Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.
Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.
The goal of this method is to determine the variation in abundance of a target protein in a cell population with single cell resolution. Single cell analysis provides a number of benefits that are not available with traditional ensemble biochemical methods.1,2,3,4,5 Firstly, working at the single cell level can capture the rich heterogeneity of a cell population that would otherwise be lost by the averaging that occurs with traditional ensemble biochemical techniques. The majority of work-horse biochemical methods work with the bulk, requiring, as they often do, millions of cells to produce a result. Of course, the consequences of assessing entire cell populations depends on a number of factors, for example, the heterogeneity in protein expression where some important features of the distribution of protein abundance may be missed. From a practical perspective, the sensitivity required of single cell techniques make them capable of working with amounts of biological material that is insufficient for even the more sensitive bulk techniques to function. A key example of this is the study of rare cell types such as circulating tumor cells (CTCs) where even for patients with a poor prognostic outlook less than 10 CTCs might be present in a single 7.5 mL blood draw.6 Here we present the methodology required to perform single cell protein measurements using a reduced volume antibody-based assay employing oil-capped droplets printed on an antibody microarray.
Microfluidic droplet platforms are high throughput, able to generate thousands of droplets per second, and capable of isolating, and even culturing, single cells in individual droplets to perform a wide array of biochemical assays. Droplet-based techniques are well suited for single cell analysis,7,8,9 with notable recent examples including DropSeq10 and inDrop11, which have been greatly aided by the power of amplification techniques. The limited amount of material and no methods of amplification for proteins make single cell proteomics especially challenging.
Droplets may be analyzed by a number of methods and fluorescence microscopy has been widely used. Single molecule techniques such as total internal reflection fluorescence (TIRF) microscopy allows fluorescent molecules to be visualized with unparalleled signal-to-noise ratio.12 Due to the exponential decay of the evanescent field, only fluorophores in high proximity to the surface (order of 100nm) are excited making TIRF a good strategy in detecting small amounts of a target molecule in a complex mixture. The inherent optical sectioning strength of TIRF also helps to avoid wash steps and limits assay time and complexity. However, TIRF requires planar surfaces and examples of TIRF microscopy applied to droplets in flow involve the formation of a planar surface of which to image.13 To this end, single cell proteomic techniques often design microfluidic chips around surface-immobilized capture agents in a microarray format.4,14
The droplets, themselves, may be formed in arrays on planar surfaces, so-called droplet microarrays.15,16,17 Spatially organizing droplets into arrays allows them to be conveniently indexed, easily monitored over time, individually addressed and, if required, retrieved. Droplet microarrays can achieve a high density of micro-reactors with thousands of elements per chip which are either free-standing or supported by microwell structures.18,19,20 They may be formed by sequential deposition by liquid handling robots, inkjet spotters, contact microarrayers21,22,23,24,25,26 or they can self-assemble on surfaces such as superhydrophillic spots patterned on a superhydrophobic surface.27,28,29
With these considerations in mind, Addressable Droplet Microarrays (ADMs) were designed to combine the versatility, spatial addressability and reduced volumes of droplet microarrays with the sensitivity of single molecule TIRF microscopy to quantitatively measure protein abundance.5 ADMs enable single cell analysis forming a droplet microarray containing single cells over an antibody microarray, which is then capped with oil to prevent evaporation. The volumes of the droplets are discrete to prevent sample loss, which would otherwise be achieved by on-chip valving in continuous flow microfluidics.30 The absolute amount of target protein from a single cell is extremely small; however, the reduced volume of the droplets allows for relatively high local concentration in order that they are detected using a sandwich antibody assay – antibody is immobilized in a distinct region, or spot, on a surface which captures protein which in turn binds to a fluorescently labelled detection antibody present in the droplet volume. As a label-free approach (i.e. protein targets do not need to be labelled directly), ADMs are generally applicable to analyzing cells from primary sources, such as processed blood, fine need aspirates and dissociated tumor biopsies, as well as cells from culture and their lysates.
