UV-induced Fluorescence Research Articles

TOWARDS OPTICAL DETECTION OF PLASMOPARA VITICOLA INFECTION IN THE FIELD

Chlorophyll fluorescence, UV-induced fluorescence and reflectance signals of grapevine leaf tissue naturally infected by Plasmopara viticola were inspected in three susceptible grapevine cultivars (Cabernet Sauvignon, Pinot Blanc and Pinot Gris). Distribution of FV/FM parameter over the leaf was found to be effective to discriminate symptomatic and asymptomatic leaf tissue. Reduction of FV/FM parameter ~ 25% was found in all infected leaf spots. Infected leaves also expressed significantly changed chlorophyll fluorescence induction kinetics expressing much slower electron transport rate on donor and acceptor site of photosystem II. Symptomatic leaves emitted high fluorescence signal under UV excitation 320nm, centered at 395nm, emanated mainly from highly fluorescent compound 2,4,6-trihydroxyphenanthrene-2-O- glucoside. Increase of this compound was found in all inspected grapevine varieties, while its concentration was strongly dependent on resveratrol concentration. Concentration of trans- resveratrol ranged in asymptomatic leaves from 0.1μg·g-1 (CS) to 0.2μg·g-1 (PB, PG). Highest increase of trans-resveratrol in infected leaf tissue was found in PB (4.4μg·g-1), followed by CS (1.6μg·g-1) and PG (1.1μg·g-1). 2,4,6-trihydroxyphenanthrene-2-O-glucoside concentrations varied from 0.41 (PB), 0.75 (PG) to 1.01μg·g-1 (CS) in asymmptomatic leaves, since its concentration rapidly increase with severity of infection [71.09 (PB), 79.37 (PG), and 26.14 μg·g- 1 (CS)]. Reflectance vegetation indices SRI R750/R700, (R780-R710)/(R780-R680) and CRI700 were the most powerful to follow changes in chlorophylls and carotenoids contents under Plasmopara viticola infection. Infected leaves also exhibited lower chlorophyll a (~50 %) and carotenoids (~70%) content. Combination of these optical signals can be used as an effective, non-invasive tool for an early detection of Plasmopara viticola in field.

Esculetin and esculin (esculetin 6-O-glucoside) occur as inclusions and are differentially distributed in the vacuole of palisade cells in Fraxinus ornus leaves: A fluorescence microscopy analysis

The location of individual coumarins in leaves of Fraxinus ornus acclimated at full solar irradiance was estimated using their specific UV- and fluorescence spectral features. Using a combination of UV-induced fluorescence and blue light-induced fluorescence of tissues stained with diphenylborinic acid 2-amino-ethylester, in wide field or confocal laser scanning microscopy, we were able to visualize the distribution of esculetin and esculetin 6-O-glucoside (esculin) in palisade cells. Coumarins are not uniformly distributed in the cell vacuole, but accumulate mostly in the adaxial portion of palisade cells. Our study indeed shows, for the first time, that coumarins in palisade cells accumulate as vacuolar inclusions, as previously reported in the pertinent literature only for anthocyanins. Furthermore, esculetin and esculin have a different vacuolar distribution: esculetin largely predominates in the first 15μm from the adaxial epidermis. This leads to hypothesize for esculetin and esculin different transport mechanisms from the endoplasmic reticulum to the vacuole as well as potentially different roles in photoprotection. Our study open to new experiments aimed at exploring the mechanisms that deliver coumarins to the vacuole using different fluorescence signatures of coumarin aglycones and coumarin glycosides.

IR-stimulated visible fluorescence in pink and brown diamond

Irradiation of natural pink and brown diamond by middle-ultraviolet light (photon energy ϵ ≥ 4.1 eV ) is seen to induce anomalous fluorescence phenomena at N3 defect centres (structure N3−V). When diamonds primed in this fashion are subsequently exposed to infrared light (even with a delay of many hours), a transient burst of blue N3 fluorescence is observed. The dependence of this IR-triggered fluorescence on pump wavelength and intensity suggest that this fluorescence phenomena is intrinsically related to pink diamond photochromism. An energy transfer process between N3 defects and other defect species can account for both the UV-induced fluorescence intensity changes, and the apparent optical upconversion of IR light. From this standpoint, we consider the implications of this N3 fluorescence behaviour for the current understanding of pink diamond photochromism kinetics.

