Visualizing Single-Cell Secretion Dynamics with Single-Protein

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Visualizing single-cell secretion dynamics with single protein sensitivity Matthew McDonald, André Gemeinhardt, Katharina König, Marek Piliarik, Stefanie Schaffer, Simon Völkl, Michael Aigner, Andreas Mackensen, and Vahid Sandoghdar Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b04494 • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 12, 2017

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Visualizing single-cell secretion dynamics with single protein sensitivity Matthew P. McDonald,1 André Gemeinhardt,1 Katharina König1,2 Marek Piliarik,3 Stefanie Schaffer,4 Simon Völkl,4 Michael Aigner,4 Andreas Mackensen,4 & Vahid Sandoghdar1,2* 1

Nano-optics Division, Max Planck Institute for the Science of Light, Staudtstraße 2, 91058 Erlangen, Germany; 2Department of Physics, Friedrich Alexander University Erlangen-Nuremberg, Schloßplatz 4, 91054 Erlangen, Germany; 3Current address: Institute of Photonics and Electronics (ASCR), Chaberská 57, 18251 Prague, Czech Republic; 4Department of Internal Medicine 5 - Hematology and Oncology, Friedrich Alexander University Erlangen-Nuremberg, Ulmenweg 18, 91054 Erlangen, Germany *To whom correspondence should be addressed. Email: [email protected] Phone: +49 9131 7133 300

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ABSTRACT: Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT), and is demonstrated with Laz388 cells—an Epstein-Barr virus (EBV) transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa – 1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general, and as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single protein sensitivity. KEYWORDS: iSCAT; label-free; single-protein; cellular secretion; dynamics; imaging

A great number of biological processes are mediated by proteins, and indeed, the field of proteomics has emerged in an effort to identify and characterize the entire set of proteins that a system produces so as to grasp its underlying biological mechanisms more fully.1,2,3 Proteomics research is traditionally conducted using methods that are based on either immunoassays, mass spectrometry (MS), or flow cytometry (FCM).1,2,4 Immunoassays, such as enzyme-linked immunosorbent assays (ELISA) and western blotting feature inherent specificity but are currently only practical for the detection of known (or suspected) analytes as each protein requires a specific immunological complement.3 MS-based techniques, on the other hand, can detect any number of distinct proteins in a sample with the added caveat that the sample must be isotopically labeled and/or separated before analysis.1 Flow cytometry is advantageous due to its processing speed (millions of cells per minute), 5 in vivo applicability,5 and live cell sorting capabilities,4 but still largely relies on antibody-linked fluorescent labeling.5 A particularly interesting and challenging application of proteomics focuses on the proteins that are secreted from cells into the extracellular space. Such species are responsible for a host of crucial cellular functions, such as intercellular communication,6 migration,7 wound healing,8 and immunological response.9,10 All cells secrete proteins and other biomolecules in an unregulated manner (constitutive secretion), while only a select few cell types are known to have regulated secretion pathways that are triggered by an external stimulus.11 In general, the secretome is measured via the proteomic techniques mentioned above with the added stipulation that only the extracellular medium is analyzed, in contrast to pure proteomic assays wherein the whole lysate is quantified. A typical measurement entails incubating a 2 ACS Paragon Plus Environment

