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Fluorescence

Wavelength

Wavelength Technology Forum

Fluorescence-based techniques are used for a wide range of applications, including biochemical, clinical, and environmental analyses. Joining us for this discussion are Ishai Nir, Horiba; Richard Larsen, Jasco; and Michael Allen, Thermo Fisher Scientific.

Which application areas will see the most growth in the field of fluorescence?

Nir: Traditional spectrofluorometry will continue to grow in many applications at a modest growth rate. Exceptions with much higher growth rates will come from both newer applications areas such as analysis of nanostructures like nanotubes and quantum dots and older applications such as water contaminants analysis where fluorescence remains the best in-situ real time technique (which unfortunately experienced a spike in use due to the BP Gulf oil spill.)

The real growth will continue to be in instruments based on fluorescence as a technique, but not necessarily thought of as fluorescence instrumentation. Examples are prevalent in high growth fields like molecular biology and techniques such as parallel rapid sequencing, enzyme-linked immunosorbent assay (ELISA), or high performance liquid chromatography (HPLC) where fluorescence is frequently the detection method of choice.

Larsen: We believe that the use of fluorescence for materials analysis will continue to grow in the near future. The analysis of new plasma display panel (PDP) and liquid crystal display (LCD) phosphors, nanomaterials, analysis of invisible anti-theft devices for monetary exchange instruments and packaging and the development/manufacture of other devices that require fluorescence characterization will continue to experience growth.

Allen: Life science analysis has the largest potential for growth. Being a sensitive and selective technique, fluorescence is well suited for the analysis of biological compounds. From microscopy to time-resolved measurements, biomolecule analysis will drive growth in fluorescence applications. Materials analysis and quality control measurements will also show steady growth as more accessories and software are capable of extending the applicability of this analysis.

Will we see any new developments in fluorescence instrumentation at Pittcon 2011?

Nir: Besides the usual cheaper, faster, newer type products, really new products will be in the area of fluorescence microscopy where various super-resolution techniques have allowed significant improvements in the resolution measurements of optical microscopy beyond what was classically held to be the limits due to diffraction.

Another area ripe for new products is fluorescence lifetime microscopy (FLIM). Just like new super-resolution microscopy goes beyond the minimum feature size limitation, FLIM breaks through conventional labeled and intrinsic fluorescence microscopy issues. In particular, since lifetime is an intrinsic property, FLIM avoids many of the loading uniformity problems that plague conventional fluorescent tag based quantitation. FLIM also gives an extra data vector for intrinsic fluorescence which can remove the autofluorescence background problem.

Larsen: I believe there will continue to be refinement of the equipment and accessories associated with fluorescence instrumentation, so I would expect some new developments to be unveiled at Pittcon.

Allen: Today’s traditional monochromator based instruments provide better sensitivity and resolution. Any hardware changes are most likely to come from accessories that extend the instrument’s existing capability. For example, temperature control, polarization measurements, and time-based measurement hardware.

Fluorescence is considered a mature technique area. Where do you see room for improvement?

Nir: Major improvements will come in two categories. The first is measurement speed, and the second is utility and ease-of-use. For many applications, the sensitivity of fluorescence cannot be fully taken advantage of due to lack of specificity. Overlapping spectral features can make composition determination difficult in applications such as dissolved organic material analysis. The solution is to acquire complete excitation-emission matrices (EEMs) and to apply chemometric analysis techniques to the data. However, acquiring EEMs using scanning spectrofluorometers can take many hours, making these measurements difficult at best, and frequently impractical. The solution comes in the form of new multichannel detector based fluorometers using CCD and NIR array detectors that reduce the acquisition time of EEMs to minutes, making the technique much more broadly applicable.

The second area for improvement of fluorescence instrumentation will be in simplicity and ease-of-use. Currently one has general purpose spectrofluorometers which are used for many applications. Because of their broad applicability, they have multiple operating modes that make their use for a specific application more challenging. We will continue to see simplification in the use of fluorometers by the creation of models with optimized hardware and software dedicated to a specific application.

Larsen: I think that new accessories for sample analysis and improvements in fluorescence signal enhancement will be sought by all manufacturers of fluorescence instrumentation.

Allen: With the detection of single molecules nearly a decade behind us, fluorescence laboratory instrumentation is poised to make great advancements in the next decade. In particular, more streamlined and simplified software for routine fluorescence analysis, better control of light sources such as LEDs for expanded use in conventional instrumentation and more advancement in time-resolved measurements. Adding the time dimension to information rich fluorescence data uncovers new opportunities for multiplexed analysis.

What do you see for the future of the fluorescence market?

Nir: Fluorescence-based detection continues to be extremely attractive because of its sensitivity and the fact that it’s based on the presence of a signal rather than a reduction in one as in absorbance. With improvements in sensitivity and selectivity (the latter obtained by methods like combining fluorescent labels with highly selective binding tags) we will someday soon see practical fluorescence-based instruments with a few, to even a single, molecule detection limits. This will expand the utility of fluorescence further in areas such as contaminant analysis, clinical diagnostics, etc. Then we will all have to get used to discussing zeptomolar (10-21) rather than femtomolar concentrations.

Allen: In most cases, instrumentation manufacturers are selectively catching up with the developments of fluorescence spectroscopy researchers. Applications are developed in research environments and instrument manufacturers work to tailor instruments to these needs and applications. As more life science, material science, and quality control assays use fluorescence, the key growth should be in the areas of polarization and time-resolved measurements. Like other mature technologies, fluorescence customers will expect smaller, application specific instruments that provide enhanced sensitivity over today’s offerings.

What do you think?

Click here to participate in our "Question of the Month" survey on Fluorescence.

If you are interested in participating in any upcoming Technology Forums please contact Group Technical Editor Steve Brown or Managing Editor Meg Evans for more information. Next month’s forum will be a discussion about FT-IR/NIR.

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