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.

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

label-free-imaging-single-proteins-secreted-from-living-cells-via

Research

JoVE Journal

Engineering

16.9K Views.  Max Planck Institute for the Science of Light (MPL). We presenteerden iSCAT voor het eerst in 2004 in het kader van detectie en spectroscopie van gouden nanodeeltjes. In de 10 jaar die volgden hebben we deze techniek verder ontwikkeld voor het opsporen en volgen van biologische nanodeeltjes zoals virussen en kleine eiwitten. De essentie van de techniek is dat elk materieel object, hoe klein ook, een eindige uitstervingsdoorsnede heeft.Het belangrijkste voordeel van deze techniek is de labelvrije detectie. Dit betekent dat als we gevoelig...

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

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Bioengineering

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Biology

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Enhancing Protein Visualization with Antibody-Mediated Live Imaging

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

Author Spotlight: Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

Developmental Biology

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Immunology and Infection

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

Live Cell Single-Molecule Imaging-Based Quantification of Protein Interactions

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

Author Spotlight: Evaluation of Protein-Condensate Dynamics in Live Human Cells

Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells.

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number ...

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

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