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large population of cells (106 per ml of culture medium) for several hours, separating the supernatant from the cellular mass and other contaminates, and finally, analyzing the specimen with either MS or an immunoassay. Each of these methods requires further processing, tagging, and separations before the sample can be fully analyzed. Furthermore, since the total secreted mass of a particular protein is generally small (µg to ng/106cells/24hr), samples are susceptible to contamination from the culture medium and/or cellular lysates. All of these factors contribute to time resolutions of several hours, thus, severely limiting the understanding of secretion dynamics—a particularly important aspect of immunological response. Single cell secretomics and proteomics studies have emerged as a way to go beyond some of the limitations of ensemble measurements.12 In the process, it has been discovered that a large degree of heterogeneity exists within a seemingly homogeneous cell population. For example, even though conventional consensus suggests that protein secretion is strictly regulated,13,14 several recent reports have shown that this is not the case at the single cell level.15,16,17,18 However, none of the existing methods can achieve single-protein sensitivity at millisecond temporal resolution free of the need for labeling. This is what we demonstrate in this work. We have recently shown that the interferometric detection of scattered light (iSCAT)19,20 is capable of sensing single small proteins with sub-second temporal resolutions.21 iSCAT works by mixing the weak field scattered by a protein with a strong coherent reference beam.19,21 Importantly, the method is labelfree and does not require immunosorbent reagents or fluorescent probes although either of these can be implemented to complement iSCAT detection.21,22,23 As a proof of concept for the application of iSCAT imaging in label-free investigation of secretion dynamics, in this work we probe the secretome of single Laz388 cells derived from B lymphocytes and immortalized with Epstein-Barr virus (EBV).24,25 EBVtransformed B cells are known to produce IgG antibodies and their pentamer aggregates (IgM).26,27,28 However, to date IgG/IgM production has not been explicitly shown for the Laz388 cell line. Figure 1a,b shows the schematics of the iSCAT optical setup and the arrangement of the detection field of view (FOV) with respect to the cell under study. Our measurement scheme is based on interrogating the region close to a cell placed on a coverglass. A diode laser beam (wavelength λ = 450 nm) focused to the back focal plane of a high numerical aperture (NA) objective generates a plane wave at the objective’s forward focus with a diameter of ca. 6 µm. The beam is directed through the coverglass and into the sample chamber under aqueous conditions. The plane wave reflected from the fluid/glass interface at the forward focus is collected back through the same objective, split with a beam splitter, and re-collimated by a second singlet lens onto a CMOS (complementary metal-oxide-semiconductor) camera chip. The spherical wave produced by a scattering protein is also collected through the objective and subsequently focused onto the CMOS chip by the same lens (see the Experimental Methods section for 3 ACS Paragon Plus Environment

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more details). In this way, the reference beam (reflected plane wave) superimposed with the scattering field is imaged on the CMOS camera. The scattered and reference beams interfere due to their coherent nature, yielding an image, where individual proteins appear as diffraction-limited shadows (Figure 1c). The depth, or darkness, of such a shadow is defined as the iSCAT contrast and indicates the amplitude of the electromagnetic field that a protein scatters.19,20,21

Figure 1. iSCAT microscopy of an individual cell’s secretome. a) Schematic of the experimental optical setup. See Experimental Methods section for more details. Abbreviations: BF, bright-field; lo mag., low magnification; hi mag., high magnification; BS, beam splitter; LPF, long-pass filter; SPDM, short-pass dichroic mirror; obj., objective; fluor., fluorescence; LED, light emitting diode; C1-4, camera 1-4. b) Cartoon of the detection region. c). Spatiotemporally filtered image (top) and a corresponding differential iSCAT image (bottom). Images were integrated over 1000 sequential frames with a total image time of 400 ms. All scale bars 1 µm.


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The total optical power ( ) hitting the CMOS chip can be written as = + + ,

where , , and are the reference, scattered, and interference powers, respectively.21 For objects

with very small scattering cross sections, fully dominates so that the detection contrast can be

described as ( − )/ ≅ / . For small proteins, this amounts to a contrast less than 10-3,

