This work is supported in part by NIH-P41 P41-RRO3155 and NIH P50-GM076516 (grant to EG), and Fondazione Monte dei Paschi di Siena (grant to FB).

1. System Calibration
<ol>
	<li>Point Spread Function (PSF) calibration
	<ol>
		<li>Dilute 10 &#181;l of 30 nm fluorescent bead solution (about 5 &#956;M) in 90 &#181;l of distilled water and then sonicate the solution for 20 min. Cut a square (1 cm x 1 cm) piece of agarose gel (3%) and deposit 10 &#181;l of the solution on the top of the gel. Overturn the piece of gel on the bottom glass of a 2 cm Petri dish and squeeze the drop on the glass.</li>
		<li>Turn on the acquisition setup, put the sample in the holder, set ...

The authors have nothing to disclose.

Single particle tracking (SPT) represents one of the most common strategies to study molecular dynamics and it has the great advantage of measuring particle trajectories. This in turn allows probing the behavior of even few labeled particles in a complex system. However, to reach this advantage SPT typically needs a low density of the probe and very bright labels. Particularly, to gain high temporal resolution (&#956;sec range) inorganic probe are usually required (e.g., quantum dots or metal nanoparticles): in ...

Starting from the original &#8220;fluid mosaic&#8221; model by Singer and Nicolson, the picture of cellular plasma membrane has been continuously updated during the last decades in order to include the emerging role of cytoskeleton and lipid domains1,2.
The first observations were obtained by fluorescent recovery after photobleaching (FRAP) unveiling that a significant fraction of membrane proteins is immobile3-5. These pioneering studies, although very informative, suffered from the relatively poor resolution in space (microns) and time (seconds) of FRAP setups. Also, being an ensemble averaging measurement, FRAP lacks in giving information on single molecule behavior.
In this context, the possibility to specifically label a single molecule with very bright tags (allowing the study of the diffusion process one molecule at a time) has been very successful. Particularly, by pushing the time resolution of the Single Particle Tracking (SPT) approach to the microseconds timescale, Kusumi, et al. gained access to unknown features of lipid and protein dynamics that greatly contributed to the recognition of the role of actin-based membrane skeleton in membrane physiology6,7. These findings generated the so-called the &#8216;picket and fence&#8217; model, in which lipid and protein diffusion is regulated by actin-based skeleton. However, in order to have access to the huge amount of information provided by SPT many experimental issues have to be addressed. Particularly, the labeling procedure is typically composed by many steps like production, purification and introduction of the labeled species into the system. Furthermore, big labels, like quantum dots or metal nanoparticles, are often required to reach the sub-millisecond timescale and the crosslinking of the target molecules by the label could not be avoided in many cases. Finally, many trajectories have to be recorded to fit statistical criteria and concomitantly a low-density of the label is required to allow tracking.
Compared to SPT, fluorescence correlation spectroscopy (FCS), overcoming many of these drawbacks, represents a very promising approach to study molecular dynamics. In fact, FCS works well also with dim and dense labels, enabling to study the dynamics of fluorescent protein-tagged molecules in transiently transfected cells. Also, it allows reaching high statistics in a limited amount of time. Finally, despite the &#8220;high&#8221; density of labels FCS provides single molecules information. Thanks to all these properties, FCS represents a very straightforward approach and has been extensively applied to study lipid and protein dynamics both in model membranes and in live-cells8-10. Many different approaches have been proposed to increase the ability of FCS to reveal the details of molecular diffusion. For instance, it was shown that by performing FCS on differently-sized observation areas one can define an &#8220;FCS diffusion law&#8221; enlightening hidden features of molecular motion11,12. Besides being varied in size, the focal area was also duplicated13, moved in space along lines14-20 or conjugated with fast cameras21,22. Using these &#8216;spatio-temporal&#8217; correlation approaches, relevant biological parameters of several membrane components were quantitatively described on both model membranes and actual biological ones, thus yielding insight into membrane spatial organization.
However, in all the FRAP and FCS applications described so far the size of the focal area represents a limit in spatial resolution that cannot be overcome. Several super-resolution imaging methods have been recently developed to bypass this limit. Some are based on localization precision, such as stochastic optical reconstruction microscopy (STORM)23,24, photoactivation localization microscopy (PALM)25, fluorescence PALM (FPALM)26, and single-particle tracking PALM (sptPALM)27: the relatively large amount of photons required at each snapshot, however, limits the time resolution of these methods to at least several milliseconds, thus hampering their applicability in vivo.
In contrast, a promising alternative for super resolution imaging have been opened by spatially modulating the fluorescence emission with stimulated emission depletion methods (STED or reversible saturable optical fluorescence transitions (RESOLFT))28,29. These approaches combine the shaping of the observation volume well below the diffraction limit with the possibility to use fast scanning microscopes and detection systems. In combination with fluorescence fluctuation analysis, STED microscopy allowed to directly probe the nanoscale spatiotemporal dynamics of lipids and proteins in live cell membranes30,31.