Fabrication of Stacked Organic Electroluminescence Devices with a Combination of Spin Coating and Electrospray Deposition

A combination of spin coating and electrospray deposition (ESD) was applied for the fabrication of an organic light-emitting diode (OLED) with a stacked structure. Two kinds of polymer materials were used to fabricate a multilayered structure. One was poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV; Sigma-Aldrich) and the other was poly[2,5-di(hexyloxy)cyanoterephthalylidene] (CN-PPV; Sigma-Aldrich). With the addition of acetone to polymer material (MEH-PPV and CN-PPV) solution, smaller droplets were produced at the emitter resulting in thin-film fabrication under a dry condition. This condition was adopted for CN-PPV film deposition on MEH-PPV surface made by the spin coating method. The double-layered OLED obtained successfully emitted light at a bias voltage of 15 V. Layer-by-layer stacking of CN-PPV and MEH-PPV was confirmed by the UV-induced fluorescence spectrum. In case of the layer-by-layer structure, the obtained spectrum showed a linear combination of spectrum from MEH-PPV and CN-PPV.

Development of a simple and high-throughput method for detecting aflatoxins production in culture media

To develop a simple, high-throughput and inexpensive procedure to detect and quantify aflatoxins into the culture media of growing mycelia. Fungal conidia (Aspergillus flavus) were inoculated into the wells of a microplate containing 200 μl of different formulations of coconut-derived liquid medium. Time-dependent production of aflatoxins in the culture media was evaluated by a procedure relying on the UV-induced fluorescence emission by the toxin, using a microplate reader. These data were validated by comparison with the outputs of a conventional HPLC-based procedure. Determinations of aflatoxin concentration, according to the fluorimetric procedure, were performed either by withdrawing samples from the plates or by direct 'in situ' readings, the latter method reinforcing the high-throughput feature of the procedure. Fluorescence enhancers (cyclodextrins) did not ameliorate the sensitivity of the procedure to low concentrations of the toxin into the medium. The efficacy of the procedure was also validated by testing the effect on toxin yield of adding an antioxidant agent (α-lipoic acid) to the medium. We give evidence that our improved procedure is reliable and suitable to analyse aflatoxin accumulation time course in coconut-derived culture medium. This study shows that our procedure may profitably be used to give insights into the mechanisms of regulation of mycotoxin production and, consequently, to implement different strategies for the containment of aflatoxin contamination of food and feed commodities.

Patterns in water quality on Canadian shores of Lake Ontario: Correspondence with proximity to land and level of urbanization

Spatially detailed surveys were conducted in 2008 over 97km of the Canadian coastline of Lake Ontario to describe patterns in water quality correlated with natural and anthropogenic features. Features of variability were used to infer lake-wide, regional, and location specific influences on nearshore water quality. Levels of solids, nutrients, major ions, Escherichia coli, and dissolved organic carbon were higher and more variable among tributary, embayment, and shoreside (0 to 1.2m depth) areas compared with the open nearshore (~3m depth to 5km offshore). A recurring pattern was a sharp drop in these features away from the shoreline with little or subtle decline beyond 1 to 3km into the nearshore. Conductivity and UV-induced fluorescence of organic material were used to detect discharges to the nearshore. Land-based influence on water quality was greatest in the urbanized Greater Toronto Area and the least over the less developed areas. Nearshore regions received inputs from the adjacent shoreline affecting water quality, the spatial extent of which was difficult to generalize, ranging from 10s of metres to several kilometres in breadth. Despite this, concentrations of total phosphorus, soluble reactive phosphorus, dissolved inorganic nitrogen, and silicate, when averaged over the entire nearshore, were typically similar to the open lake reflecting the broad distribution of the nearshore relative to the areas most influenced by adjacent lands. The development of environmental management to address water resource problems in the nearshore should proceed with awareness of the strong spatial dimensions that define nearshore water quality.