which is usually smaller than the residual contrast arising from the variations of the refractive index or topography on the glass substrate (see Figure 1c, top). To visualize the arrival of new proteins, subsequent video frames are subtracted from each other resulting in an image shown in Figure 1c (bottom). As shown in Figure 1a, we augment our iSCAT microscope with low-magnification bright-field imaging capability from the opposite side in order to view the whole cell. In our experiment, we inject about 100 – 200 washed cells into a sample cuvette composed of a plastic dish, which is clamped to a plasma-cleaned microscope coverglass (Figure 1a). After the cells are allowed to settle to the bottom of the sample chamber, a healthy cell is selected, positioned close to the iSCAT FOV with piezoelectric positioners and is monitored via iSCAT, fluorescence, and bright-field microscopies (see Figure 1a). To monitor the viability of the cells under study, we also add propidium iodide (PI). Because PI fluoresces upon binding to DNA, a fluorescent cell is an indicator of PI leakage through a dysfunctional membrane.29 Figure 2a shows a bright-field image of a single Laz388 cell positioned about 4 µm away from the iSCAT laser beam (FOV marked by a white square on the image). The cell is viable as indicated by the absence of fluorescence signal from the PI viability stain (Figure 2a, inset). A corresponding iSCAT image is displayed in Figure 2b where several particles are observed as they adsorb to the coverglass. The acquisition time is 400 ms, and the image was acquired within minutes of injecting the cell into the sample cuvette. Figure 2c presents cross sections of the detected spots with peak iSCAT contrasts in the range of 3.5×10-4 – 3.6×10-3. Assuming that different proteins have very similar indices of refraction and neglecting shape factors in the electrostatic polarizability of proteins, we can relate the iSCAT contrast to the molecular weight (MW) of the detected particle. We find the detected MWs to be in the range of 100 – 1100 kDa as determined from an empirical calibration of iSCAT contrast versus molecular weight.21 We note that a single IgG antibody with an effective scattering radius of about 4 nm (Ref. 30) and MW ~ 150 kDa has an estimated iSCAT contrast of about 8×10-4 in our setup (more details of this estimate and analysis can be found in the Supporting Information, SI).


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Figure 2. In vitro secretomics with single protein sensitivity. a) Bright-field image of a single Laz388 cell about 4 µm away from the iSCAT FOV (indicated by a white square). The corresponding fluorescence image is shown in the inset, with a white line indicating the cell’s position. FL: Fluorescence; BF: bright-field. Scale bar 10 µm. b) iSCAT snapshot with a frame time of 400 ms. Scale bar 1 µm. c) Cross sections of detected iSCAT spots marked in (b). To obtain an overview of the distribution of the molecular weights of the secreted particles, we recorded iSCAT images from a single cell over a period of 125 s. These movies were then scrutinized frame by frame with an analysis algorithm that searches for contrast dips in each iSCAT image. Figure 3a shows a contrast histogram of more than 500 proteins that were detected from a Laz388 cell over the course of 125 s (spaced ~4 µm from FOV; diagonal-hatched blue bars). For comparison, we superimpose the contrast distribution of a purified IgG sample reported in Ref. 21 (diagonal-hatched rose bars). The strong correlation with the lower edge of the secreted protein distribution provides a first indication that we detect IgG antibodies actively secreted by a Laz388 cell.


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Figure 3. Secretome quantification and identification. a) Histogram of detected proteins during a 125 s long measurement period where detected contrasts are counted in bins of 1×10-4 contrast (diagonalhatched blue bars). 503 distinct proteins were counted in this period. The red arrow indicates the expected contrast corresponding to an IgG dimer. Superimposed over the cell secretion data is the contrast distribution of a purified IgG solution injected into the iSCAT FOV with a micropipette (diagonalhatched rose bars, normalized for clarity).21 The detected secretion events from a single Laz388 cell on an anti-human IgG-functionalized coverglass are also shown (125 s integration, hollow black bars). The antihuman IgG surface selectively binds all four IgG antibody subtypes and resists the adsorption of other proteins. This measurement was replicated 10 times with similar results. b) Comparison of 2.5 fps (400 ms) and 25 fps (40 ms) image acquisitions of a single Laz388 cell’s secretions. The right column shows three sequential 40 ms images, while the left displays the 400 ms image composed of ten total images, including the three shown on the right. Scale bar 1 µm. It should be noted that continuously acquiring data at 25 fps is indeed possible throughout the entirety of a typical 30 – 90 minute measurement, however it was impractical in our current setup as the produced data volume is significant. To confirm the secretion of IgG, we performed experiments using an anti-human IgG-functionalized coverglass to avoid the detection of other proteins. In this scheme, the surface selectively binds IgG antibodies released by the cell and discriminates against the adsorption of other proteins to the surface.21


