Name: label-free imaging of single proteins secreted from living cells via iscat microscopy
Uploaded: 2018-11-20T14:00:00
Duration: 10 min 55 s
Description: Watch this Scientific Journal Video about Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy at JoVE.com

This work was supported by the Max Planck Society, an Alexander-von-Humboldt Professorship, and the Deutsche Forschungsgemeinschaft (CRC 1181). We thank Stefanie Schaffer at Universit&#228;tsklinikum Erlangen for providing Laz388 cells and for useful discussions. We thank Simone Ihloff and Maksim Schwab at MPL for technical support.

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The authors have nothing to disclose.

One of the most crucial aspects to obtaining useful iSCAT data is the ability to find the correct focal position at the coverslip surface, and, furthermore, to hold this position for long periods of time. Failing to do so will result in broadened PSFs, weak iSCAT signals, and drift-associated artifacts in dynamics analyses. It turns out that finding the focal plane on a clean, bare coverslip surface is not an easy task as surface features are not visible against the large reference beam background (see Figure 4d).
Raw iSCAT images are often obscured by background signals that arise from wavefront impurities in the excitation source, and can hinder one&#39;s ability to find the correct imaging plane. Active wavefront subtraction is a useful way to circumvent this issue and subsequently monitor the iSCAT focus during a measurement16. One way to accomplish this is through spatial sample modulation. In brief, a function generator applies a 50 Hz square wave to the external control port of the piezo stage, resulting in a spatial sample modulation at the applied frequency (290 nm amplitude). Synchronous camera acquisitions are triggered from the same source, and, when combined through lock-in principles, result in a wavefront-compensated image14,16. The resulting image typically shows the surface roughness of the coverslip (Figure 4e). Small features remaining on the glass after cleaning can be used to bring the microscope into focus. Parameters used for this active background subtraction step may be changed according to the frame rate, exposure time, or hardware.
As mentioned above, the use of a high-quality beam splitter in the iSCAT setup (step 1.2.1.) is recommended, as imaging artefacts like ghosting or interference arising from thin planar beam splitters will influence the image and disturb the measurement. Figure 7 shows a comparison between a high-quality and low-quality beam splitter. Both raw iSCAT images show the same area on the coverslip containing some residual particles. The same iSCAT setup was used to capture both images, only the beam splitter was exchanged. Figure 7a shows the image formed on the camera by use of a thicker (5 mm), AR-coated, and wedged beam splitter. Due to the wedged design, the reflected beam from the back surface of the beam splitter is anti-parallel to the reflection arising from the front surface and is not entering the objective. No interference artefacts occur. Figure 7b shows the same field of view on the sample but this time a thinner (1 mm) planar beam splitter was used. The two reflections from front and back surfaces of the beam splitter are parallel and propagate to the camera. Interference artefacts are clearly visible.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58486/58486fig7.jpg" /> 
Figure 7: Comparison of iSCAT images produced with high- and low-quality beam splitters. (a) Resulting raw iSCAT image by use of a 5 mm thick, AR-coated, and wedged beam splitter. (b) Resulting raw iSCAT image of the same area by use of a 1 mm thick planar beam splitter. Both beam splitters have the same splitting ratio (50% reflection, 50% transmission). Interference artefacts arising from Fresnel reflections are clearly observed in the image produced with the 1 mm thick planar beam splitter. Scale bars: 2 &#181;m. <a href="https://www.jove.com/files/ftp_upload/58486/58486fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In this protocol we describe a wide-field illumination scheme for iSCAT as it is fast, easy to realize and allows for parallel sensing over a large area14. Another common approach is to use acousto-optic deflectors (AODs) and scan a confocal beam across the sample12,17. This approach avoids the need for high-quality wavefronts but is more experimentally complex than conventional wide-field imaging. Furthermore, the speed of confocal illumination is limited by that of AODs. Depending on the desired experimental parameters, either confocal or wide-field illumination schemes can, in principle, be utilized to detect single proteins secreted from living cells.