Measuring the variation in protein abundance across a cell population is important in determining the heterogeneity in response, for example, to a drug and will help in providing insight into cellular functions and pathways, assessing subpopulations and their behavior as well as identify rare events that would otherwise be masked by bulk methods. This protocol describes how to produce and use addressable droplet microarrays to quantitatively determine the abundance of the transcription factor p53 in human cancer cells and may be used to investigate the role of p53 in response to chemotherapeutic drugs. The target protein is determined by the choice of capture and detection antibodies and may be modified to include more or different targets. Instructions are provided to build a simple apparatus incorporating a concentric nozzle from general lab consumables to manually array 10 nL droplets capped with oil. The full experimental process is described whereby each droplet is then loaded with a single cell, which is then lysed and the expression of protein determined with single molecule resolution using TIRF microscopy.
1. Preparation
2. Form Addressable Droplets and Load with Single Cells
3. Data Analysis
The absolute basal protein copy number of p53 was determined with single cell resolution in a human colon cancer cell line, BE cells. We demonstrate how p53 expression can vary over several orders of magnitude and show a weakly positive correlation between cell size and protein copy number within the resting BE cell population.
Addressable Droplet Microarrays are formed when aqueous droplets are dispensed at antibody spot locations and capped with oil. Here, the droplets support a sensitive p53 protein assay. Coverslips are arrayed with capture antibody spots using a contact microarrayer and a pin. The anti-p53 capture antibody is taken from a commercial ELISA test kit. The printing conditions were such that capture spots were approximately 100 µm in diameter with a maximum capture capacity of 6.2 ± 0.5 x 105 proteins per spot.32
The use of an isolator bonded to the coverslip allows a higher density of droplets to be formed since it limits the spread of oil to nearby antibody capture spots. In each well of the isolator droplets are formed by dispensing an aqueous solution, which is then capped with oil to prevent evaporation (Figure 1A). A minimal amount of oil may be dispensed to cap the droplet; however, it is useful to dispense an amount which fills each isolator well exactly such that the oil surface is flat for imaging. Isolators are either commercially sourced or made by laser cutting acrylic sheets. Silicone isolators are available pre-cut wells in a 4 x 6 array (n = 24) but may be modified to a 4 x 11 array (n = 44) using a biopsy or multi-hole leather punch. The laser cut isolator in Figure 1B shows an array of 100 hexagonal wells each with a maximal diameter of 2.89 mm, a center-to-center separation of 3.9mm and a height of 0.5mm. The droplets may be stored and remain stable for extended periods of time (observed up to 3 months).
Droplets are created manually using a home-built apparatus, which translates a concentric tubing nozzle (Figure 1C), which is connected to two syringe pumps (Figure 1A). The concentric tubing nozzle is comprised of a fused silica capillary, which dispenses the aqueous phase, fed through a short length of PEEK tubing, which dispenses the oil phase. Using a microscope, the nozzle is aligned to an antibody spot and the pumps would first dispense the aqueous solution immediately followed by the oil (Figure 1E, steps 1-4). Once all droplets in the ADM are formed, a microinjector and micromanipulator (see the Table of Materials) are used to load each droplet with a single cell (Figure 1D). The micropipette would capture a cell from a reservoir of cells, dispensed in one of the wells on the chip, be translated to a droplet well and then penetrate the oil cap to deliver the cell into the aqueous droplet (Figure 1D, steps 5-8).
All spots within the droplets are imaged prior to lysis and will be used to determine the degree of non-specific binding to the spots (Figure 2A). Single cells are fully lysed (including nucleus) using optical methods.31 Optical lysis relies on the shearing force of an expanding laser pulse-induced cavitation bubble to disrupt the cells. Care must be made that the oil-water interface not be disturbed by these processes by limiting the pulse energies and depositing cells away from the oil/water interface. Once cells are lysed, the array is imaged by TIRF microscopy using an automated microscope; frames are acquired every 10 min for 30 min and then every 20 min for a further 60 min. The conditions, such as antibody affinity, protein diffusion and droplet volume, are such that binding equilibrium is reached within the 90 min acquisition. A typical single cell pulldown is shown in Figure 2A.