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The hollow black bars in Fig. 3a present the contrast histogram of secretions from a single Laz388 cell over the course of 125 s utilizing an anti-human IgG-functionalized coverglass. We find that the large particles detected on a bare coverglass (diagonal-hatched blue bars) are greatly suppressed, and a single peak at IgG’s expected contrast is evident, in excellent agreement with the measurements on purified IgG samples (diagonal-hatched rose bars). This confirms that IgG is actively secreted by Laz388 cells. As described in the Supporting Information, we have also corroborated this finding with an immunoglobulin ELISA measurement of the Laz388 cell culture medium. The extension of the histogram towards higher iSCAT contrast points to the secretion of more massive particles. In particular, the peak at ~ 1.5×10-3 (marked by a red arrow) could originate from a dimer species with MW of 300 kDa. Larger particles and proteins ( / ~ 2.5 – 5×10-3) represent the smallest fraction of secreted biomass, and are likely due to larger oligomers or small exosomes. We remark that EBV-transformed cells are also known to produce viral proteins31 although this has not been explicitly reported for Laz388 cells in the literature. This suggests that many of the detected adsorption events might be EBV-related. Another possible explanation for large contrasts might be that many smaller proteins land in the same diffraction-limited spot on the coverglass within the 400 ms imaging window. To examine this, we synchronously collected both 400 ms (2.5 fps) and 40 ms (25 fps) images of a Laz388 cell’s secretions over the course of a few minutes. Figure 3b shows a 400 ms image along with three of the sequential 40 ms counterpart images on the right. A single adsorption event is observed in both the 25 fps and the 2.5 fps images (indicated with a red arrow), and spans only one 40 ms differential frame. The adsorbed protein is invisible in the third 40 ms frame due to the differential nature of the imaging scheme (see Figure 1). Such observations suggest that the large contrasts observed in 400 ms iSCAT images are, indeed, due to single particle adsorption events that stem from larger secretomes.


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Figure 4. Real-time secretion dynamics of single Laz388 cells. a) iSCAT contrast histograms of a single Laz388 cell positioned ~5 µm from the FOV over the course of ~90 minutes. The color indicates total protein count. The cell is in pure RPMI 1640 medium (without HEPES pH stabilizer). Each vertical pixel line represents a ~140 s histogram of the temporal data. The false-color count scale is increased after 68 min (the break is indicated by a dashed red line) due to the enormous efflux of proteins after cell death. b) Bright-field and fluorescence (inset) images of the cell at different stages of the acquisition period. Scale bar 10 µm. c) Average fluorescence signal emitted by the cell throughout the acquisition period (red dashed line) compared with the pH of an identical aliquot of RPMI 1640 medium throughout the acquisition period (blue dots). In addition to its exquisite sensitivity, an important advantage of our method is its ability to monitor secretion in real time. In Figure 4, we present the secretion dynamics of a Laz388 cell, where iSCAT images of the cell’s secretome were collected every 400 ms for approximately 1.5 hours. A side-by-side movie of the iSCAT, fluorescence, and bright-field microscopies is provided in the SI. Histograms of detected proteins are displayed in the false-color map in Figure 4a. Here, each horizontal pixel represents 9 ACS Paragon Plus Environment

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a ~140 s binning window, and the color denotes the number of detected protein binding events within a specific contrast bin. The cell is observed to secrete proteins actively for about 20 minutes, at which point the secretions drastically slow. In tandem, the cell slightly expands in the bright-field image as shown in Figure 4b, hinting that the cell is under external stress although at this point the cell does not show any signal from the PI stain (Figures 4b, c). Such cellular swelling in the absence of membrane permeability is associated with stage 1 oncosis, or cellular swelling due to external injury.32 In other words, the cell is viable and its membrane is not compromised. The stress is due to the medium slowly becoming more alkaline in ambient conditions. This is shown in Figure 4c where the pH of an identical RPMI aliquot in a separate cuvette was measured. We see that the pH increases above 8.5 after 80 min in ambient conditions. Usually, this alkalization is arrested by use of a buffering agent (such as HEPES),33 but it is not included here to induce stress and cell death in Laz388 cells. This second phase lasts approximately 50 min during which the pH increases from approximately 8.1 to 8.4 (Figure 4c). The third phase is marked by an intense fluorescence signal from the PI stain (Figure 4c) and an enormous burst of activity in the iSCAT signal. The swelling, catastrophic rupture, and death after 70 min in the experimental chamber are indicative of an “ischemic cell death” via oncosis as opposed to apoptosis. 34 We will return to this phenomenon below. An important aspect of iSCAT’s capabilities is its ability to sense the spatial concentration gradient induced by cellular secretions and thereby ascertain a cell’s inherent secretion rate ( ). The concentration gradient induced by a constantly secreting cell can be described by

