I am a Researcher at the University of Strathclyde and working with an industrial partner Horiba Scientific IBH. I work in the field of Fluorescence Spectroscopy and Fluorescence Imaging. My PhD thesis, Nanometrology using Time-Resolved Fluorescence can be found here:
Below I will discuss my research, in more basic terms using pictures and animations and try to avoid the use of any equations. Please feel free to ask questions in the comments, if anything doesn’t make sense.
Basics: Let’s Look at Light
Me holding a DVD and shining White Light onto it using a Webcam. The White Light interacts with the Diffraction Grating (DVD) and splits the light out into it’s individual colours:
You will see that the DVD splits the white light into a rainbow like pattern. Red light has the longest wavelength so is at the edge or the DVD, followed by orange, yellow, green, turquoise, blue and purple.
A set of slits, can be used with a motorised Diffraction Grating to select the desired colour of light.
Light can be described by it’s colour, visible light is described as Red, Orange, Yellow, Green, Blue, Violet as it can be seen by the human eye and the individual colours can be distinguished. Light can however be more accurately by it’s wavelength where…
Ultraviolet light and Near Infrared cannot be seen by the human eye.
Light can be thought of both as a particle and as a wave. The top image shows light that has a wavelength of 700 nm (Red) where 1 nanometre = 1 nm = 1 millionth of a mm and the second image shows light that has a wavelength of 350 nm (UV).
If we look at a point object moving along each wave, think of it as yourself walking up and down a mountain, into a valley and back to the same height. Comparing the two, we can see that in the time it takes the point object to move from start to finish on the 700 nm (Red) wave, it has done this twice already with the 350 nm (UV) wave. It takes you twice as much energy to walk up and down a mountain into a valley and back to the same height twice than it does a single time. So in other words the 350 nm (UV) wave has twice as much energy as the 700 nm (Red) wave.
Light can Interact with Matter
The sun radiates white light and a proportion of this white light reaches the earth’s atmosphere. The earth’s atmosphere contains primarily diatomic Nitrogen (triply bonded) and diatomic Oxygen (doubly bonded). Both molecules are extremely stable however the triple bond on the Nitrogen makes it even more stable than diatomic Oxygen.
The high energy UV light emitted from the sun is not strong enough to interact with diatomic Nitrogen but it is strong enough to promote an outer electron to an excited state in diatomic Oxygen (O2) forming Ozone (O3) and a free radical. These species are highly reactive and interact with nearby diatomic Oxygen molecules.
Light can also scatter off of the air molecules. Rayleigh scattering is an elastic process meaning the colour of light is unchanged upon collision with the air molecule. Rayleigh scattering follows the relationship 1/(wavelength^4). Thus the lower wavelength of light such as Blue is substantially more likely to scatter than higher wavelength of light. It is for this reason the Sky looks Blue.
Rayleigh scattering is a relatively instantaneous process occurring on the femtosecond time scale. 1 fs =1×10^-15 s. Rayleigh scattering is more prominent for larger objects, as they have a larger cross-section for the light to interact with.
Molecules vibrate and an incident photon may interact with a molecule as it vibrates, transferring some energy across to the molecule. This type of scattering is inelastic as the photon’s colour is changed slightly due to the energy lost as a result of the interaction with the molecule. This type of scattering is called Raman scattering. Raman scattering is a relatively rare interaction, with only 1 in 1 million photons exhibiting Raman scattering opposed to Rayleigh scattering. Raman scattering is normally too weak to see by eye.
Fluorescence Dyes, for example Rhodamine 6G are molecules which usually have large aromatic rings. These aromatic rings contain delocalised electronics which can interact with visible light.
One of these molecules, may absorb a photon of visible light. The absorbance process again is instantaneous and occurs on the fs regime. The conjugation of the aromatic rings stabilises the excited state, thus the molecule may remain in the excited state for some time, typically a time within the ns regime. Some of the additional energy is lost as heat. Spontaneous emission (the emission itself is on a fs regime) of a photon occurs. The ns process of fluorescence is measurable with modern day electronics.
There is another process called phosphorescence, the emission pathway is quantumly unfavourable and thus it takes a far longer time to spontaneously emit a photon. Phosphorescence occurs on the µs to ms timescale.
Molecular Interaction with Light:
- Rayleigh is elastic scattering meaning that it’s the same colour (wavelength) as incident light and occurs on a fs time scale.
- Raman is inelastic scattering leading to slight energy loss and thus is slightly higher in wavelength than the incident light. Raman scattering is much rarer than Rayleigh scattering but also occurs on the fs time scale.
- Fluorescence emission due to a molecular interaction is a different colour (higher wavelength) and has a moderate energy loss. It occurs on the ns timescale.
- Phosphorescence emission also due to a molecular interaction is a different colour (higher wavelength) and likewise has a moderate energy loss. It occurs on the µs to ms timescale.