The process of image analysis to determine the single molecule counts is schematically depicted in Figure 2B. Images of spots are either considered to be non-congested, where target protein concentration is such that single molecules are well separated and easily distinguished, or congested, where target protein concentration is higher and single molecule images overlap and are no longer individually distinguishable. Since the TIRF excitation profile is uneven, it is crucial that all acquired images be flat-field corrected. A Gaussian blur is applied to a non-congested frame, which acts as a smoothing filter to remove detail and noise and produce an image with the average profile of the TIRF excitation profile. This processed image may be used to 'flatten' the original image. For non-congested images, where, even at equilibrium, there are few single molecules, a frame may be processed to correct itself. This approach is not applicable to a congested spot, where single molecules densely occupy the spot and requires a background image to be acquired for flattening. Flattened frames are then thresholded for pixels with intensities at least 3 times the background standard deviation plus its mean value. Single molecules are then automatically detected by identifying 4-9 clustered pixels with circularity greater than 0.5 using the Particle Analysis features of Fiji. From the results, the average intensity of a single molecule may be calculated. It is used to determine the number of single molecules on a congested spot by dividing the antibody spot total intensity by the known average intensity of a single molecule. These steps may be automated using Fiji's script editor.
A concentration series is performed to determine the single molecule count at equilibrium in the droplets with known concentrations of recombinant p53 protein (Figure 3A). The conditions are such that the capture antibody spots capture a fraction of the total target protein, approximately 1% in the region of linearity (for 105-108 proteins per droplet R2 = 0.99). The level of non-specific binding in the droplets is 187 ± 60 molecules. The results of the single cell pulldowns may then be converted to achieve distributions of absolute protein copy number per cell. Figure 3B shows the distribution of absolute p53 protein expression in single BE cells using ADMs. P53 protein expression in the BE cells, which may be assumed to be genetically identical or very similar, is a stochastic process. The distribution is Gamma-like – asymmetric and unimodal with a long tail. Consequently, the mean (1.82 × 106 proteins) can overestimate the modal protein abundance (bin 0 – 5.0 × 105 proteins), and the standard deviation (1.88 × 106 proteins) doesn't fully capture the features of the distribution. This distribution is an example of the importance of single cell measurements to capture the heterogeneity of protein expression in the cell population, which would otherwise be lost when averaging with bulk measurements. Additional parameters for each cell may be measured. Here, the cell volume is estimated by measuring each cell diameter prior to lysis in the droplet and assuming the cell is approximately spherical. Figure 3C shows a comparison of p53 absolute protein copy number in single BE cells as a function of cell size. There is a tendency for larger cells to have a higher total p53 copy number. Interestingly, it is possible from the distribution that there is coordination between p53 expression and cell size since there appears to be a minimum p53 copy number per cell; however, the mechanisms by which this arises requires further investigation.
Figure 1: Addressable Droplet Microarray Apparatus and Chip. (a) Droplets are dispensed manually using a 3-axis manipulator to translate a concentric tubing nozzle. The aqueous droplet is dispensed at specific locations in an antibody microarray followed by capping oil. (b) The isolator enables a higher density of addressable droplets on the substrate by limiting the spread of the oil. Isolators may be produced by laser cutting 0.5 mm acrylic sheets. To aid visualization of the droplets, blue food coloring dye is deposited using the apparatus in a). Scale bar 10 mm, inset scale bar 1 mm. (c) The concentric tubing nozzle allows addressable droplets to be formed, assembled as in step 1.2 of the protocol. (d) A microinjector and micromanipulator are used to isolate cells into individual addressable droplets, prepared as in step 1.3 of the protocol. (e) The major steps in the process of creating and loading addressable droplets is shown. An antibody spot is located using an encoded translation stage (1) where the tip of the capillary tubing is aligned (2) and the aqueous (3) then oil (4) components are dispensed. Single cells may be loaded using a glass microcapillary (5-8) which can repeatedly address the aqueous droplet (7) and deposit a cell (8). Scale bars 100 µm. The red arrow in (5) and (8) highlights the isolated single cell and the inset of (8) shows the isolated single cell at high magnification (scale bar 10 µm). Portions of this figure has been modified from reference12, reproduced by permission of The Royal Society of Chemistry. Please click here to view a larger version of this figure.
