Applications of Advanced Fluorescence Spectroscopy

Interview conducted by Jake WilkinsonMay 18 2017

Fluorescence spectroscopy is a highly versatile technique that can be used to examine and characterize a wide range of unusual materials. In addition to this the technique is also used frequently in the life sciences as a means of identifying and observing cellular features and processes. Advances in the instrumentation used and the continuing availability of novel fluorescent materials means that the scope and power of fluorescence spectroscopy continues to grow.

AZoM spoke to Maria Tesa, Applications Specialist at Edinburgh Instruments, about the science behind fluorescence spectroscopy, novel applications for the technique and the advanced instruments that Edinburgh Instruments supply.

What is fluorescence spectroscopy? What can fluorescence spectroscopy tell us about samples that conventional spectroscopy cannot?

Fluorescence spectroscopy measures the photoluminescence of a sample, that is, the light emitted by it upon excitation by photons. When a molecule absorbs radiation, it is excited to an upper electronic state. As it decays back to its ground state, it emits a photon of a particular energy.

The intensity of light as a function of excitation wavelength (excitation spectrum) or as a function of emission wavelength (emission spectrum) provides information on the nature and concentration of the sample.

The great advantage of fluorescence spectroscopy over other techniques such as absorption spectroscopy is that fluorescence is a background-free technique, so very small concentrations of analyte can be detected. It is also more selective; you can easily distinguish between analytes by varying the excitation and emission wavelengths. In fluorescence spectrometers, these wavelengths are selected by monochromators after the source and before the detector.

A unique feature of some fluorescence spectrometers is the possibility to measure time-resolved luminescence. This has a wide range of potential applications, since the fluorescence lifetime depends on the molecule studied and on its chemical environment.

A fluorescent sample viewed under the microscope. Image Credit: Jeerasak Meeraka/Shutterstock.com

What different types of materials and systems is fluorescence spectroscopy used to study?

Any type of sample can be studied by fluorescence spectroscopy: solutions, powders, crystals, thin-films, etc. The only condition of course is that the sample must be luminescent.

Examples of the use of fluorescence spectroscopy include the study of fluorescent dyes that are widely used with biological samples, both in routine assays and in advanced research. It is also employed in material science to characterize luminescent materials.

Developing new phosphors, LEDs, or laser gain media requires full characterization of their luminescence, so fluorescence spectrometers are widely used by researchers in this field. In particular, the spectroscopy of luminescent nanomaterials has experienced a rise in the last few years and we are seeing increasing demand for instruments configured for quantum dot and nanotube research.

Quantum dots in solution. Luminescent materials can be characterized using fluorescence spectroscopy. Image Credit: Tayfun Ruzgar/Shutterstock.com

How can fluorescence spectroscopy be used to analyse nanotubes? What information about nanotubes can be determined?

Fluorescence spectroscopy is a great tool for the structural characterization of carbon nanotubes. The peak excitation and emission wavelengths of semiconducting single-walled nanotubes are in the near-infrared (NIR), and depend on their diameter and chirality.

Acquiring the fluorescence intensity as a function of both wavelengths yields an excitation-emission map, which reveals all the different types of nanotubes that are present in the mixture and their relative abundance. This technique is very simple and fast, and is often used to check the purity of carbon nanotube dispersions.

What practical applications can the fluorescent properties of nanotubes be used in?

One of the main applications of nanotubes is in the field of bio-imaging. Fluorescent dyes are often used to label biomolecules and then probe their location in a sample using fluorescence microscopy. One drawback of this technique is that biological tissue itself absorbs visible light and may be fluorescent itself, so the detection limit of visible dyes is affected by auto-fluorescence.

Using carbon nanotubes as the probe avoids this problem because their emission is in the NIR, where this background is not present. For this reason there is a growing interest in using nanotubes for both in-vivo and in-vitro imaging.

Another property of the NIR fluorescence of nanotubes is that it changes depending on the nanotube’s environment: interaction with other molecules induces a change in bandgap, and therefore a change in the fluorescence spectrum. A lot of recent research on nanotube-based chemical sensors exploits this idea. Sensors can be based on changes in the NIR fluorescence spectrum, or in the intensity of emission.

Diagram of a Multi-Walled Carbon Nanotube (MWCNT). Fluorescence spectroscopy can be used to characterize nanotubes which can then be used in biological imaging. Image Credit: Angel Soler Gollonet/Shutterstock.com

Does fluorescence spectroscopy provide additional information on any other types of nanomaterials?

Of course, any nanomaterial that is luminescent can be studied by fluorescence spectroscopy. The photoluminescence characteristics of quantum dots and nanoparticles depend strongly on their size and their electrical properties, so fluorescence spectroscopy can be used for characterizing these materials.

For example, it can reveal the distribution of particle sizes in a quantum dot mixture, or can help understand the electronic structure of a new molecule via fluorescence lifetime studies.

How can the FLS1000 fluorescence spectrometer be used for nanomaterials analysis?

Many nanomaterials have weak emission and therefore require a very sensitive spectrometer to detect it. The FLS1000 offers industry-leading sensitivity: it can detect single photons emitted from the sample, reaching the quantum limit of detection.

