Published: November 20th, 2018
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 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.
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’s weak scattered light via interferometric mixing with a secondary reference wave. The detected intensity () in an iSCAT microscope is described by
where is the incident intensity, is a coefficient for the contribution of the reference wave, signifies the scattering strength of the nano-object under study, and 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 accounts for the transmissivity or reflectivity of the sample chamber, respectively. The term is proportional to the protein’s scattering cross section and can be neglected compared to the cross term. Thus, setting for complete destructive interference, the detected light is given by where is the reference intensity and 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 — an Epstein-Barr virus (EBV) transformed B lymphocyte cell line20,21 — 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.
CAUTION: Please read all relevant material safety data sheets (MSDS) before using any chemicals, observe all appropriate safety practices, and wear personal protective equipment (laser safety goggles, eye protection, gloves, laboratory coats) as needed.
1. Building the iSCAT Microscope16,18
NOTE: The iSCAT microscope typically consists of a modified inverted microscope setup. In brief, a laser is focused onto the back focal plane of a high numerical aperture (NA) objective and an imaging lens is used to focus the particle’s back-scattered light onto a camera chip. In general, this wide-field microscope can be built from scratch or based on an existing inverted microscope. This protocol covers the essential steps to realize the setup, while changes in the used hardware are possible. A more detailed description of the assembly of an iSCAT microscope can be found in the work of Arroyo et al.18.
CAUTION: An iSCAT microscope involves a Class IIIB to Class IV laser light source. Appropriate eye protection is necessary when assembling and aligning the microscope optics. During the microscope assembly, make sure that the laser beam path remains straight and is not deflected as new optical components are added.
Figure 1: iSCAT sample stage. The photograph shows the massive aluminum block on which the piezo translation unit (black) is mounted as well as the centered 100x objective. The 3-axis piezo stage allows for precise positioning of the sample in the focal plane of the objective. Coarse focusing is carried out by rotating a threaded tube on which the objective is mounted (not depicted). The block is positioned on the optical table with four steel pedestals above the 45° coupling mirror. Please click here to view a larger version of this figure.
Figure 2: Coarse focusing of the iSCAT microscope. The schematic shows the arrangement of the optics to help bring the system into focus. The AR-coated back side of the beam splitter (70/30 BS) is marked in red. Important distances are provided in green. The focal lengths (f) of the lenses used are denoted. Components in the blue dashed box are added in steps 1.2.6 - 1.2.7. The concave lens (used to re-collimate the converging iSCAT beam) and the screen are removed later. Please click here to view a larger version of this figure.
Figure 3: iSCAT microscope. The schematic shows the completely assembled iSCAT microscope. The AR-coated back side of the beam splitter (70/30 BS) is marked in red. Important distances are provided in green. The focal lengths (f) of the lenses used are denoted. Please click here to view a larger version of this figure.
Figure 4: iSCAT microscopy of proteins secreted by single cells. (a) Schematic of the microscope described in the protocol. See section 1 for more information. Abbreviations: LED, light-emitting diode; SPF, short-pass filter; obj, objective; SPDM, short-pass dichroic mirror; BS, beam splitter; LPF, long-pass filter; BF, bright-field; fluor, fluorescence; C1-C3, camera 1-3. (b) Bright-field image of a single Laz388 cell about 4 µm away from the iSCAT field of view (depicted by a white square). Image taken by camera C3, scale bar: 10 µm. (c) Fluorescence image of the same region shown in (b) with the position of the cell marked by a white circle. The absence of fluorescence indicates that the cell is viable. Image taken by camera C2, scale bar: 10 µm. (d) Raw iSCAT camera image snapshot with 80 µs exposure time. Image taken by camera C1. (e) iSCAT image of the same region after spatiotemporal background subtraction as described in the discussion section. The image was integrated over 1000 sequential raw frames (d) with a final frame time of 400 ms and reveals the surface roughness of the glass coverslip. (f) Corresponding differential iSCAT image that shows the binding event of 2 proteins onto the coverslip. The image was constructed by subtracting two consecutive filtered images (e). Scale bars in (d), (e), and (f): 1 µm. This figure has been adapted from McDonald, M.P. et al.16. Copyright 2018 American Chemical Society. Please click here to view a larger version of this figure.
2. Preparation of the Experiment
Figure 5: Custom-built sample holder. (a) Sample holder components: (1) acrylic cuvette dish; (2) aluminum base plate; (3) holding screws; (4) silicone O-ring; (5) coverslip. (b) Fully assembled sample holder. Please click here to view a larger version of this figure.
3. iSCAT Microscopy of Secreting Cells
4. Data Analysis
NOTE: Experimental data is inherently noisy, and iSCAT images are no different. There are several sources of noise in a typical iSCAT measurement, including wavefront distortions in the incident light source, surface roughness of the coverslip, and camera noise. The section below presents some ways in which these noise sources are remedied via post processing. Additionally, lateral mechanical instabilities of the setup lead to noisy data and must be addressed accordingly, as described in the discussion section below. The described analyses are performed with custom MATLAB scripts.
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.
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. Please click here to view a larger version of this figure.
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'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.
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 µm. Please click here to view a larger version of this figure.
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'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's ability to scatter light—a property that is intrinsic to all proteins—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 & 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.
The authors have nothing to disclose.
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 Universitätsklinikum Erlangen for providing Laz388 cells and for useful discussions. We thank Simone Ihloff and Maksim Schwab at MPL for technical support.
|100x / 1.46 NA objective
|alpha Plan Apochromat oil immersion
|20x / 0.4 NA objective
|3-axis stage with 100x100x10µm range
|Diode laser (445 nm)
|lenses, mirrors, posts, mounts
|LED light source
|Shortpass filter (580 nm)
|to modify the spectrum of the LED for fluorescence excitation
|Longpass filter (500 nm)
|to modify the spectrum of the LED for fluorescence excitation
|70R/30T beam splitter
|Economy beam splitter
|used for the comparison of fringe effects
|Wedged plate beam splitter
|used for the comparison of fringe effects
|Shortpass dichroic mirror (550 nm)
|8R/92T beam splitter
|for iSCAT aquisition
|for bright field / fluorescence aquisition
|Longpass filter (600 nm)
|Core i7 Processor, 16 GB RAM, SSD
|LabVIEW 2016 Suite
|Matlab 2014 Suite
|RPMI 1640 medium
|without phenol red
|HEPES Buffer Solution (1M)
|Glass bottom culture dishes
|used for calibration
|Immersol immersion oil
|Propidium iodide stain
|Small pipette tips
|Flexible pipette tips
|Sodium hydroxide, pellets
|for preparing 0.2M NaOH solution
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