The same physical quantities of STED-based microscopy can be obtained by a modified spatio-temporal image correlation spectroscopy (STICS32,33) method that is suitable for the study of the dynamics of fluorescently-tagged membrane proteins and/or lipids in live cells and by a commercial microscope. The experimental protocol presented here is composed by few steps. The first one requires a fast imaging of the region of interest on the membrane. Then, the resulting stack of images is used to calculate the average spatial-temporal correlation functions. By fitting the series of correlation functions, the molecular &#8216;diffusion law&#8217; can be obtained directly from imaging in the form of an apparent diffusivity (Dapp)-vs-average displacement plot. This plot critically depends on the environment explored by the molecules and allows recognizing directly the actual diffusion modes of the lipid/protein of interest.
In more details, as previously shown34, the spatio-temporal auto-correlation function of the acquired image series critically depends on the dynamics of the molecules moving in the collected image series (please note that the same reasoning can be applied in a line acquisition where just one dimension in space is considered). In particular, we define the correlation function as:
<img fo:content-width="2.3in" src="/files/ftp_upload/51994/51994eq1.jpg" width="230" />(1)
where <img fo:content-width=".5in" src="/files/ftp_upload/51994/xyt.jpg" width="50" /> represents the measured fluorescence intensity in the position x, y and at time t, <img fo:content-width=".08in" src="/files/ftp_upload/51994/e.jpg" width="8" /> and <img fo:content-width=".1in" src="/files/ftp_upload/51994/x.jpg" width="10" /> represents the distance in the x and y directions respectively,<img fo:content-width=".1in" src="/files/ftp_upload/51994/t.jpg" width="10" /> represents the time lag, and <img fo:content-width=".2in" src="/files/ftp_upload/51994/brackets.jpg" width="20" /> represents the average. This function can be expressed as:
<img fo:content-width="2.3in" src="/files/ftp_upload/51994/51994eq2.jpg" width="230" />(2)
where &#8216;N&#8217; represents the average number of molecules in the observation area, <img fo:content-width=".10in" src="/files/ftp_upload/51994/circlex.jpg" width="10" /> represents the convolution operation in space, and <img fo:content-width=".5in" src="/files/ftp_upload/51994/ex.jpg" width="50" /> represents the autocorrelation of the instrumental waist. This latter can be interpreted as a measure of how the photons of a single emitter are spread out in space due to the optical/recording setup (the so called Point Spread Function, PSF, generally well approximated by a Gaussian function). Finally, <img fo:content-width=".5in" src="/files/ftp_upload/51994/ext.jpg" width="50" /> represents the probability to find a particle at a distance <img fo:content-width=".10in" src="/files/ftp_upload/51994/e.jpg" width="10" /> and <img fo:content-width=".10in" src="/files/ftp_upload/51994/x.jpg" width="10" /> after a time delay <img fo:content-width=".10in" src="/files/ftp_upload/51994/t.jpg" width="10" />. If we consider a diffusive dynamics, in which particles move randomly in all directions and net fluxes are not present, this function is also well approximated by a Gaussian function where the variance can be identified as the Mean Square Displacement (MSD) of the moving particle. Thus, the waist of the correlation function (also referred as <img fo:content-width=".3in" src="/files/ftp_upload/51994/2t.jpg" width="30" />), can be defined as the sum of the particle MSDs and the instrumental waist and can be measured by a Gaussian fitting of the correlation function for each time delay. The measured iMSD can be used to calculate an apparent diffusivity of the moving molecules <img fo:content-width=".4in" src="/files/ftp_upload/51994/dappt.jpg" width="40" /> and an average displacement<img fo:content-width=".4in" src="/files/ftp_upload/51994/rt.jpg" width="40" /> as:
<img fo:content-width="1.7in" src="/files/ftp_upload/51994/51994eq3.jpg" width="170" />(3)
<img fo:content-width="1.7in" src="/files/ftp_upload/51994/51994eq4.jpg" width="170" />(4)
Few considerations on the experimental setup used can guide the reader throughout the following sections. In order to selectively excite the fluorophores on the basal membrane of living cells we will use a total internal reflection (TIR) illumination, using a commercial TIR fluorescence (TIRF) microscope (details can be found in the material section). Moreover, in order to collect the fluorescence we will use a high magnification objective (100X NA 1.47, high numerical aperture is required for TIRF illumination) and an EMCCD camera (physical size of the pixel on the chip 16 &#956;m). To reach a pixel size of 100 nm we apply an additional magnification lens of 1.6X. As discussed below, a time resolution below 1 msec would be required to properly describe the dynamics of fast membrane lipids below 100 nm. In order to reach this temporal resolution we need to select a region of interest (ROI) smaller than the whole chip of the camera (512 x 512). In this way, the camera will read a reduced number of lines increasing the time resolution. However, in this readout regime the frame time would be limited by the time required to shift the charges from the exposure to the readout chip on the camera and is usually in the order of milliseconds for 512 x 512 pixel EMCCD. To beat this limit, an emerging technology allows shifting the ROI-lines only instead of the whole frame, with a practical effective reduction of the exposed chip size (called Cropped Sensor Mode in our EMCCD). For this configuration to be effective, the chip outside of the ROI must be covered by a couple of slits mounted in the optical path. Thanks to this setup a time resolution down to 10-4 seconds can be achieved. Please note, however, that this approach can be coupled with many different experimental setups, as explained in the &#8216;discussion&#8217; section.
Demonstration of the method will be provided in live cells, by using both an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (ATTO488-PPE) and a GFP-labeled variant of the Transferrin Receptor (GFP-TfR). In the case of ATTO488-PPE this approach can successfully recover an almost constant Dapp as a function of average displacement indicating a mostly free diffusion, as previously reported30,35. By contrast, TfR-GFP shows a decreasing Dapp as a function of average displacement, suggesting partially-confined diffusion6. Moreover, in the latter case it is possible to quantify the local diffusion constant and the average confinement area over many microns on the membrane plane.