(,)

(,)

=

+

(,) −

(, ) where (, ) is the concentration at a distance and time , is the diffusion constant of the protein of interest, and is the rate of particle adsorption to the coverglass.7 This gradient can be related to the iSCAT measurement with the flux of particles () per unit time at a distance ( − ) from the surface of a cell with radius , which is given by

() = Assuming = 39 µm2 s-1 and

! √ + #

4% ! √ + #

&

) '( (+',) * .

Eq. (1)

= 10 µm (see SI for more information and details of the derivation), the

parameter can be extracted by fitting spatially dependent iSCAT data with Eq. (1) .


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Figure 5. IgG secretion rates and secretion heterogeneity. An example of IgG flux () measurement as a function of distance ( − ) from a Laz388 cell’s surface (black squares). A cartoon of the measurement scheme is shown in the inset. Here, the concentration of proteins (green dots) is shown to diminish with increasing . An iSCAT FOV is illustrated with a red square. The flux was measured in 3 µm increments of r at 10 total positions with 30 s integration per location. The dashed curve plots a theoretical fit, allowing one to extract the value of = 62 IgG s-1. The measurement was replicated 9 times, giving a mean of 91 ± 85 IgG s-1 (see Table S1 for individual values, standard deviation reported). Figure 5 shows an example of the measured iSCAT flux of IgG as a function of distance to an individual Laz388 cell (black squares). The scheme for the measurement is shown as an inset. Here, we placed the iSCAT FOV at different separations from the cell surface whereupon detected IgG adsorption events (contrasts between 6×10-4 and 1×10-3) were counted over a 30 s integration period and converted into () by dividing the total number of events by a factor of (30 s × 25 µm2). This was repeated at 3 µm

increments extending radially outward from the cell for a total of ten () measurements. To account for

residual flow and convection in the cuvette, this measurement was repeated in the four cardinal directions and finally averaged into one () trace. We remark that in our measurements on many different cells, we did not observe any dependence on the orientation of the cell so that here we assume a radially symmetric secretome concentration gradient. We emphasize, however, that our methodology is ideally suited for investigating secretion anisotropy in other systems.35 By fitting the data shown in Figure 5 with Eq. (1), we extract a value of 62 IgG s-1. Repeating

such measurements on eight other cells yielded values ranging from 2 to 200 with an average value of 91 ± 85 IgG s-1 (see SI). These data are in good agreement with typical values of 80 ± 40 IgG s-1

measured in large populations of other EBV-transformed lymphoblastoid cell lines (2 ± 1


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µg/ml/106cells/24hr)36 but clearly reveal the large heterogeneity of individual cells. In the SI, we also report on commercial ELISA kit measurements on large cell populations.

Figure 6. Single cell lysate analysis. a) Two dimensional histogram of a single Laz388 cell’s leakage during cell death. Each vertical line of pixels represents approximately 18 s. b) Histogram of the total collected lysate. Here, contrast is mapped into molecular weight (MW) on the horizontal axis using a calibration curve adapted from Ref. 21. The data in Figure 4 indicate that our methodology could also lend itself to the investigation of single cell lysis. In Figure 6, we present an iSCAT analysis of the secreted biomass throughout a cell’s ischemic death.34 As with the cell featured in Figure 4, this cell was subjected to high pH conditions for about one hour prior to the start of the measurement. Figure 6a shows that after an initial rupture at approximately 2.5 min, the cell’s contents are observed to continuously leak into the extracellular space for over 30 minutes. This sudden evacuation of cell contents, along with the swelling observed in bright-field images (see Figure 4b), strongly suggests that these cells are undergoing oncosis, or “cell death characterized by