Ultraviolet-induced red fluorescence of patients with acne reflects regional casual sebum level and acne lesion distribution: qualitative and quantitative analyses of facial fluorescence

The ultraviolet (UV)-induced red fluorescence of patients with acne has been considered to be caused by Propionibacterium acnes. To study the correlation of the facial red fluorescence with the casual sebum level and the number of acne lesions and to investigate the difference in clinical features, according to both distribution and proportion of fluorescence. A total of 878 patients clinically diagnosed with acne vulgaris were included. Inflammatory and noninflammatory acne lesions were counted separately. UV fluorescent photography and casual sebum level measurements were performed. UV-induced fluorescence patterns were classified according to the facial distribution. The proportions of UV-induced red fluorescence were calculated. We identified six different fluorescence distribution patterns in the T-zone (the forehead, nose and chin) and three different patterns in the U-zone (both cheeks). The proportion of fluorescence in the U-zone showed a positive correlation with the casual sebum level and the number of acne lesions. In the T-zone, the fluorescence proportion correlated with the casual sebum level, but not with the number of acne lesions. As the patients' age and the age at onset increased, the distribution of fluorescence changed from the upper part of the T-zone to the lower part, and to the centre of the face in the U-zone. Our results support the hypothesis that the origin of facial red fluorescence is sebum. In patients with acne, analyses of the pattern and proportion of UV-induced red fluorescence can be useful for evaluating the sebum secretion and selecting efficient treatment modalities.

UV-induced fluorescence spectra and lifetime determination for detection of leaf rust (Puccinia triticina) in susceptible and resistant wheat (Triticum aestivum) cultivars.

In modern agriculture, the use of cultivars that are resistant against specific stresses, e.g. pathogen infections, is an integral component. Considering the great demand for a rapid and objective screening method for stress resistance of new cultivars, the question arises, whether time resolved fluorescence spectroscopy is suitable for such purposes. Amongst others, infected plants might accumulate specific compounds such as salicylic acid and phenylpropanoid compounds as key substances in plant disease resistance, whereas synthesis and accumulation may influence fluorescence parameters such as absolute intensity of single peaks, ratios between peaks and lifetime. Experiments were conducted in a controlled-environment cabinet cultivating four leaf rust susceptible and three leaf rust resistant genotypes. Fluorescence measurements were conducted using a compact fibre-optic fluorescence spectrometer with a nanosecond time-resolution. Results of experiments revealed that UV-induced measurements of spectral characteristics as well as determination of fluorescence lifetime are suited to detect leaf rust (Puccinia triticina) in wheat (Triticum aestivum L.) cultivars as early as 2 days after inoculation (dai). For this purpose several parameters such as the fluorescence (F) amplitude ratios F451/F522, F451/F687, F451/F736, F522/F687, F522/F736 as well as fluorescence mean lifetime especially at 470nm, might be used. Discrimination between resistant and susceptible cultivars to the leaf rust pathogen could be accomplished 3dai by using the ratio of fluorescence amplitude between the blue (F451nm) and red (F687nm) peak, and mean lifetime at 440, 500 and 530nm. Our results indicate that the combination of spectrally and time-resolved fluorescence could be an additional tool in plant breeding programs for an automatic and precise high-throughput system for evaluation of the pathogen resistance of new genotypes.

Discrimination of Corn from Monocotyledonous Weeds with Ultraviolet (UV) Induced Fluorescence

In production agriculture, savings in herbicides can be achieved if weeds can be discriminated from crop, allowing the targeting of weed control to weed-infested areas only. Previous studies demonstrated the potential of ultraviolet (UV) induced fluorescence to discriminate corn from weeds and recently, robust models have been obtained for the discrimination between monocots (including corn) and dicots. Here, we developed a new approach to achieve robust discrimination of monocot weeds from corn. To this end, four corn hybrids (Elite 60T05, Monsanto DKC 26-78, Pioneer 39Y85 (RR), and Syngenta N2555 (Bt, LL)) and four monocot weeds (Digitaria ischaemum (Schreb.) I, Echinochloa crus-galli (L.) Beauv., Panicum capillare (L.), and Setaria glauca (L.) Beauv.) were grown either in a greenhouse or in a growth cabinet and UV (327 nm) induced fluorescence spectra (400 to 755 nm) were measured under controlled or uncontrolled ambient light intensity and temperature. This resulted in three contrasting data sets suitable for testing the robustness of discrimination models. In the blue-green region (400 to 550 nm), the shape of the spectra did not contain any useful information for discrimination. Therefore, the integral of the blue-green region (415 to 455 nm) was used as a normalizing factor for the red fluorescence intensity (670 to 755 nm). The shape of the normalized red fluorescence spectra did not contribute to the discrimination and in the end, only the integral of the normalized red fluorescence intensity was left as a single discriminant variable. Applying a threshold on this variable minimizing the classification error resulted in calibration errors ranging from 14.2% to 15.8%, but this threshold varied largely between data sets. Therefore, to achieve robustness, a model calibration scheme was developed based on the collection of a calibration data set from 75 corn plants. From this set, a new threshold can be estimated as the 85% quantile on the cumulative frequency curve of the integral of the normalized red fluorescence. With this approach the classification error was nearly constant (16.0% to 18.5%), thereby indicating the potential of UV-induced fluorescence to reliably discriminate corn from monocot weeds.