Figure 2: Image analysis steps. Each spot in the array is periodically imaged by TIRF microscopy until equilibrium (tEq) is reached (90 min for p53 assay). The equilibrium time depends on t in solution as well as the affinities of the antibodies' pair for the targeted protein. a) Example single molecule TIRF microscopy images of the background as well as an example single cell pulldown (scale bars 20 μm). Inset image shows a magnified portion of the background image with some single molecules highlighted by red arrows (scale bar 5 μm). b) Outline of image analysis detailed in step 3 of the protocol. Briefly, there are two broad regimes where the spots may be considered non-congested, single molecules are sparse, and congested, where the density of single molecules is such that there is significant overlap and may no longer be singly identifiable. A crucial step in the image analysis is field flattening to remove the effect of the non-uniform intensity profile of the excitation laser. In the digital counting regime, single molecules are identified directly, based on their intensity and shape parameters. In the analogue counting regime, the number of single molecules is calculated by measuring the total spot intensity at equilibrium and dividing by the average intensity of a single molecule, which is known from the digital counting regime. Steps may be straightforwardly automated in ImageJ to analyze the data. The bottom panel sequence of images shows accumulation of target protein on the antibody capture spot; initially, spots are not congested (t1) whereas as target protein accumulates on the antibody spot, the single molecules can become increasingly congested (tn) until equilibrium is reached (teq). Note that whether a spot remains non-congested or becomes congested depends on a number of factors, in particular the abundance of target protein in the analysis volume. Please click here to view a larger version of this figure.
Figure 3: Single cell data. Absolute quantification is achieved using a calibration curve. (a) Single molecule counts are made using known concentrations of recombinant protein. This is a calibration curve and is used to convert single molecule counted on each spot to number of target proteins in the analysis volume and hence single cell copy number. The horizontal red dashed line indicates the level of non-specific binding of 187 ± 60 molecules. The error bars are one standard deviation of the mean of 3 experimental runs. The dashed line represents 100% detection of protein. (b) A distribution of the single cell basal p53 protein expression in BE cancer cells is shown showing a long-tailed gamma-like distribution where p53 protein expression in some cells is significantly higher than the modal value. (c) Scatter plot of p53 protein copy number per cell as a function of cell volume showing how protein expression varies as a function of cell size. Portions of this figure have been modified from 12 and reproduced with permission. Please click here to view a larger version of this figure.
Addressable Droplet Microarrays are a sensitive and extensible method for quantitatively determining the absolute copy number of protein within a single cell.
Limiting the level of non-specific binding (NSB) is critical within the protocol to achieving as low a limit of detection as possible. Proteins and other biochemical species may non-specifically bind to a number of interfaces present within the droplets — the coverslip surface, the antibody spot and the oil/water interface. Proteins can be lost by partitioning into the interface or the oil itself. We have shown that 4% BSA present in the aqueous droplet is capable of limiting NSB of proteins using the antibodies specified in the protocol. Alternative protein targets and the antibodies used to detect them may require alternative blocking approaches, such non-ionic detergents or compatible surfactants. Fluorinated oils may also be tested for their suitability in serving as the capping oil. In such cases, additional considerations such as the critical micelle concentration and the density of the capping oil must be made.
The unwashed printing buffer, which remains at each spot location after arraying aids in alignment. When initially depositing the droplets, care must be taken to not scratch or damage the antibody spot by the tip of the concentric tubing nozzle. In developing the method further, a fluorescent dye may be doped into the antibody printing buffer which becomes immobilized in addition to the capture antibody. This would allow for wash and blocking steps to be performed prior to droplet arraying; however, such a step would not necessarily be required if using automated methods of droplet arraying that such as inkjet printing methods.
The surface of the commercially sourced coverslips upon which droplets are formed is coated with a hydrophilic polymer and the aqueous droplets produced upon them with a volume of 10 nL have a diameter of 751 ± 42 mm. There is a limit to how much the deposited droplet should wet the surface or spreads due to the weight of the capping oil since below a minimum thickness there is increased optical scatter from the TIRF excitation laser. The scatter increases the average level of background and can drown out signal when detecting single molecules. The antibody spot must also be located away from the droplet edge since the excitation laser light may partially or fully strike an area outside of the aqueous droplet. The critical angle condition will no longer be met for light striking the glass-oil interface as opposed to the glass-water interface.
To quantitatively determine the abundance of protein the number of single molecules bound to a spot must be determined through image analysis. To determine the total amount of protein in solution from the single molecule count a calibration curve is required and enables the technique to be absolutely quantitative.
To minimize photobleaching, spots are not imaged continuously and the TIRF excitation laser source power is minimized. The protocol as presented has been used successfully for photostable fluorophores such as the Alexa Fluor dyes. Alternative fluorophores may be used but photobleaching must be assessed and the imaging protocol adapted if necessary. The total number of single molecules per frame may be plotted against time to reproduce the binding curve although this is certainly not necessary if binding kinetics are not sought and imaging a single frame at equilibrium will suffice.
