Back to topPrinciple
ESE fluorescence measurement systems, such as the ESElog, work with impinging light based on a confocal measurement principle. In contrast to the off-axis principle, the excitation and emission beam in confocal systems have the same, parallel course (see figure Comparison of the off-axis and confocal principle). In the detector, the measurement signal is extracted by a precise system of beam splitters and filters. This measurement principle works on solid surfaces as well as in liquids. One of the major advantages of the confocal principle compared to the off-axis principle is the much higher flexibility regarding detector and sample positioning. When using the off-axis principle, accurate positioning of the sample is highly critical in order to get comparable results. In contrast, when using the confocal principle, the positioning of the sample is less critical.
Back to topProcedure
The detector comes in a solid housing and can be easily mounted to standard optical benches such as LINOS or Thorlabs. Together with a range of accessories, this unique detector is highly suited as a measurement tool for laboratory tests. Direct measurement of liquids or solids can easily be achieved by a small measurement window. The detector can be connected to a PC via the integrated USB or RS485 interface. No additional control units are necessary. To evaluate data, a software tool provided by ESE may be used or, for more detailed evaluation, a connection to labVIEW can be made.
Complete fluorescence measurement deviceBack to top
With precise micro-optics, powerful excitation light sources, highly sensitive sensors, and microprocessor-controlled electronics, the compact and robust module is a complete measurement device. The ESElog is also highly suited as a development tool for measurement device developers. After first trials or prototyping with the ESElog, users can easily switch to the Fluo Sens Integrated for the development and production of a serial fluorescence measurement device. A wide range of accessories is available to enable researchers to quickly start the development process.
Back to topApplications
The ESElog is ideally suited to a range of applications, including:
- Research and development
- Environmental testing
- Food testing
- Brand security
Oxygen in air
An oxygen sensor based on heavy metal chelates and a collisional quenching mechanism of fluorescence by oxygen was used to determine the oxygen content in air. In a closed metal cylinder with a glass window and gas in- and outflow pipes, the oxygen sensor (small surface, approximately 0.8 mm x 0.8 mm) was exposed to a variety of oxygen concentrations (1% O2 in N2, 21% O2 (air), 100% O2). The confocal fluorescence sensor (ESElog) was used to excite the oxygen sensor (470 nm) and detect light emitted by it (625 nm). The response of the signal is very fast, in the ms range, and is reversible (see figure Fast and reversible signal response).
Oil and algae in water
Fluorescence has been used for the analysis of oils for the past 60 years. One of the goals of this analysis is the analysis of core samples, for example water, to detect the presence of oils in order to determine possible contamination in water reservoirs or lakes.
A small mobile, handheld system is routinely used by first responders to check water quality in lakes that are used as drinking water reservoirs. An oil-contaminated sample, an algal sample, and a water blank sample were measured (see figure Effective differentiation of algal fluorescence from oils), with the signal being recorded continuously.
The black trace shows strong responses for both lube oil and algae: the response to algae is undoubtedly due to NADPH fluorescence, which is used routinely as a marker for aquatic biomass. Clearly this measurement is open to interference from oils and other aromatic hydrocarbon products: The additional use of the second channel (sensitive to generic algal chlorophylls) differentiates algal fluorescence from oils effectively. The use of chlorophyll-specific wavelength sets could be used to differentiate different algal types.
Viable cell test
Traditionally, the toxic effects of unknown compounds have been measured in vitro by counting viable cells after staining with a vital dye. The resazurin system measures the metabolic activity of living cells. Viable cells take up resazurin and reduce it internally to resorufin. This fluorescent compound and the redox reaction can be monitored. Only viable cells can reduce resazurin, therefore change in fluorescence intensity is due to viable cells.
A dilution series of E. coli K12 cells was prepared, resazurin was added, and the experiment carried out according to the manufacturer’s instructions. Fluorescence was measured in a 1 cm quartz cuvette. A total of 27 cells in a total volume of 300 µl were detected. Extrapolation of the data to the ±3 times standard deviation data show that the limit of detection is around 15 cells in total in a 300 µl volume and this can be distinguished from the blank (see figure Efficient detection of viable cells). The blank was resazurin containing buffer without cells.
Chlorophyll fluorescence analysis has become one of the most powerful and widely used techniques available to plant physiologists and ecophysiologists. No investigation into photosynthetic performance of plants under field conditions seems complete without some fluorescence data. The goal of measuring the yield of chlorophyll fluorescence is to gain information about changes in the efficiency of photochemistry and heat dissipation.
A dilution series of chlorophyll A in ethanol was prepared and measured in a 1 cm quartz cuvette using a laser diode-based sensor. The fluorescence intensity was recorded for each concentration of chlorophyll A and linear regression analysis of the data was performed (see figures Fluorescence intensity of different chlorophyll A concentrations). A 4 pM (0.0035 ng/ml chlorophyll) solution of chlorophyll A could still be distinguished from the blank. The blank was ethanol without chlorophyll A.