Visualising Molecular Processes by Eye
The following samples are:
- 1 Water
- 2 Silica Solution (small glass nanoparticles suspended in water)
- 3 Fluorescein Dye in water
- 4 Rhodamine 6G Dye in water
- 5 ADOTA Dye in water
I will look at these samples using a Horiba Scientific DeltaPro equipped with a Green Laser:
Here is the DeltaPro sample chamber (without a sample). The Green Light from the laser can be seen to bounce off the card:
Let’s look at water again the Green Light from the laser can be seen to bounce off the card but a small amount of light scatters off the water molecules:
Let’s now look at the Silica Solution, the green light from the laser can be seen to bounce highly off the silica nanoparticles, it remains the same colour meaning no further interaction has occurred with the silica nanoparticles.
Let’s now look at the Fluorescein Dye Solution, one can see the tinge of green going through the Fluorescein is slightly different in colour to the Green Laser. This is fluorescence, a small fraction of the light has interacted with the dye molecule and the dye molecule has emitted a photon at a higher wavelength. In this case a green laser is not the optimal colour to excite this dye. A Blue Laser would be more suited.
Fluorescein is commonly used by opticians in eye tests. They stick some Fluorescein Dye in your eye and then shine Blue Light onto your eye. They then use a microscope and camera to image your eye.
The dye readily flows to damaged areas such as this abrasion and thus easily highlights the issue to your optician.
Let’s now look at the Rhodamine 6G Dye Solution, one can see that Green Light is incident on the sample but the sample glows Yellow instead. Green Light interacts with the Rhodamine 6G Dye Molecule to promote an electron from the Ground State to the Excited State. The Excited Dye Molecule interacts with the surrounding water molecules, losing some energy to the surroundings in the form of heat. After some time (~4 ns) after Excitation the Dye Molecules emits a Yellow Fluorescence Photon which is slightly lower in energy than the Green Excitation Photon. Due to the difference in colours between the Green Excitation Photon and Yellow Fluorescence Photon one may use a Filter or Monochromator to separate out these two signals.
Let’s now look at the ADOTA Dye Solution, one can also see Yellow Fluorescence in response to Green Excitation in a similar manner to the Rhodamine 6G Dye Solution.
The above laser can be coupled to a microscope as shown. Fluorescence can be viewed from a microscope slide:
Fluorescence microscopy is commonly used to examine biological objects. For instance the following petal is examined using a fluorescence microscope at varying excitation wavelengths:
More details can be seen when using blue and red light opposed to green light. This is because the petal contains a high amount of chlorophyll which reacts with the blue and red light respectively. Leafs, which have a higher abundance of chlorphyll tend to be green because they reflect the green light while absorbing the blue and red light.
Detectors and Timing
The examples above were strongly fluorescent samples and the detector used was the human eye. The human eye cannot see in the UV or NIR however cameras or detectors can be made to measure within these regimes. Here for instance is a picture of me, viewed under a NIR camera:
Many high sensitive detectors use a photocathode and a dynode. The signal coming from an incident photon is converted into an electrical current at the cathode and enhanced along the dynode chain.
Dark counts may also be computed as a result of an electron shaking lose near the start of the dynode. This noise is temperature dependent so can be reduced by cooling the detector. For a reliable measurement one needs to acquire a good signal to noise ratio.
Up until now, high laser power and bright samples were shown which could be visualised by eye. In most applications a more sensitive instrument is used, which allows for analysis of a far more dilute sample. The Horiba Scientific Duetta for instance can rapidly excite the sample using white light coming from a monochromator, it can compare the ratio of light coming into the sample, with that passing through (to get an absorbance spectrum) and it can use a highly sensitive CCD camera to measure the emission spectrum at each excitation wavelength. This plot creates an EEM which is a spectral fingerprint of the sample. This type of measurement is commonly used to determine analytes and their relative concentration and is very important for instance in detecting contaminants in water treatment plants.
Three individual examples were measured, Rhodamine 6G, Rubene and a mixture of the above two solutions. In the Rubene sample a contaminant can be picked up which has a different excitation and emission wavelength. The mixture of Rubene and Rhodamine 6G have an overlaying spectral fingerprint and cannot be readily separated using this technique.
These two molecules however have different decay times. Fluorescence decays can be measured using a Horiba DeltaFlex with a Blue Excitation source at varying Emission wavelength.
As each specie has it’s own decay time, with one decay time (Rubene) about twice as long as the other (Rhodamine 6G). Earlier time slices of the data should have a higher ratio of short lived:long lived component than later time-slices.
The data can also be fitted to two exponentials and their relative contribute plotted with respect to emission wavelength. As can be seen the Decay Associated Spectrum of the mixture resembles the individual steady state spectra of the three components.
Time is therefore another important parameter of the spectral fingerprint of a sample.