June 14, 2009
June 14, 2009
June 17, 2009
14.635.1 - 14.635.7
Fluorescence Correlation Spectroscopy with Confocal Detection
Fluorescence correlation spectroscopy (FCS) is one of the many different methods of high-resolution spatial and temporal analysis of low concentrated bio-molecules. One outstanding feature of this method is its comparable noninvasiveness, which makes it perfectly suited for measurements inside living cells. Comparing to other fluorescence techniques, the parameter of primary interest is not the emission intensity itself, but spontaneous intensity fluctuations caused by the minute deviations of the small system from thermal equilibrium. Traditional FCS measurements suffered from poor signal-to- noise ratios, mainly because of large ensemble numbers of molecules flowing in and out of the detection area. Researchers managed to reduce the concentrations and observation volumes such that only few molecules are simultaneously detected. The final breakthrough was achieved by Rigler and his coworkers by combining the FCS techniques with confocal detection. The purpose of this paper is to describe the design/development of the FCS setup. The setup is modeled to collect the fluorescence light from the sample and is passed through the emission filter. The pinhole in the image plane blocks any fluorescence light not originating from the focal region. Afterwards, the light is focused onto the detector, preferably a photomultiplier with single photon sensitivity. Future plan is to incorporate this research experience into our Summer Undergraduate Research Training Program (SURTP) for our students.
Spectroscopy (1) is originally the study of the interaction between radiation and matter as a function of wavelength. In fact, historically, spectroscopy referred to the use of visible light dispersed according to its wavelength. Later the concept was expanded greatly to comprise any measurement of a quantity as function of either wavelength or frequency. Spectrometry is the spectroscopic technique used to assess the concentration or amount of a given species. In those cases, the instrument that performs such measurements is a spectrometer. Spectroscopy/spectrometry (2,3,4) is often used in physical and analytical chemistry for the identification of substances through the spectrum emitted from or absorbed by them. The science of spectroscopy can be part of the curriculum for chemistry and biochemistry engineering major. The spectroscopy course should include an introduction to light and its properties, the electromagnetic spectrum, types of light both visible and invisible to the human eye, and the effects of light interaction with matter. Also the descriptions of common spectroscopic analysis techniques and applications of spectroscopy should be included.
Fluorescence spectroscopy uses higher energy photons to excite a sample, which will then emit lower energy photons. This technique has become popular for its biochemical and medical applications. Fluorescence correlation spectroscopy (FCS) (5,6,7) is one of the
Yousuf, A., & Zeng, Y. (2009, June), Fluorescence Correlation Spectroscopy With Confocal Detection Paper presented at 2009 Annual Conference & Exposition, Austin, Texas. https://peer.asee.org/5011
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