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swelling”.34 Many different proteins, particles, and vesicles are released by the bursting cell as can be seen by the varied iSCAT contrasts observed in the total event histogram shown in Figure 6b (3×10-5 – 5×10-3). Here, every protein is counted into 2.5×10-5 contrast bins from the entire 30 minute measurement period. We can surmise from the histogram that the majority of a Laz388 cell’s biomass is in the range of 100 – 300 kDa (3×10-4 – 1.5×10-3 contrast), but the measurements also clearly demonstrate our ability to detect and calibrate larger particles, in this case up to about 1 MDa. Our work provides a glimpse into a single cell’s lysate37 and could readily be integrated into a flow-based system38 so as to fully capture and detect all lysate products with single protein sensitivity. We emphasize that iSCAT can be intrinsically extremely fast, only limited by the speed of CMOS cameras, which can currently reach about 1 MHz.22 In this work, we have used Laz388 cells to illustrate the ability of iSCAT microscopy for real-time monitoring and quantitative characterization of cellular secretion or lysis with single protein sensitivity. In our specific study, we found a single Laz388 cell to actively secrete IgG at a rate of the order of 100 molecules per second, which is in good agreement with ensemble secretion measurements from other EBV-transformed cells.36 Moreover, we showed that single Laz388 cells secrete many different particles spanning a molecular weight range of 100 kDa – 1 MDa. An interesting future development would be to combine these experiments with different separation and selection procedures, e.g. via specific surface functionalization or immunoassays,15,16,17 for multiplexed detection of different proteins in the secretome. Another promising direction is the extension of our work to a well-array system16 for improving the throughput. Compared to the established methods such as mass spectrometry1 or immunoassays3, iSCAT detection provides orders of magnitude higher temporal resolution and is suitable for real-time investigation of living, active cells with single-protein sensitivity. Notably, this method removes the need for labeling and long incubation periods. Thus, the method presented here opens doors to a new class of biomedical studies such as eavesdropping between two cells, e.g., by measuring cytokine flow through an immunological synapse or within the immediate area of a stimulated cell.

Laz388 cell culture: Laz388 cells are cultured at 37 °C and 5% CO2 in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), amino acids, pyruvate, and antibiotics. The cells are split and provided with fresh medium every 2 - 3 days. Before an experiment, medium containing ca. 1 million cells is extracted from the culture flask and washed with 10 ml room temperature RPMI 1640 and centrifuged at 300g for 7 minutes, at which point the supernatant is discarded. This washing step is repeated, and the cells are re-suspended in 500 µl RPMI 1640 and immediately used in an experiment. The cells are sourced from the Dana-Farber Cancer Institute in Boston, MA, USA and have not been authenticated 13 ACS Paragon Plus Environment

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(RRID: CVCL_GY04). They are routinely tested for mycoplasma and squirrel monkey retrovirus (SMRV) contamination by polymerase chain reaction (PCR) analysis. Both are negative.

Anti-human IgG functionalized coverglass: N-hydroxysuccinimide (NHS) coated glass coverslips were purchased from Microsurfaces Inc. and stored at -20 °C in an argon atmosphere. Monoclonal anti-human IgG antibodies were purchased from Abcam (catalog number ab181236) and stored at -20 °C. First, an NHS-coated coverglass is removed from -20 °C storage and allowed to warm to room temperature under continuous N2 flow. The antibodies are warmed to room temperature and diluted to 10 µg/ml in 200 µl sodium acetate buffer, at which point the solution is distributed on top of the NHS-coated coverglass. A wet tissue is draped over the coverglass to avoid evaporation. After 20 minutes, the coverglass is rinsed five times with 1 ml aliquots of phosphate buffer solution (PBS), making sure to never let the surface dry completely. 600 µl of deactivation buffer is then introduced onto the coverglass and allowed to sit for 30 minutes under a wet tissue. The coverglass is then rinsed five times with 1 ml PBS, and then loaded with 1 ml RPMI 1640 to be immediately used in an iSCAT experiment.