UV-induced fluorescence recovery and solubility modulation of photocaged conjugated oligomers

This article describes photoactivatable conjugated oligomers that show both increased fluorescence quantum yield and pH-dependant solubility upon irradiation with UV light. Sonogashira coupling between ester-substituted phenylacetylenes and alkoxy-substituted diiodo phenylene-vinylenes yielded conjugated phenylene-ethynylene/phenylene-vinylene oligomers. Oligomers with nitrobenzyl ester moieties had quenched fluorescence in polar solvents; UV irradiation restored their quantum yield of fluorescence to that of corresponding alkyl ester-substituted oligomers. These photocaged oligomers also exhibited UV-induced changes in solubility consistent with photogeneration of carboxylic acids. This approach is therefore effective at tuning the properties of conjugated organics with light after traditional synthetic operations, and has potential for use in photoactivatable fluorophores or solution-processable multilayer devices.

Contributions of Visible and Ultraviolet Parts of Sunlight to Photoinhibition

Photoinhibition is light-induced inactivation of PSII, and action spectrum measurements have shown that UV light causes photoinhibition much more efficiently than visible light. In the present study, we quantified the contribution of the UV part of sunlight in photoinhibition of PSII in leaves. Greenhouse-grown pumpkin leaves were pretreated with lincomycin to block the repair of photoinhibited PSII, and exposed to sunlight behind a UV-permeable or UV-blocking filter. Oxygen evolution and Chl fluorescence measurements showed that photoinhibition proceeds 35% more slowly under the UV-blocking than under the UV-permeable filter. Experiments with a filter that blocks UV-B but transmits UV-A and visible light revealed that UV-A light is almost fully responsible for the UV effect. The difference between leaves illuminated through a UV-blocking and UV-transparent filter disappeared when leaves of field-grown pumpkin plants were used. Thylakoids isolated from field-grown and greenhouse-grown plants were equally sensitive to UV light, and measurements of UV-induced fluorescence from leaves indicated that the protection of the field-grown plants was caused by substances that block the passage of UV light to the chloroplasts. Thus, the UV part of sunlight, especially the UV-A part, is potentially highly important in photoinhibition of PSII but the UV-screening compounds of plant leaves may offer almost complete protection against UV-induced photoinhibition.

Detection of Cotton Lint Trash within the Ultraviolet—Visible Spectral Range

Cotton lint trash is a serious problem in the textile industry. The principle upon which this research is based is that different materials have different spectral absorption, excitation, and emission characteristics. Although white-light imaging is widely used to detect colored foreign-matter contaminants, or "trash", it is almost useless for detecting white trash. The objective of the research described in this paper was to achieve the best trash detection result possible in the spectral range from 250 to 850 nm. Diffuse reflection spectroscopy indicated that the differences in gray value between lint and white trash become significant in the ultraviolet (UV) range, especially from 250 to 350 nm. Fluorescence spectroscopy gave reliable evidence that the UV-induced fluorescence intensity of white trash is much stronger than that of lint. To detect several types of trash simultaneously, the interaction of white-light imaging and UV-induced fluorescence imaging was studied. To avoid the spectral interference caused by white light in fluorescence imaging, a novel method--an alternating imaging detection method--is proposed. Experiments indicated that the advantages of both white-light imaging and UV-induced fluorescence imaging were preserved in the method. The novel method could effectively detect both colored and white trash in real time. This method can also be applied to trash detection in seed cotton, wool, tea leaf, and tobacco leaf.

