![]() ![]() Where λ λ is the wavelength in vacuum and n is the medium’s index of refraction. As it is characteristic of wave behavior, interference is observed for water waves, sound waves, and light waves. Here we see the beam spreading out horizontally into a pattern of bright and dark regions that are caused by systematic constructive and destructive interference. Passing a pure, one-wavelength beam through vertical slits with a width close to the wavelength of the beam reveals the wave character of light. The laser beam emitted by the observatory represents ray behavior, as it travels in a straight line. In Figure 17.2, both the ray and wave characteristics of light can be seen. Interference is the identifying behavior of a wave. However, when it interacts with smaller objects, it displays its wave characteristics prominently. As is true for all waves, light travels in straight lines and acts like a ray when it interacts with objects several times as large as its wavelength. The range of visible wavelengths is approximately 380 to 750 nm. When the genes are expressed, the protein will be attached to GFP and can be identified in the cell by its fluorescence.Where c = 3.00 × 10 8 c = 3.00 × 10 8 m/s is the speed of light in vacuum, f is the frequency of the electromagnetic wave in Hz (or s –1), and λ λ is its wavelength in m. The gene encoding GFP can be inserted next to a gene encoding a protein that will be studied. Green fluorescence protein (GFP) is used in molecular biology to monitor the activity of proteins. The presence and placement of the gene in the organism then fluoresces when shined with ultraviolet light. Single stranded DNA encoding a gene of interest is covalently bonded to a fluorescent molecule and washed over the organism's chromosome, binding to its complementary sequence. For example, fluorescence in situ hybridization (FISH) is a method of determining what genes are present in an organism's genome. Fluorescent molecules can also be used as tags. To obtain an excitation spectrum, the excitation monochromator varies while the emission monochromator is fixed.įluorescence spectroscopy can be used to measure the concentration of a compound because the fluorescence intensity is linearly proportional to the concentration of the fluorescent molecule. To obtain an emission spectrum, the excitation monochromator is fixed and the emission monochromator varies. The electrical outputs from the two transducers are then processed by an analog to digital converter to compute the ratio of the sample to reference intensities, which can then be used for qualitative and quantitative analysis. The emitted radiation passes through a second filter and then is focused on the sample photomultiplier tube. The sample beam is focused on the sample by a pair of lenses and causes fluorescence emission. Both beams pass through the primary filter, with the reference beam being reflected to the reference photomultiplier tube. The reference beam is attenuated by the aperture disk so that its intensity is roughly the same as the fluorescence intensity. The source beam is split near the source into a reference beam and a sample beam. from OpenStax (CC-BY-3.0)įigure 2 is a schematic of a typical filter fluorometer that uses a source beam for fluorescence excitation and a pair of photomultiplier tubes as transducers. Figure 2: Schematic representation of a fluorescence spectrometer. The average lifetime is 10 -7 to 10 -9 seconds for ?, ?* states and 10 -5 to 10 -7 seconds for n, π* states. Molar absorptivity of π → π* transitions is 100-1000 fold greater. Quantum yield (\(\Phi\)) is greater for \(\pi^* \rightarrow \pi\) transition because these excited states show short average lifetimes (larger \(k_f\)) and because deactivation processes that compete with fluorescence is not as likely to happen. Fluorescence commonly occurs from a transition from the lowest vibrational level of the first excited electronic state to the one of the vibrational levels of the electronic ground state. Instead such emission is confined to the less energetic \(\pi^* \rightarrow \pi\) and \(\pi^* \rightarrow n\) processes. Consequently, fluorescence due to \(\\sigma^* \rightarrow \sigma\) transitions is rarely observed. Most organic molecules have at least some bonds that can be ruptured by energies of this strength. This relationship shows that fluorescence intensity is proportional to concentration.įluorescence rarely results from absorption of UV-radiation of wavelengths shorter than 250 nm because this type of radiation is sufficiently energetic to cause deactivation of the excited state by predissociation or dissociation. ![]()
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