Femtosecond spectroscopy relies on ultrafast light pulses from a laser both to start the reaction and also as a means of detecting short lived transitions states and the end products of the reaction. As the name suggests, femtosecond spectroscopy requires the light pulse length to be in the order of magnitude of femtoseconds, 10-15 seconds. Lasers which could deliver pulses of this length were first designed and created in the mid 1980s (W.H Knox et al. (1985), Charles Shank (1986)). The Pump-Probe technique, which had previously been used to observe chemical reactions taking place on a microsecond timescale in the 1950s and 1960s by Norrish and Porter, could now be used to observe chemical bonds breaking and forming on a femtosecond timescale. Often referred to as the “father of femtochemistry”, Ahmed Zewail was the first to monitor chemical reactions on this timescale and received the Nobel prize for chemistry for his work in 1999. In the years since then, femtochemistry has grown into a vast discipline of its own with more applications and techniques being discovered all the time.
The word laser stands for Light Amplified by a Source of Electromagnetic Radiation. It is light which is focussed on one spot, with a very arrow beam and can also emit light with a very narrow spectrum. It is this property of lasers which allow them to deliver pulses of light only femtoseconds in length at approximately 800 nm in length. Light of 800 nm lies in the infra-red region of the electromagnetic spectrum.
The pump-probe technique can be used to detect fleeting transition states. Pulses of light from a femtosecond laser are given at regular intervals to provide image-like information on the species formed over the course of the reaction. The first pulse at the beginning of the reaction is used to promote electrons to an excited state and the subsequent pulses are used to monitor the electronic states of electrons in the molecule. The initial pump pulse is usually stronger than the probing pulses. Electrons require energy to be excited to a higher electronic state, in this case provided by the laser, and release energy when they fall back down to the ground state. The optical constants of a molecule are specific to each different molecule and include properties such as refractive index, adsorption coefficient and reflectivity at a particular wavelength of light. These optical constants are determined in the probing stage of this reaction and, when plotted against time, they provide information on the electronic state of the electrons in the molecule.
During a chemical reaction, the bonds in a molecule are broken and reformed by the movement of electrons between atoms. The species present when bonds are in the process of being broken and formed is called the transition state of a reaction and is only present for an amount of time in the region of femtoseconds which is why it was impossible to detect these species before the development of femtosecond spectroscopy.
There are many applications of the pump-probe technique ranging across many different disciplines of science from chemistry and physics to biology and medicine.
Optical transfection – The process of using titanium:sapphire femtosecond lasers to make microscopic holes in cell membranes to allow surrounding DNA or RNA can enter the cell. This is very useful in a biological sense because it allows scientists to study the effects of disease on human and animal cells. In the future it may become very important in regards to stem cell gene therapy. Stem cell gene therapy is a medical technique through which stem cells are inserted into a patients’ tissue or organs to remove or repair diseased cells. It is used to treat such conditions as diseases of the blood and to restore blood levels after being treated for certain types of cancer. Current methods for transplanting stem cells into patients have several disadvantages which optical transfection may get around. When using femtosecond lasers to insert stem cells, the nanohole created is self repairing and relatively non-invasive compared to current methods for inserting stem cells so there is no chance of the cell or surrounding tissue being further damaged by the insertion of stem cells in this manner. Since this process happens one stem cell at a time, it is a process that is very easy to control the introduction of specific genes.
STE-SRS microscopy – Spectrally-tailored excitation-stimulated Raman scattering microscopy is a process which can be used in deep-tissue imaging and brain scanning in a clinical setting. While not as widespread in its usage as techniques such as Magnetic Resonance Imaging, it is nevertheless useful in the diagnoses of medical conditions. The technique is based upon that of vibrational spectroscopy. A femtosecond laser is used as in the pump-probe process. It can be used to detect molecules in the body as well as their concentrations. As with optical transfections, STE-SRS microscopy has significant advantages over the more widespread techniques. No labelling of the tissue is required for its imaging which makes this technique ideal for molecules such as drugs which may undergo photobleaching upon contact with fluorophores. This means that the molecules alter the fluorophores so that they are unable to emit light when they are excited with photons as fluorophores normally do. STE-SRS microscopy could be a very useful medical imaging technique in the future as it can be used with great specificity and sensitivity.