘-diiodo-2,2‘-bisoctoxy-1,1‘-binaphthyl (S-M-2) and (S)-6,6‘-dibromo-3,3‘-di(4-methylphenyl)-2,2‘-bisoctoxy-1,1‘-binaphthyl (S-M-3) with 5,5‘-divinyl-2,2‘- bipyridine (M-1) under Heck cross-coupling reaction, respectively. The chiral polymers P-1 and P-2 can show strong blue-green fluorescence. The responsive properties of chiral polymers on transition metal ions and H+ were investigated by fluorescent spectra. Cu2+ and Ni2+ lead to nearly complete fluorescent quenching of two polymers, and Zn2+, Cd2+, and H+ can cause slight fluorescent quenching. Such distinct ion responsive behaviors revealed the obvious difference of coordination abilities of transition metal ions with 2,2‘-bipyridyl ligand receptors. The obvious quenching effect of P-1 and P-2 also suggested that the chiral conjugated polymers backbone structure and the efficiencies of energy or electron transfer between backbone and receptor ions in the main chain play important roles in fluorescence quenching. The results show that the chiral conjugated polybinaphthyls incorporating the 2,2‘-bipyridyl moiety as the recognition site can act as promising active materials for application as fluorescent chemosensors.

Effective switch-on fluorescence sensing of zinc(II) ion by 8-aminoquinolino-β-cyclodextrin/adamantaneacetic acid system in water

A water-soluble 8-aminoquinolino-β-cyclodextrin/1-adamantaneacetic acid ( 1/ADA) system is prepared in situ and exhibits a unique switch-on fluorescence response to Zn 2+ over other common metal ions. Spectrophotometric studies demonstrate that this system can strongly coordinate Zn 2+ through a cyclodextrin/substrate/metal triple recognition mode, and the resulting 1/ADA/Zn 2+ ternary complex emits the blue-green fluorescence ( λ = 490 nm) that can be easily distinguished by eyes in aqueous solution. Significantly, the switch-on fluorescence response of 1/ADA to Zn 2+ is barely affected by various metal ions except Cu 2+. As a result, this system can behave as an efficient supramolecular fluorescence sensor for Zn 2+ in water.

First Report of Bacterial Blight of Rutabaga (Brassica napus var. napobrassica) Caused by Pseudomonas syringae pv. alisalensis in California.

In 2005, commercial, organically grown rutabaga (Brassica napus var. napobrassica) in San Benito County, CA showed symptoms of a previously undescribed disease on approximately 30% of the plants. Initial symptoms consisted of small (1 to 2 mm in diameter), angular, water-soaked flecks that often were surrounded by chlorotic haloes. These flecks enlarged and coalesced into large, irregularly shaped, gray brown lesions that could be as long as 10 mm. Lesions were visible from both adaxial and abaxial leaf surfaces and generally retained the chlorotic borders. A blue-green fluorescing pseudomonad was consistently isolated from lesions on King's medium B. Eight isolates were characterized and were levan positive, oxidase negative, and arginine dihydrolase negative. Isolates did not rot potato slices but induced a hypersensitive reaction in tobacco (Nicotiana tabacum cv. Samsun). These data indicated that the bacteria belonged to Lelliot's LOPAT group 1 (2). This was confirmed with data from fatty acid methyl ester analysis (MIS-TSBA version 4.10; MIDI, Inc., Newark, DE) that showed that the isolates were highly similar (similarity = 0.922 or greater) to Pseudomonas syringae. Amplification of repetitive bacterial sequences (rep-PCR) using the BOXA1R primer and the polymerase chain reaction resulted in identical banding patterns for the rutabaga isolates and the P. syringae pv. alisalensis pathotype strain. Pathogenicity was demonstrated by growing inocula of six isolates in nutrient broth shake cultures for 48 h (24°C), adjusting the bacterial suspension to 106 CFU/ml, and misting the resulting suspensions onto rutabaga (cv. American Purple Top). Plants were enclosed in plastic bags for 24 h and then incubated in a greenhouse (24 to 26°C). Control plants were misted with sterile water and treated the same way. After 5 to 7 days, foliar symptoms similar to symptoms seen in the field developed on all inoculated plants, and reisolated bacteria were characterized and found to be P. syringae pv. alisalensis. Control plants remained symptomless. The results of two sets of pathogenicity tests were the same. To our knowledge, this is the first report of commercially grown rutabaga as a host of P. syringae pv. alisalensis and the first report of a B. napus host of this pathogen. This bacterial pathogen has previously been reported on commercial plantings of arugula (Eruca sativa), broccoli (Brassica oleracea var. botrytis), and broccoli raab (Brassica rapa var. rapa) in California and under experimental conditions it causes disease on additional hosts, including members of the Poaceae (1).

Monitoring and screening plant populations with combined thermal and chlorophyll fluorescence imaging

Thermal and chlorophyll fluorescence imaging are powerful tools for the study of spatial and temporal heterogeneity of leaf transpiration and photosynthetic performance. The relative advantages and disadvantages of these techniques are discussed. When combined, they can highlight pre-symptomatic responses not yet apparent in visual spectrum images and provide specific signatures for diagnosis of distinct diseases and abiotic stresses. In addition, their use for diagnosis and for selection for stomatal or photosynthetic mutants, these techniques can be applied for stress tolerance screening. For example, rapid screening for stomatal responses can be achieved by thermal imaging, while, combined with fluorescence imaging to study photosynthesis, they can potentially be used to derive leaf water use efficiency as a screening parameter. A particular advantage of imaging is that it allows continuous automated monitoring of dynamic spatial variation. Examples of applications include the study of growth and development of plant lines differing in stress resistance, yield, circadian clock-controlled responses, and the possible interactions between these parameters. In the future, such dual-imaging systems could be extended with complementary techniques such as hyperspectral and blue-green fluorescence imaging. This would result in an increased number of quantified parameters which will increase the power of stress diagnosis and the potential for screening of stress-tolerant genotypes.