Optical setup: A diode laser beam at wavelength λ = 450 nm is spatially filtered with a 35 µm pinhole and then focused on to the back focal plane of a high-NA oil-immersion objective (100×/1.46NA) with an f = 500 mm singlet lens. This produces a collimated plane wave at the objective’s forward focus that is ~ 6 µm in diameter (fwhm). The light reflected off of the coverglass/medium interface is collected back through the same objective, split with a 70R:30T beam splitter, and imaged with a second f = 500 mm singlet lens onto a CMOS camera chip (Figure 1a, C1). The scattered light is also collected through the same objective, split with the beam splitter, and focused onto the camera chip by the f = 500 mm lens. The coherent nature of the scattered and reflected beams creates an interference pattern that is imaged by the CMOS camera. The bright-field trans-illumination source (λ ~ 560 ± 25 nm LED) is condensed onto the sample with a 20×/0.4NA objective. The reflected LED light is re-collected through the same 20x objective and focused onto a CMOS camera chip for low magnification bright-field imaging (Figure 1a, C4). The transmitted LED light is collected through the 100x/1.46NA objective, split from the iSCAT laser beam with a λ = 550 short-pass dichroic mirror (SPDM) and further split into fluorescence and bright-field channels with a 10R:90T beam splitter. Both channels are then imaged onto separate CMOS (Figure 1a, C2 and C3) chips via achromatic doublet lenses. Fluorescence and high magnification bright-field images are acquired every 16 seconds within an experiment, during which period the iSCAT laser is shuttered to ensure that it does not interfere with the fluorescence imaging. Likewise, the bright-field/fluorescence


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excitation source is shuttered during iSCAT acquisition. Imaging, shuttering, and timing are controlled by home-written LabVIEW acquisition software.

iSCAT imaging: The iSCAT CMOS camera (Figure 1a, C1) operates at a 5 kHz constant frame rate, with an integration time of 30 µs and an area of 128 × 128 pixels (5 × 5 µm). The camera is controlled by a frame grabber computer card (National Instruments), which is triggered by an external function generator operating a 50 Hz square wave. Upon receiving a TTL pulse from the function generator, the frame grabber triggers the camera to acquire 100 images at a 5 kHz frame rate. The 50 Hz square wave output of the function generator is fed into the external control port of the piezo controller. In this way, the piezoelectric positioner’s location along the y-axis is modulated between 2 positions at 50 Hz (290 nm amplitude), and the camera synchronously records 100 frames per period. This is followed by a 20 ms dead time, for a total frame time of 40 ms. This process is cycled over 1 or 10 iterations, at which point / is extracted and combined into a single iSCAT contrast image (1 cycle, 40 ms image; 10 cycles, 400 ms image). / is extracted in each frame using lock-in principles.21 In practice, this is

accomplished by averaging the 50 images associated with each piezo position within one cycle, and then subtracting one from the other. This essentially removes all aspects of the image that did not move with the piezoelectric positioner, such as wavefront non-uniformities.

Data analysis: Raw iSCAT images are filtered with a 2-dimensional (2D) Fourier filter that excludes high spatial frequencies. This attenuates effects stemming from extraneous sources, such as camera read out noise. Next, each sequential frame is normalized by subtracting its preceding frame, resulting in a rolling differential image. Finally, each frame is then subjected to a peak-seeking algorithm that finds protein bindings with a 2D Bessel function convolution and catalogues their position, contrast, and index. All statistical analyses presented herein follow from these data.

SUPPORTING INFORMATION: Supporting Information is available in the online version of this paper and includes: a movie of a Laz388 cell secretions as measured by bright-field, fluorescence, and iSCAT microscopies; iSCAT contrast of an antibody; total secretion of a single cell; ensemble ELISA measurements.

CONFLICT OF INTEREST: The authors declare no competing financial interest.

ACKNOWLEDGEMENTS: 15 ACS Paragon Plus Environment

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This work was supported by the Max Planck Society, an Alexander-von-Humboldt Professorship and the Deutsche Forschungsgemeinschaft (CRC 1181). We thank Simone Ihloff and Maksim Schwab for technical support.

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Visualizing Single-Cell Secretion Dynamics with Single-Protein

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