As discussed throughout the protocol, it is imperative to minimize lateral mechanical fluctuations in the sample stage of the microscope. Even nanometer deviations in the position of the sample can lead to variations in consecutive camera frames and induce significant extraneous noise in the differential image. It is therefore recommended to use a mechanically stable microscope stage and a damped optical table (step 1.1.1.) and to cover the setup with optical curtains or panels during an experiment (step 3.5.).
An active focus stabilization scheme could also be considered for long-term measurements. In this approach, a second laser is incorporated into the microscope in a total internal reflection (TIR) arrangement, and subsequently imaged onto a quadrant photodiode. Changes in the system&#39;s focus translate into lateral displacements of the TIR laser spot on the quadrant diode, which can then be used in an active feedback loop to control the z-axis of the piezo stage26. Long-term vertical drift effects are thus eliminated.
Several modifications and extensions can be applied to the presented technique to address specific experimental needs. For example, commercial microscope stage incubators are available that could readily be incorporated into the iSCAT microscope for long-term imaging of cells. Other techniques can also be implemented to complement iSCAT imaging, such as confocal or TIR fluorescence microscopies17. To adapt on the system under study, iSCAT secretion measurements can be carried out in other cell media such as DMEM or DPBS, however, the pH indicator phenol red should be avoided as it can disturb the experiment due to absorption of the laser light. Additionally, supplements like fetal calf serum (FCS) or human platelet lysate (hPL) contain proteins that may interfere with iSCAT detection. Depending on the desired sensitivity of the experiment, these supplements should be excluded from the microscopy medium.
iSCAT relies on an analyte&#39;s ability to scatter light&#8212;a property that is intrinsic to all proteins&#8212;and is thus inherently nonspecific. Nevertheless, some degree of specificity is possible as iSCAT signals scale linearly with protein mass14,27,28. This allows for the calibration of an iSCAT system using standard protein samples, such as bovine serum albumin (BSA) and fibrinogen14,27,28. In fact, very recently, Young et al.28 have extended on the work of Piliarik &#38; Sandoghdar14 and have shown that iSCAT can be used to determine the molecular weight of proteins as small as streptavidin (53 kDa) with a mass resolution of 19 kDa and an accuracy of about 5 kDa. Several conventional approaches can further complement iSCAT by providing an extra level of specificity. As an example, enzyme-linked immunosorbent assays (ELISA), and/or other surface modifications, restrict protein binding events so that only the target protein is detected16.
In this protocol, we described how iSCAT microscopy can be used to investigate cellular secretions at the single protein level with subsecond temporal resolution16. The technique is general and can be implemented on any commercial or home-built microscope. In contrast to single-molecule fluorescence approaches, the method does not suffer from photobleaching or blinking effects but it still achieves single-protein sensitivity. These features make iSCAT a powerful tool in the field of biosensing and microscopy. Future applications will focus on elucidating complex cellular interactions such as immunological response to a stimulus or cellular communication.