Although there is a requirement of two high affinity antibodies, this results in a highly sensitive and highly specific assay, crucial for measurements at the single cell level. The p53 antibodies used in this protocol are well optimized in detecting free p53 from single cells. The target protein is determined by the choice of antibodies and may be modified in a number of ways: 1, both the capture and detection antibodies may be replaced to bind alternative targets; 2, the detection antibody may be replaced to probe protein-protein interactions or post-translational modifications to the protein bound to the capture antibody, e.g. determine the phosphorylation status of captured p53; 3, multiplexed assays may be achieved by printing multiple capture antibody spots in close proximity — printing a 3 × 3 spot array is possible within 10nL droplets. Of course, for any change of target protein a number of antibody screens, controls and optimizations must be undertaken.
Several methods for lysing single cells have been reported.33 Optical lysis is a chemical-free approach to rapidly lyse individual cells without altering the contents of cells and maintaining the integrity of proteins and their complexes. Chemical lysis using detergents poses a problem to the integrity of the droplets when using mineral oil and can interfere with interactions between molecules.34 However, for assays where chemical lysis is compatible it is attractive approach since it is not reliant on equipment such as lasers or micropatterned electrodes.35
The droplets show comparable performance with alternative single cell methods. In the current generation of droplets using the suggested p53 antibodies, the level of NSB is 187 ± 60 molecules per spot. The single molecule counts exhibit strong linearity (R2 = 0.99) in the region spanning 105–108 recombinant p53 proteins per droplet. This suggests that while lower levels of protein may be detected in cells, there is a limit of quantitation of 105 p53 proteins; this corresponds to a single molecule count on the spots of 901 ± 83. This is predominantly limited by the volume of the droplets and adsorption to the interface. The performance of the p53 assay is such that when performed in a 10 nL volume only 1.1 ± 0.2% of the total target protein is captured from solution; this increases to 85 ± 9% when reducing the analysis volume to 0.2 nL.36 By reducing the volume of the droplets to below 1nL and reducing adsorption, there is potential using the specified antibodies to p53 in improving the limit of detection to <50 proteins per cell. Therefore, addressable droplets have the potential to be used to measure very low abundance proteins from single cells.
The addressability of the droplets afforded by the easily penetrable oil cap permits the cell analysis volume to be sequentially challenged. The use of a micropipette enables the injection of a desired number of cells into each droplet in a plug of 10-20 pL, which does not significantly increase the volume. For the chip shown in Figure 1B with 100 wells, to form droplets and load them with single cells typically takes 75-90 min. Loading cells may also be achieved by addressing the droplet using the concentric tubing nozzle instead of the micropipette or using a solution containing cells when initially forming the droplets. The latter would be useful in limiting the number of steps in the protocol but, in both cases, the occupancy per droplet would follow a Poissonian distribution. This would limit the proportion of single cell data per chip; however, droplets with zero or multiple cells would serve as controls. An additional limitation in using the concentric nozzle to address droplets is the droplet volume would increase in 10nL increments. With the suggested modifications above this could be improved. On-chip droplet microfluidics are capable of producing droplets at high frequency and have potential in being automatically combined with ADMs to produce and manipulate droplets and sequentially deposit them in a planar array. This would certainly overcome the limitations in production of ADMs while also enabling single molecule readout of droplets.
The ability to address each individual droplet has a range of possible uses. We have demonstrated the ability to sequentially load single cells. It also permits removal of the unbound cellular material post lysis and post analysis by pipette for analysis using other methods. Examples of subsequent analysis would include quantitative real-time PCR or mass-spectrometry.
One of the current bottlenecks for laboratories wanting to take a quantitative approach to single cell protein analysis is the high technical barrier and the need for specialized equipment. With ADMs, miniaturized, high sensitivity analyses may be achieved without the need for clean room facilities to fabricate microfluidic lab-on-a-chip devices. Experiments are not limited by the design of the chip and their volume and position may be altered without the need for fabricating new masters. Certainly, there is scope for improvement in simplifying the technique and lowering the barrier to entry for single cell analysis to just a microarrayer, to print both antibody and droplet microarrays, and a fluorescence microplate reader, to image.
