1Physics Department, Boston University, 2Department of Biomedical Engineering, Boston University
Di Fiori, N., Meller, A. Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules. J. Vis. Exp. (33), e1542, doi:10.3791/1542 (2009).
1 - Assembling the microfluidic cell
The microfluidic cell is made by fusing a patterned, 2 mm thick, polydimethylsiloxane (PDMS) disk to a freshly cleaned quartz cover slip. The PDMS disk contains four 30 μl rectangular chambers that are accessed by inlet and outlet flexible capillaries connected to a buffer reservoir and a syringe pump, respectively, for the controlled flow of buffer. The ﬂow cell is supported on its back side by a glass window and placed on a custom-made cell holder containing a water cooled thermoelectric element to regulate temperature.
* Piranha is a solution of H2O2 and H2SO4 in 1:2.5 proportions. To clean the cover slips, immerse them in piranha at 90° C for 20 min.
2 - Activating the surface for molecule immobilization
For nucleic acid studies, the simplest surface immobilization scheme consists of first coating a glass or quartz cover slip with biotinylated bovine serum albumin (BSA) and then with streptavidin. This allows the biotinylated sample to be immobilized with high specificity for days at room temperature.
The microfluidic cell is mounted on a motorized x-y stage allowing coarse (up to 25 mm) motion using optically encoded DC motors, and ﬁne (1 nm) motion using two closed-loop piezo actuators (Physik Instrumente model M-014 or similar). This can be done either before or after activating the surface for sample immobilization.
3 - Preparing the Imaging Buffer (IB)
The IB consists of an enzymatic oxygen scavenging system and a triplet quencher, such as Trolox. Since the IB is flushed through constantly overnight, at least 10 ml should be prepared in advance.
The surface scanning, molecule localization and acquisition of single molecule intensity traces is controlled by a LabView program that permits automated data collection1. The program scans 20x20 μm2 areas at 0.2 μm resolution, with a rate of 0.1 μm/ms. Data is immediately processed to yield intensity-weighted locations of all pixels above the background counts. Once the immobilized molecules are found, they are moved one by one into the confocal volume and the intensities of the donor and acceptor fluorophore are recorded as a function of time until both fluorophores photobleach. The stage is then programmed to move to a new origin 100 μm away, and the scanning process is repeated.
4 - Representative Results
Below are some images showing the assembled microfluidic cell before surface activation and after being mounted on the microscope ready for molecule immobilization. If the channel is activated successfully, surface scans should be similar to the scans depicted below showing emission of the donor dye (green) and acceptor (red) after direct donor excitation. These molecules are moved one by one into the probing volume and the fluorescence is recorded over time until both dyes photobleach, as shown in the single molecule traces. From traces like these one can obtain the FRET efficiency which informs on the instantaneous donor-acceptor separation, which in turn is used to explore the structure and dynamics of molecules of biological interest.
Figure 1: Microfluidic cell with four channels, each with inlet/outlet capillaries. Please click here to see a larger version of figure 1.
Figure 2: Cell mounted on the microscope ready for measurements. Please click here to see a larger version of figure 2.
Figure 3: Setup for sm-FRET measurements. Please click here to see a larger version of figure 3.
Figures 6, 7, 8, and 9: Intensity trajectories of donor (green) and acceptor (red) fluorophores labeled to a DNA molecule 8, 10, 16 and 18 basepairs apart. Please click to see a larger version of figure 6, figure 7,figure 8, or figure 9.
Although the above protocol has been optimized for our particular system and for a specific choice of dyes (TMR and ATTO647N), it is by no means the only proved method. Recently, an alternative oxygen scavenging system consisting of protocatechuic acid (PCA)/protocatechuate-3,4-dioxygenase (PCD) was shown to increase the photostability of certain dyes4. Likewise, other triplet-state quenchers such as β-mercaptoethanol (BME) can be used instead of Trolox, although these might not suppress long-lasting blinking of some dyes, in particular Cy55.
Although immobilization via biotin-streptavidin-biotinylated bovine serum albumin (BSA) is the preferred choice for nucleic acids studies, a polyethylene glycol (PEG)-coated surface is better suited for studies of proteins since it prevents non-specific adhesion1. Additionally, sub-diffraction-limited size vesicles can be used to encapsulate the biomolecule of interest in cases where it can be affected by surface interactions6.
In this article we have used a confocal setup equipped with silicone avalanche photodiodes to acquire the fluorescence intensity traces. This protocol works equally well with Total Internal Reflection Fluorescence microscopes (TIRF) equipped with CCD cameras. Regardless of the microscopy used, sm-FRET can inform on spatial resolutions approaching the angstrom when the donor and acceptor signal are properly corrected7.
We thank Chandran Sabanayagam for his help in developing the system, Joshua Edel for advice on designing the microfluidic device and the staff at the Rowland Institute at Harvard University for machining parts of the setup. We acknowledge useful discussions with members of Meller’s group at the Rowland Institute and at Boston University, and members of T. Ha’s group at UIUC for helpful information. Finally, we appreciate the financial support from the National Science Foundation (PHY-0701207) and the National Institute of Health (GM075893).
|Fused silica capillary tubing||Polymicro Technologies||TSP150375|
|Fused silica cover slip||Esco Products||R425000|
|Streptavidin||Thermo Fisher Scientific, Inc.||0021122|
|Sylgard 184 Elastomer Kit||Dow Corning||3097366-1004||Silicone Elastomer Kit|
|Kwik-Cast Sealant||World Precision Instruments, Inc.||Kwik-Cast||Silicone Casting Compound|