Secreted proteins play a significant role in various physiological processes1. Because of this, they are routinely studied as a collective ensemble (proteomics) or as individual entities2,3. Proteomics traditionally investigates the entire set of proteins present in a particular biological system by way of e.g., enzyme-linked immunosorbent assays (ELISA), flow cytometry, or mass spectrometry4,5,6. Single proteins, on the other hand, are generally detected using a variety of techniques that are based on fluorescence7,8, plasmonics9,10, or cryogenic electron11 microscopies. All of these techniques use complex instruments, labeling, or both and often lack dynamics information as they only deliver long-term information about the system under study.
Here we use iSCAT12,13 microscopy to sense individual secretory proteins with sub-second temporal resolution14. Importantly, the technique detects the weak scattered signal intrinsic to every protein12,14. The amount of light that a small bioparticle scatters scales with its polarizability. Assuming that the shape of a protein can be approximated by an effective scattering sphere14,15,16, and that different proteins have very similar refractive indices, the measured signal can be directly connected to the molecular weight (MW) of the protein. The empirical calibration of iSCAT contrast versus molecular weight by reference measurements allows one to distinguish proteins of different sizes. iSCAT experiments can readily be complemented by fluorescence microscopy17,18, immunosorbent reagents, as well as fluorescent or scattering labels to allow for a specific detection of any protein of interest14,17,19.
In principle, iSCAT functions by amplifying a protein&#8217;s weak scattered light via interferometric mixing with a secondary reference wave. The detected intensity (<img alt="Equation 1" src="/files/ftp_upload/58486/58486eq1.jpg" />) in an iSCAT microscope is described by
<img alt="Equation 2" src="/files/ftp_upload/58486/58486eq2.jpg" />
where <img alt="Equation 3" src="/files/ftp_upload/58486/58486eq3.jpg" />&#160;is the incident intensity, <img alt="Equation 4" src="/files/ftp_upload/58486/58486eq4.jpg" />&#160;is a coefficient for the contribution of the reference wave, <img alt="Equation 5" src="/files/ftp_upload/58486/58486eq5.jpg" />&#160;signifies the scattering strength of the nano-object under study, and <img alt="Equation 6" src="/files/ftp_upload/58486/58486eq6.jpg" />&#160;is the phase shift between the scattered and reference waves14. Either the transmitted or back-reflected incident light is typically used as a reference wave, where in each case <img alt="Equation 7" src="/files/ftp_upload/58486/58486eq7.jpg" />&#160;accounts for the transmissivity or reflectivity of the sample chamber, respectively. The term <img alt="Equation 8" src="/files/ftp_upload/58486/58486eq8.jpg" />&#160;is proportional to the protein&#8217;s scattering cross section and can be neglected compared to the cross term. Thus, setting <img alt="Equation 9" src="/files/ftp_upload/58486/58486eq9.jpg" />&#160;for complete destructive interference, the detected light is given by <img alt="Equation 10" src="/files/ftp_upload/58486/58486eq10.jpg" />&#160;where <img alt="Equation 11" src="/files/ftp_upload/58486/58486eq11.jpg" />&#160;is the reference intensity and <img alt="Equation 12" src="/files/ftp_upload/58486/58486eq12.jpg" />&#160;is the interference intensity.
iSCAT microscopy offers an excellent method to study biological processes at the single-molecule level. As an example, we investigate Laz388 cells &#8212; an Epstein-Barr virus (EBV) transformed B lymphocyte cell line20,21 &#8212; as they secrete proteins such as IgG antibodies16. However, the method is general and can be applied to a variety of other biological systems. iSCAT is inherently unspecific and can detect any protein or nanoparticle or it can be extended with common surface functionalization methods for specific or multiplexed detection. Its simplicity and ability to be combined with other optical techniques, such as fluorescence microscopy, make iSCAT a valuable complementary tool in cell biology.

