This work was supported by a Lundbeck Junior Group Leader Fellowship to LNN. PWW acknowledges grant funding support from the Natural Sciences and Engineering Research Council of Canada (NSERC). We also thank the Danish Molecular Bioimaging Center at University of Southern Denmark for access to spinning disk microscopy.

Acquisition of EGFP-tagged proteins in the plasma membrane for kICS analysis can be done on an epifluorescence microscope, a TIRF setup or on a spinning disk microscope set to acquire images of the membrane of interest. The camera pixel size as well as the time between image frames is needed for the analysis. Image sequences of 100-1,200 frames at a frame rate of 4-30 Hz can be used for the analysis. During the acquisition, it is important to keep the membrane in focus throughout the duration of the imaging and focus on ...

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This paper presented a detailed step-by-step overview of how to apply the kICS analysis method to determine diffusion coefficients from microscopy images of proteins tagged with fluorescent proteins. The analysis is independent of probe choice with a very broad dynamic range in labeling density and can thus also be applied to proteins labeled with quantum dots as well as dyes and fluorescent proteins such as EGFP.
To calculate valid diffusion coefficients for the proteins, there are several cr...

The four dimensional spatiotemporal organization and lateral mobility of plasma membrane proteins are tightly regulated and may play a role in protein function, activity and protein-protein interactions. Lateral diffusion of plasma membrane proteins, has traditionally been studied by calculating diffusion coefficients for single particles from time-lapse imaging of quantum dot or dye labeled plasma membrane proteins2-4. This approach requires insertion of an extracellular tag in the plasma membrane protein for quantum dot or dye labeling, which can compromise protein folding and function and thus, cannot be achieved for some proteins. The steric volume of a quantum dot has been shown to slow down diffusion of the protein5 and moreover, only a tiny fraction of the proteins in the population are labeled with quantum dots, and it is not known if this fraction is representative for the total pool of plasma membrane proteins. Single particle tracking (SPT) measurements of diffusion coefficients from image series of quantum dot labeled proteins involves mapping the imaged peak positions of the particles with two dimensional Gaussian fits followed by extensive analysis algorithms. The analysis is computationally very intensive, requiring extensive non-linear curve fitting in each image frame of a time-lapse sequence to approximations of the microscope point spread function (typically two dimensional spatial Gaussians) and subsequent linking of particle positions into particle trajectories describing the motion of single molecules6,7.
A recently developed image correlation technique, k-Space Image Correlation Spectroscopy (kICS) enables relative simple measurements of diffusion coefficients of fluorescently tagged plasma membrane proteins. The possibility to routinely calculate diffusion coefficients of membrane proteins labeled with fluorescent proteins by kICS is a unique tool which holds several advantages over traditional quantum dot SPT analysis: No insertion of extracellular tags and time consuming extracellular labeling is required (cell lines expressing fluorescent proteins may be used); diffusion coefficients are extracted from the total pool of fluorescent proteins compared to a subset labeled with quantum dots; analysis is simple without the need to track single proteins and the analysis can be performed using an existing code with no requirement for additional user programming. The method is rapid because it is an averaging technique which enables fast computation and calculation of diffusion coefficients. These rapid diffusion measurements for the protein population complement the more detailed molecular transport information obtained for a subset of the population by the painstaking SPT methods.
kICS time correlates fluorescence microscopy image sequences that have first been transformed to Fourier space, and thereby separates fluorescence fluctuations due to photophysics from those due to molecular transport1. As a result, kICS can determine the number density, flow speed, and diffusion of fluorescently labeled molecules while being unbiased by complex photobleaching or blinking of the fluorophores. This makes kICS a useful tool for quickly determining the diffusion dynamics of fluorescently-labeled cell membrane proteins without the need to custom write algorithms. Besides fluorescently labeled proteins, kICS can also be applied to quantum dot-labeled membrane proteins8.
This paper provides a step-by-step introduction of how to use kICS to extract diffusion coefficients of EGFP-tagged plasma membrane proteins by demonstrating how to place the crop, how to use the code and how to evaluate the generated plots, from which diffusion coefficients are extracted. As an example, data obtained by spinning disk-microscopy set in semi-total internal reflection fluorescence (TIRF) mode, of a renal water channel protein aquaporin-3 (AQP3) tagged with EGFP is presented.

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			<td height="21" style="height:21px;width:216px;">DMEM</td>
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			<td height="42" style="height:42px;width:216px;">FBS</td>
			<td style="width:71px;">Invitrogen</td>
			<td>10082147</td>
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			<td height="21" style="height:21px;width:216px;">Penicilin&#160;&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Kanamycin</td>
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			<td height="21" style="height:21px;width:216px;">Streptomycin</td>
			<td style="width:71px;">Gibco</td>
			<td>11860038</td>
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			<td height="21" style="height:21px;width:216px;">Phenol red free medium</td>
			<td style="width:71px;">Gibco</td>
			<td>11880-028</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:71px;">Sigma</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/d8418?lang=en&amp;region=DK">D8418&#160;</a></td>
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			<td height="21" style="height:21px;width:216px;">HEPES</td>
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			<td>15630056</td>
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			<td height="21" style="height:21px;width:216px;">Apparatus</td>
			<td style="width:71px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Spinning Disk Microscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">Ti Eclipse</td>
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			<td height="21" style="height:21px;width:216px;">EMCCD Camera</td>
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</table>

easy measurement of diffusion coefficients of egfp-tagged plasma membrane proteins using k-space image correlation spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

To acquire suitable image stacks to be analyzed with kICS, different microscope systems can be used. It is possible to analyze image sequences from an epi-fluorescence microscope as well as a TIRF&#160;or spinning disk setup. The membrane must be flat without any large moving cell organelles or moving vesicles/objects. This paper presents a time lapse image sequence of AQP3 tagged with EGFP in live MDCK cells imaged on a spinning disk microscope with focus set to the plasma membrane&#160;at a frame rate of 9.2 Hz. The fo...

Watch this Scientific Journal Video about Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy at JoVE.com

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

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10.6K vistas.  Aarhus University.  Este documento proporciona una guía paso a paso a la técnica de análisis de fluctuación de imagen k-espacio espectroscopia de correlación (CCI) para la medición de los coeficientes de difusión de la etiqueta fluorescente proteínas de la membrana plasmática en células de mamíferos vivos. 