<table border="0" cellpadding="0" cellspacing="0" width="1142">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:480px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">iXon Ultra 897</td>
			<td style="width:183px;">Andor</td>
			<td>DU-897U-CS0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Solis</td>
			<td style="width:183px;">Andor</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">CHO-K1</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:119px;">CCL-61</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATTO 488 labeled PPE</td>
			<td style="width:183px;">ATTO-TEC GmbH</td>
			<td>AD 488-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">DOPE</td>
			<td style="width:183px;">Avanti Polar Lipids, Inc.</td>
			<td style="width:119px;">850725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">DOTAP</td>
			<td style="width:183px;">Avanti Polar Lipids, Inc.</td>
			<td style="width:119px;">890890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">100X Penicillin-Streptomycin-Glutamine</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">FBS</td>
			<td>Gibco</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">HEPES</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:119px;">15630-106&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">PBS</td>
			<td style="width:183px;">Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">SimFCS 3.0</td>
			<td style="width:183px;">Globals Software</td>
			<td style="width:119px;"></td>
			<td>the software can be downloaded here: http://www.lfd.uci.edu/globals/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMI6000 with TIRF modulus</td>
			<td style="width:183px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">LAS AF</td>
			<td style="width:183px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Lipofectamine 2000</td>
			<td style="width:183px;">Lipofectamine</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Matlab</td>
			<td style="width:183px;">MathWork</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Imagej</td>
			<td style="width:183px;">NIH</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C-terminal GFP tagged Tranferrin Receptor</td>
			<td style="width:183px;">OriGene</td>
			<td>RG200980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Agar</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td>A5306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Chloroform</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:119px;">528730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Latex beads, fluorescent yellow-green, 30 nm</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td>L5155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">SONICA Ultrasonic Cleaners</td>
			<td>SOLTEC</td>
			<td style="width:119px;">ETH S3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Petri Dishes</td>
			<td style="width:183px;">Willco</td>
			<td>GWSt-3522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Bio-Format importer for Matlab</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.openmicroscopy.org/site/support/bio-formats5/users/matlab/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">ICS-MatLab Tools</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td><a href="https://www.cellmigration.org/resource/imaging/software/ICSMATLAB_28-02-06.zip">https://www.cellmigration.org/resource/imaging/software/ICSMATLAB_28-02-06.zip</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Simulation by Matlab Tutorial</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td>https://www.cellmigration.org/resource/imaging/icsmatlab/ICSTutorial.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Simulation by SimFCS Tutorial</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td>https://www.cellmigration.org/resource/imaging/ppt-pdf/RICS%20Simulations.ppt</td>
		</tr>
	</tbody>
</table>

from fast fluorescence imaging to molecular diffusion law on live cell membranes in a commercial microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

In order to calibrate the instrumental waist, the image of a single fluorescent nano-bead can be measure as described in Protocol step 1.1. A typical fluorescent image of these beads is presented in Figure 1. The fitting of intensity distribution by a 2D Gaussian function gives back good residuals and allows measuring the instrumental waist at 270 nm. This value is in good agreement with the expected diffraction limit estimated by the Rayleigh equation. This calibration is not necessary for the measureme...

Watch this Scientific Journal Video about From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope at JoVE.com

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

from-fast-fluorescence-imaging-to-molecular-diffusion-law-on-live

Research

JoVE Journal

Bioengineering

11.3K Görüntüleme.  Scuola Normale Superiore. Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.