A number of clinical applications of single cell analysis are emerging, particularly in the treatment of cancer. Tumor heterogeneity is a major challenge to effective chemotherapy. In tumor development, mutations arise with increasing frequency and heterogeneity can result in morphological, genetic, and proteomic variability. Single cell analysis is able to resolve cellular heterogeneity within a tumor and provide a platform to help better understand and predict drug resistance and guide therapy.
The authors have nothing to disclose.
ASR designed experiments, developed protocols and analyzed data. SC and PS performed cell size experiments. ASR and OC wrote the manuscript. The authors wish to gratefully acknowledge the support of Prof. David R. Klug for providing access to equipment. The authors wish to thank the Imperial College Advanced Hackspace for access to fabrication and prototyping facilities.
Cell culture | |||
Phosphate-Buffered Saline (PBS) | Life Technologies | 10010015 | |
DMEM high glucose | Sigma | D6429 | |
Foetal Bovine Serum (FBS) | Biochrom | S0115 | |
cell culture flasks | Corning | SIAL0639 | |
Trypsin/EDTA | Biochrom | L2153 | |
Name | Company | Catalog Number | Comments |
Microarray | |||
Microcontact Arrayer | DigiLab, UK | OmniGrid Micro | |
Microcontact pin | ArrayIt, USA | 946MP2 | |
Coverslips (Nexterion) | Schott, Europe | 1098523 | Size (mm): 65.0 x 25.0; Thickness (mm) 0.17 |
p53 capture antibody | Enzo | ADI-960-070 | |
p53 detection antibody, Alexa Fluor 488 labelled | Santa Cruz | sc-126 | stock concentration 200μg/mL |
Saline-sodium citrate buffer | Gibco | 15557-044 | |
Betaine | Sigma | 61962 | |
Sodium dodecyl sulphate | Sigma | L3771 | |
384 well plate (low volume) | Sigma | CLS4511 | |
Nitrogen gas cylinder | BOC | Industrial grade, oxygen-free | |
Name | Company | Catalog Number | Comments |
Droplets | |||
Micromanipulator | Eppendorf | Patchman NP2 | |
Manual Microinjector | Eppendorf | CellTram Vario | |
Micropipette | Origio, Denmark | MBB-FP-L-0 | |
Syringe pumps | KD Scientific | KDS-210 | |
100 μL syringe | Hamilton | 81020 | Gas tight, PTFE Luer lock |
1 mL syringe | Hamilton | 81327 | Gas tight, PTFE Luer lock |
Silicone isolator | Grace Bio-Labs | JTR24R-A-0.5 | 6×4 well silicone isolator with adhesive |
Laser cutter | VersaLASE | VLS2.30 CO2 Laser 3W | for laser cutting of custom isolators |
1mm thick acrylic sheet | Weatherall-UK | Clarex Precision Sheet 001 | for laser cutting of custom isolators |
Adhesive sheet | 3M | used to adhere custom isolators to microarrayed coverslips | |
Super glue | Loctite | LOCPFG3T | |
150 μm ID/360 μm OD fused silica tubing | IDEX | FS-115 | |
1.0 mm ID/1/16” OD PFA tubing | IDEX | 1503 | |
0.014” ID/0.062” OD PTFE tubing | Kinesis | 008T16-100 | |
1.0 mm ID/2.0 mm OD FEP tubing | IDEX | 1673 | |
Bovine Serum Albumen (BSA) | Fisher Scientific | BP9700100 | |
Mineral oil | Sigma | M5904 | |
Ultra-pure water | Millipore, Germany | MilliQ | |
Name | Company | Catalog Number | Comments |
Microscopy & Optics | |||
TIRF microscope with encoded XY stage | Nikon, Japan | Nikon Ti-E | |
EM-CCD | Andor Technologies, Ireland | IXON DU-897E | |
Laser excitation source | Vortran, USA | Stradus 488-50 | |
Optical lysis laser source | Continuum, USA | Surelite SLI-10 | |
Microscope filter cube for TIRF | Chroma, USA | z488bp | |
Microscope filter cube for Optical Lysis | Laser 2000, UK | LPD01-532R-25 | |
Name | Company | Catalog Number | Comments |
Software | |||
Fiji | Open Source | Image analysis software | |
Matlab | Mathworks | version 7.14 or higher | Image analysis software |