Another thing to consider is that much nanomaterial research is carried out on samples such as thin films that are highly scattering, and the excitation light can therefore interfere with the measured spectrum. In these cases you need a system with the best possible stray light rejection, such as the FLS1000.

The most important feature of the FLS1000 is that it can be configured to the end user’s needs. Depending on your application, you might be interested in the steady state fluorescence, time-resolved fluorescence or quantum yield of a nanomaterial. The instrument can be easily configured for all of these measurements. You can also fit different types of detectors, for example NIR detectors if your samples are luminescent in this region.

In short, the instrument provides flexibility through its modular construction to explore different avenues in the future, and this is important in cutting-edge areas like nanoscience where the materials are fairly unexplored.

The FLS1000 Fluroesence Spectrometer from Edinburgh Instruments

How can fluorescence spectroscopy be used alongside fluorescent dyes in the life sciences? Why is a high-resolution fluorescence spectrometer important for this application?

As I briefly mentioned earlier, fluorescent dyes are a basic tool in bioscience. Their luminescent properties depend on their chemical environment, and therefore are used to probe changes in the biological medium.

Fluorescence lifetime measurements of dyes are the basis of Förster resonance energy transfer (FRET), which is a widespread technique to characterize interactions between biomolecules. The emission of a fluorescent dye typically decays in a few nanoseconds, so you need fast and precise detection electronics to characterize it. Most high-end fluorescence spectrometers such as the FLS1000 use the technique of time-correlated single photon counting (TCSPC) to provide the necessary temporal resolution.

Going back to the applications in bio-imaging, dyes can be chemically modified to target specific molecules and employed to image their position in a live cell. This requires coupling your fluorescence spectrometer to a microscope, so a high-end modular system like the FLS1000 is often an advantage.

Cortical neurons marked with a fluorescent dye. Image Credit: Juan Gaertner/Shutterstock.com

What impact has the rising availability of nanomaterials had on the dyes used in fluorescence cell imaging?

Many traditional organic dyes are not very photo-stable and they degrade and lose intensity as you are measuring the sample. A great advantage of some materials like nanoparticles is that they are typically very stable.

Another advantage is that many nanomaterials emit strongly in the NIR, and this is important in for bio-imaging applications. Since there are not many photo-stable NIR-emitting organic dyes, the availability of nanomaterials has sparked an interest in NIR fluorescence microscopy.

What features of the FLS1000 make it useful in cell imaging applications?

The FLS1000 offers a fibre launch sample holder that allows easy coupling to a microscope via optical fibres or liquid light guides. It allows the user to perform wide-field excitation and observe a large area of the sample using a CCD camera on the microscope. You can also couple the emission light guide to the instrument’s emission monochromator to perform single-photon detection. It is also possible to perform point excitation with a pulsed laser and scan the microscope stage. This option in combination with the TCSPC electronics allows you to perform fluorescence lifetime imaging microscopy (FLIM) experiments.

One advantage of the FLS1000 over other commercial fluorescence microscopy systems is that you can configure it with several detectors in different spectral regions, and switching detectors is just one click in the software. Therefore, both visible and NIR-emitting dyes can easily be studied with the same instrument

Fluorescence spectroscopy allows cellular features such as microfilaments, mitochondria, and nuclei to be observed. Image Credit: Heiti Paves/Shutterstock.com

How can fluorescence spectroscopy be used to develop NIR lasers? What information on the state of the lasing medium can it be used to determine?

Research on near and mid-IR laser materials is another important application of the FLS1000. There is increasing demand for these types of lasers in the market, and one of the first steps towards developing them is of course a detailed characterization of the gain medium’s emission.

This requires a high-resolution fluorescence spectrometer such as the FLS1000. This instrument can be configured for single photon detection up to 1650 nm, or with analogue detectors up to 5.5 μm, so it is ideal for these types of studies.

What features of the FLS1000 make it a strong candidate for any fluorescence spectroscopy application?

The key feature of the FLS1000 is the instrument’s flexibility: it can be configured for any type of experiments through selection of sources, monochromators, sample holders and detectors.

Advanced accessories such as cryostats, microscopes and plate readers can be integrated; as well as third-party lasers. Thanks to this flexibility, upgrades are easy so it is the instrument of choice for any type of advanced fluorescence research, whether it is in biology, physics, material science or chemistry.

An energy level diagram demonstrating the electronic mechanism of fluorescence. Image Credit: Wikimedia

Where can our readers find out more about fluorescence spectroscopy, the FLS1000 and Edinburgh instruments?

If you want to learn about fluorescence spectroscopy or about Edinburgh Instruments you can visit our website, www.edinst.com, for information on our products, technical support and application notes.

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About Maria Tesa

Maria Tesa is an Applications and Product Specialist at Edinburgh Instruments in Scotland. She joined the company in May 2016 after completing a PhD on spectroscopy at Heriot-Watt University (Edinburgh).

Her role at Edinburgh Instruments involves providing technical support in the sales of fluorescence spectrometers, transient absorption spectrometers, and lasers to customers in both Industry and Academia.