Discrimination of corn, grasses and dicot weeds by their UV-induced fluorescence spectral signature

Real-time spot spraying of weed patches requires the development of sensors for the automatic detection of weeds within a crop. In this context, the potential of UV-induced fluorescence of green plants for corn-weed discrimination was evaluated. A total of 1 440 spectral signatures of fluorescence were recorded in a greenhouse from three plant groups (four corn hybrids, four dicotyledonous weed species and four monocotyledonous weed species) grown in a growth chamber. With multi-variate analysis, the full information contained in each spectrum was first reduced to the scores calculated from five principal components. Then, a linear discriminant analysis was applied on these scores to classify spectra on a species/hybrids basis and, subsequently, the resulting classes were aggregated according to the three plant groups. This two-step process minimized the error generated by heterogeneous groups such as dicotyledonous weeds. The output of this classification shows the significant potential of UV-induced fluorescence for plant group discrimination as the success rate reached 91.8%. No error was observed between corn and dicot weeds and most of the errors between corn and grasses came from confusion between the hybrid Pioneer 39Y85 and Setaria glauca L. (Beauv.). Analysis also determined that the position of the fluorescence sensor on the leaf and the plant age had negligible effects on the efficiency of fluorescence to discriminate plant groups. The factors to consider for transferring the results about UV-induced fluoro-sensing from laboratory to the field are discussed.

Developmental changes in spatial distribution of in vivo fluorescence and epidermal UV absorbance over Quercus petraea leaves.

Epidermal phenolic compounds (mainly flavonoids) constitute a vital screen that protects the leaf from damage by natural ultraviolet (UV) radiation. The effectiveness of epidermal UV-screening depends on leaf anatomy, the content of UV-screening compounds and their spatial uniformity over the leaf area. To investigate in vivo the spatial pattern of the epidermal UV-screen during leaf development, a fluorescence imaging method was developed to map the epidermal UV-absorbance at a microscopic scale. This study was done on oak (Quercus petraea) leaves that were used as a model of woody dicotyledonous leaves. The leaf development of 2-year-old trees, grown outdoors, was monitored, at a macroscopic scale, by in vivo measurements of chlorophyll content per unit area and epidermal UV-absorbance using two optical leaf-clip meters. The distribution of pigments within leaves was assessed in vivo spectroscopically. The microscopic images of UV-induced fluorescence and UV-absorbance acquired in vivo during leaf development were interpreted from spectral characteristics of leaves. At a macroscopic scale, epidermal UV-absorbance was high on the upper leaf side during leaf development, while it increased on the lower leaf side during leaf expansion and reached the adaxial value at maturity. At a microscopic scale, in immature leaves, for both leaf sides, the spatial distribution of epidermal UV-absorbance was heterogeneous, with a pattern depending on the flavonoid content of vacuoles in developing epidermal cells. At maturity, epidermal UV-absorbance was uniform. The spatial pattern of epidermal UV-screen over the area of oak leaves is related to leaf anatomy during development. In vivo spectroscopy and fluorescence imaging of the leaf surface showed the distribution of pigments within the leaf and hence can provide a tool to monitor optically the leaf development in nature.

Shear- and UV-Induced Fluorescence Switching in Stilbenic π-Dimer Crystals Powered by Reversible [2 + 2] Cycloaddition

We have designed and synthesized asymmetric cyano-stilbene derivatives containing trifluoromethyl (-CF(3)) substituents with the aim of producing tightly packed pi-dimer systems that as crystals exhibit reversible [2 + 2] cycloaddition with characteristic fluorescence modulation. (Z)-3-(3',5'-Bis(trifluoromethyl)biphenyl-4-yl)-2-(4'-(trifluoromethyl)biphenyl-4-yl)acrylonitrile (CN(L)-TrFMBE) and its derivatives were found to form antiparallel pi-dimer stacks in crystals due to their specific intermolecular interactions, including C-F...H and C-F...pi interactions. The CN(L)-TrFMBE pi-dimer crystals (and powder) are not at all fluorescent initially but switch to a highly fluorescent state (Phi(PL) = 24%) when an external shear-strain and/or prolonged UV (365 nm) irradiation is applied. Our experimental and theoretical investigations show that the fluorescence modulation in this particular system is due to the external and/or internal (in the case of UV irradiation) shear-induced lateral displacement of the pi-dimer molecular pair, which effectively turns the fluorescence emission on at the cost of frustrated [2 + 2] cycloaddition. Further, the fluorescence 'off' state can be restored by thermal annealing, which regenerates the tightly packed pi-dimer by reverse displacement together with the thermal dissociation of the [2 + 2] cycloaddition product. This system provides a very rare example of high-contrast reversible fluorescence switching that is driven by a change in the molecular packing mode in the solid state, which enables piezochromic and photochromic responses.

