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WIREs Syst Biol Med
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Live cell single‐molecule detection in systems biology

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Abstract In this review, we describe technology and use of single‐fluorophore imaging and detection in living cells with regard to application in systems biology and medicine. Because all biological reactions occur under aqueous conditions, the realization of single‐fluorophore imaging using an optical microscope has led to the direct observation of biological molecules at work. Today, we can observe single molecules in individual living cells and even higher multicellular organisms. Using single‐molecule imaging, we can determine the absolute values of kinetic and dynamic parameters of molecular reactions as a whole and during fluctuations and distribution. In addition, identification of the coordinate of single molecules has enabled super‐localization techniques to virtually improve spatial resolution of optical microscopy. Single‐molecule detection that depends on point detection instead of imaging is also useful in detecting concentrations, diffusive movements, and molecular interactions in living cells, especially in the cytoplasm. The precise and absolute values of positional, kinetic, and dynamic parameters that are determined by single‐molecule imaging and detection in living cells constitute valuable data on unitary biological reactions, because they are obtained without destroying the integrity of complex cellular systems. Moreover, most parameters that are determined by single‐molecule measurements can be substituted directly into equations that describe kinetic and dynamic models in systems biology and medicine. WIREs Syst Biol Med 2012, 4:183–192. doi: 10.1002/wsbm.161 This article is categorized under: Laboratory Methods and Technologies > Imaging

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Types of fluorescence microscope used for single‐molecule imaging in living cells. (a) Total internal reflection microscope. (b) Low‐angle oblique illumination (LOI) microscope. (c) Epi‐illumination microscope. The upper panels show the illumination path, and the lower panels are single‐molecule images of teteramethyl rhodamine‐labeled epidermal growth factor (EGF) on the surface of a living HeLa cell, acquired using each illumination mode. Focus on the bottom (a, c) or top (b) of the same single cell.

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Super localization microscopy. (a) Centroid of a single‐molecule image can be determined with nanometer‐range accuracy by Gaussian fitting. Here, an image of a membrane protein (ErbB1) fused with a photo‐convertible fluorescent protein (mKikGR) is shown. (b) Before photo‐conversion, mKikGR emits green fluorescence under blue light irradiation. By weak violet irradiation, mKikGR is altered to emit red fluorescence under yellow light irradiation. (c) Procedure of photo‐activation localization microscopy (PALM). In stochastic optical reconstruction microscopy (STORM), a single fluorophore is converted between two colors repeatedly. (d) A PALM image (yellow) of ErbB1‐mKikGR expressed on the plasma membrane of a CHO cell is superimposed onto the total internal reflection (TIR) illumination image (red) acquired in the same field of view.

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Fluorescence correlation spectroscopy (FCS) and related technologies. Principle of measurement (upper panels) and examples of raw data (middle panels) are shown with autocorrelation function (bottom left), cross‐correlation function (bottom middle), and histograms of intensity ratio (bottom right) for (a) FCS, (b) fluorescence cross‐correlation spectroscopy (FCCS), and (c) photon‐counting histogram (PCH), respectively. (a) Lateral diffusion of GFP‐ERK in the cytoplasm of MCF‐7 cell was measured using FCS. Total concentration and the diffusion coefficient of two mobile components were determined. (b) Colocalization between tetramethyl rhodamine‐labeled heregulin (Rh‐HRG) bound to its receptor in the plasma membrane and GFP‐PI3K in the cytoplasm was detected by FCCS. FCCS suggests that 16% of PI3K molecules are associated with the HRG receptor. (c) Intramolecular fluorescence resonance energy transfer (FRET) signal from RAF molecule fused with GFP and YFP at the N‐ and C‐termini (GFP‐RAF‐YFP)21 was detected using PCH in the cytoplasm of HeLa cells. GFP‐RAF and RAF‐YFP are molecules fused only to GFP and YFP, respectively, as control. A peak of approximately 0.6, shown in the distribution of GFP‐RAF‐YFP suggests a closed conformation of RAF showing FRET from GFP to YFP.

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