<table border="0" cellpadding="0" cellspacing="0" style="width:1081px;" width="1083">
	
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Item/Device</td>
			<td style="width:152px;"></td>
			<td style="width:161px;"></td>
			<td style="width:513px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100x / 1.46 NA objective</td>
			<td>Zeiss</td>
			<td>420792-9800-000</td>
			<td>alpha Plan Apochromat oil immersion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20x / 0.4 NA objective</td>
			<td>Leica</td>
			<td>566049</td>
			<td>N Plan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Piezo Stage</td>
			<td>PI</td>
			<td>P-517k020</td>
			<td>3-axis stage with 100x100x10&#181;m range</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diode laser (445 nm)</td>
			<td>Lasertack</td>
			<td>PD-01236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optics/Optomechanics</td>
			<td>Thorlabs/Newport</td>
			<td>-</td>
			<td>lenses, mirrors, posts, mounts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pinhole</td>
			<td>Thorlabs</td>
			<td>P30H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LED light source</td>
			<td>Thorlabs</td>
			<td>MCWHL5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shortpass filter (580 nm)</td>
			<td>Omega Optical</td>
			<td>580SP</td>
			<td>to modify the spectrum of the LED for fluorescence excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Longpass filter (500 nm)</td>
			<td>Thorlabs</td>
			<td>FEL0500</td>
			<td>to modify the spectrum of the LED for fluorescence excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70R/30T beam splitter</td>
			<td>Newport</td>
			<td>20Q20BS.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Economy beam splitter</td>
			<td>Thorlabs</td>
			<td>EBS1</td>
			<td>used for the comparison of fringe effects</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wedged plate beam splitter</td>
			<td>Thorlabs</td>
			<td>BSW26</td>
			<td>used for the comparison of fringe effects</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shortpass dichroic mirror (550 nm)</td>
			<td>Edmund Optics</td>
			<td>66249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8R/92T beam splitter</td>
			<td>Thorlabs</td>
			<td>BP108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CMOS camera</td>
			<td>Photonfocus</td>
			<td>MV1-D1024E-160-CL</td>
			<td>for iSCAT aquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CMOS cameras</td>
			<td>Mightex</td>
			<td>SCE-B013-U</td>
			<td>for bright field / fluorescence aquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Longpass filter (600 nm)</td>
			<td>Thorlabs</td>
			<td>FELH0600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td>Fujitsu Siemens&#160;</td>
			<td>-</td>
			<td>Core i7 Processor, 16 GB RAM, SSD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acquisition Software</td>
			<td>LabVIEW</td>
			<td>-</td>
			<td>LabVIEW 2016 Suite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Analysis Software</td>
			<td>Matlab</td>
			<td>-</td>
			<td>Matlab 2014 Suite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasma Cleaner</td>
			<td>Diener</td>
			<td>Diener pico</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Binder</td>
			<td>Model CB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reagent/Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 medium</td>
			<td>Gibco</td>
			<td>11835063</td>
			<td>without phenol red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES Buffer Solution (1M)</td>
			<td>Sigma Aldrich</td>
			<td>59205C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slides</td>
			<td>Marienfeld</td>
			<td>107052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottom culture dishes</td>
			<td>ibidi</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescent Microspheres</td>
			<td>Invitrogen</td>
			<td>F8821</td>
			<td>used for calibration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersol immersion oil</td>
			<td>Zeiss</td>
			<td>444960</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propidium iodide stain</td>
			<td>Invitrogen</td>
			<td>R37108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small pipette tips</td>
			<td>Eppendorf</td>
			<td>30075005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flexible pipette tips</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol, 99.8%</td>
			<td>Fisher Scientific</td>
			<td>E/0650DF/15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide, pellets</td>
			<td>Sigma Aldrich</td>
			<td>221465</td>
			<td>for preparing 0.2M NaOH solution</td>
		</tr>
	</tbody>
</table>

label-free imaging of single proteins secreted from living cells via iscat microscopy

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

A schematic of an iSCAT microscope is shown in Figure 4a. Representative bright-field, fluorescence, and raw iSCAT images are shown in Figure 4b, 4c, and 4d, respectively16. Figure 4e and 4f show the results of background removal and differential post processing; two adsorbed proteins are visible as diffraction-limited spots in Figure 4f. Figure 6 shows a histogram of the detected proteins over the course of 125 s. These data were obtained by applying a peak-seeking algorithm to the captured images to count the binding events and catalog their contrast16. A total number of 503 proteins were detected.
Next, secreted species are identified by comparison with reference measurements carried out on purified protein solutions, or through additional measurements with functionalized glass surfaces14,16. The iSCAT data, thus, directly visualize cellular secretion dynamics on a subsecond scale16. As an example, we have previously found that IgG antibodies are a major fraction of the Laz388 secretome and are released from the cell at a rate of ca. 100 molecules per second16. Additionally, other particles spanning a range of 100 kDa - 1000 kDa are secreted by the cells16. The described method can be further employed e.g., to investigate the spatial concentration gradient of secretions surrounding a cell16, or to determine the temporal dynamics of cellular lysis16.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58486/58486fig6.jpg" /> 
Figure 6: Quantification of secreted proteins by a single Laz388 cell. The histogram shows detected proteins during a time period of 125 s. Contrast values are accumulated in 1 x 10-4 contrast bins (blue bars). A total of 503 individual proteins were counted during this measurement. The experiment was repeated 10 times with similar results. This figure has been adapted from McDonald, M.P. et al.16. Copyright 2018 American Chemical Society. <a href="https://www.jove.com/files/ftp_upload/58486/58486fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy at JoVE.com

Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

We present a protocol for the real-time optical detection of single unlabeled proteins as they are secreted from living cells. This is based on interferometric scattering (iSCAT) microscopy, which can be applied to a variety of different biological systems and configurations.

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living ...

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