A versatile strategy for achieving the second-order advantage when applying different artificial neural networks to non-linear second-order data: Unfolded principal component analysis/residual bilinearization

Second-order instrumental signals showing a non-linear behaviour with respect to analyte concentration can still be adequately processed in order to achieve the important second-order advantage. The combination of unfolded principal component analysis with residual bilinearization, followed by application of a variety of neural network models, allows one to obtain the second-order advantage. While principal component analysis models the training data, residual bilinearization models the contribution of the potential interferents which may be present in the test samples. Neural networks such as multilayer perceptron, radial basis functions and support vector machines, are all able to model the non-linear relationship between analyte concentrations and sample principal component scores. Three different experimental systems have been analyzed, all requiring the second-order advantage: 1) pH–UV absorbance matrices for the determination of two active principles in pharmaceutical preparations, 2) fluorescence excitation–emission matrices for the determination of polycyclic aromatic hydrocarbons, and 3) UV-induced fluorescence excitation–emission matrices for the determination of amoxicillin in the presence of salicylate. In all cases, reasonably accurate predictions can be made with the proposed techniques, which cannot be reached using traditional methods for processing second-order data.

SPIDER SEDUCTION REQUIRES UV LIGHT

WHEN SOME SPIDERS desire a little romance in their lives, they use ultraviolet light as the aphrodisiac. Biologists in Singapore and England have now added the ornate jumping spider Cosmophasis umbratica to a growing list of critters—including parrots, butterflies, and fish—that rely on UV reflection and fluorescence to socialize ( Science 2007,315,481). What makes these spiders' use of UV light stand out—besides the afterglow—is that UV reflectance and UV-induced fluorescence are employed on a sex-specific basis. Without UV wavelengths, these spiders get no love. Males reflect UV light off scales on their faces and bodies during the mating game, whereas females have sensory organs called palps, typically used to capture prey, that fluoresce bright green when excited by UV light. In the absence of UV light, the success rate for consummating courtships decreases, explains senior author Daiqin Li of the National University of Singapore. Although he suspected the jumping spider might ...

ELASTIC SCATTERING SPECTROSCOPY FOR NONINVASIVE DETECTION OF CANCER

Optical spectroscopy mediated by fibre-optic probes can be used to perform noninvasive, or minimally-invasive, real-time assessment of tissue pathology in-situ. The most common approaches have been based on UV-induced fluorescence spectroscopy and Raman spectroscopy, which are assumed to be responsive to biochemical changes in cells. On the other hand, our method of elastic-scattering spectroscopy (ESS) is sensitive to the sub-cellular architectural changes, such as nuclear grade or nuclear to cytoplasm ratio, mitochondrial size and density, etc., which correlate with features used in histological assessment. The ESS method senses those morphology changes in a semi-quantitative manner, without actually imaging the microscopic structure. To aid in the design of optical probes and the understanding of the resulting spectroscopic signals, modelling of photon scattering and migration in tissue is carried out using Mie theory and Monte Carlo simulations. Clinical demonstrations of ESS have been conducted in a variety of organ sites, and promising results have been obtained. Larger-scale clinical studies are now starting.

One- and two-dimensional miniaturized electrophoresis of proteins with native fluorescence detection.

Miniaturized electrophoresis was successfully coupled with native fluorescence detection for direct analysis of proteins in one- and two-dimensional separations. The detection setup was based on direct observation of the UV-induced fluorescence of proteins using a CCD camera and a Hg (Xe) lamp for sample excitation. Protein mixtures were readily separated by size on a 1-cm segment of the one-dimensional gel in 8 min, and a detection limit of 0.04 ng per band was achieved. The dynamic range of the system was larger than 2 orders of magnitude. Miniaturized slab gel electrophoresis was performed on a special holder designed to couple isoelectric focusing with SDS-PAGE. Two-dimensional separation, including rehydration of IEF strip and fluorescence detection was completed in 2.5 h. Approximately 200 protein spots from Escherichia coli were detected on a 1 cm(2) area. A detection limit of 0.1 microg of total protein was achieved. The operation should be amenable to total automation.

Capillary isoelectric focusing with UV-induced fluorescence detection

Low-molecular-mass fluorescent compounds excitable in the near UV region with suitable acidobasic and electrophoretic properties are suggested as isoelectric point (p I) markers for isoelectric focusing (IEF) with UV photometric and UV excited fluorometric detection. The experimental set-up of capillary IEF with UV excited fluorometric detection and properties of new UV-induced fluorescent p I markers are given. The p I values of 18 new p I markers determined independently of IEF methods range from 2.1 to 10.3. The examples of separation of new p I markers together with derivatized proteins by capillary IEF with photometric or fluorometric detection are presented.