Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Summary

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

Abstract

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Introduction

The ability to visualize protein-DNA interactions at the single-molecule level and in real-time has provided significant insight into genome stability^1,2. In addition to working with single molecules of DNA one at a time, the ability to view transactions between individual molecules nearby provides additional insight^3,4,5. The manipulation of additional DNA molecules requires both additional optical traps as well as high-quality, multi-channel, microfluidic flow cells⁶.

There are several methods available to generate more than one optical trap. These include galvanometer scanning mirrors, acoustic optic modulators, and diffractive optics, which generate holographic optical tweezers^4,7,8,9. Often, scanning mirrors and acoustic optic modulators produce traps that timeshare. In the setup described here, the beam of a single Nd:YAG laser is split on polarization, and then galvanometer laser scanning mirrors control the position of what is termed the mobile trap (Figure 1)⁴. To facilitate the positioning of mirrors and filters to direct the trapping beam to the back aperture of the microscope objective, a HeNe laser is used. This makes overall alignment easier as the HeNe beam is visible to the naked eye, whereas infrared beams are not. The HeNe beam is also safer to work with making the positioning of mirrors and other components less stressful. Initially, the beam path for this laser is separate from the 1064 nm beam, but is introduced into the same beam path, and then into the microscope objective. Once physical alignment is achieved, positioning the 1064 nm beam on top of the HeNe beam is done and this is facilitated by the use of an infrared viewer and various beam imaging tools to visualize beam position and quality. Then, the beam expander is introduced, and the resulting expanded infrared beam is aligned onto the back aperture of the objective. Finally, the objective is removed and the power in each polarized beam is measured and adjusted using λ/2 waveplates to be equal (Figure 1C). Power measurements are also done once the objective is returned and typically there is a 53% power loss. There is, however, sufficient power to form stable fixed and moving optical traps in the focal plane (Figure 1D).

To image DNA transactions, microfluidic flow cells play a key role as they permit controlled measurements at the single-molecule level with high spatial and temporal resolution (Figure 2). The term microfluidic refers to the ability to manipulate fluids in one or more channels with dimensions ranging from 5-500 μm^10,11. The term stream refers to the actual fluid within a channel and the channel refers to the physical channel in which a fluid stream or streams move. Single-channel flow cell design has a common, physical channel where reactions are observed and there is typically only one fluid stream present. Thus, these designs are known as single-stream flow cells. In contrast, multi-stream flow cells are defined as a microfluidic device in which two or more entry channels converge into a single, common, physical channel (Figure 2A). Within the common channel, the fluid streams that originate from the individual channels flow parallel to one another, and remain separated with only minimal mixing between them occurring due to diffusion (Figure 2B). In the majority of experimental setups, a single pump pushes fluids into each channel at the same speed. In contrast, when boundary steering is used, three or more, independently controlled pumps push fluids through the channels. However, each pump operates at a different speed but the net flow rate in the common channel is constant¹². This permits the rapid exchange of main channel components simply by altering pump speed.

In addition to laminar flow, another critical factor is the parabolic velocity profile within the laminar fluid stream. The highest flow velocity occurs in the middle of the stream and the slowest occurs next to the surfaces (Figure 2C)¹³. This profile must be considered to fully stretch a DNA molecule that is attached to a bead held in the stream for precise visualization of fluorescent DNA and accurate single-molecule analyses. Here, the DNA is stretched to B-form and is held in place under 0 pN of force. To achieve this, focusing of the optical trap position should be to a position 10-20 μm from the bottom coverslip surface (Figure 2D). Care must be taken so that the DNA molecule is not stretched beyond B-form as this can inhibit enzyme reactions. Under typical buffer conditions, 1 μm = 3,000 bp of DNA¹⁴. Furthermore, by trapping 10-20 μm from the coverslip, the DNA complex is positioned far from the surface thereby minimizing surface interactions.

Many methods have been used to create microfluidic device channels and these can be done in the laboratory or flow cells can be purchased from commercial sources^6,15,16,17. The optimal materials used to construct the flow cells must be mechanically rigid, optically transparent with low fluorescence, and impervious to organic solvents⁶. Frequently, borosilicate float glass, or fused silica are used to provide a stable flow environment for an extended time that is suitable for optical trapping, visualization, and force detection. These materials also permit the use of non-aqueous solvents (e.g., spectrophotometric grade methanol) to simplify surface wetting and removal of air bubbles, and denaturants (e.g., 6M guanidinium hydrochloride) or detergents to clean the flow cell. Finally, the methods used to introduce fluids into flow cells vary from complex vacuum pump systems to single syringe pumps^{14,18,19,20,21,22,23,24,25,26,27}. In the approach described here, a syringe pump that can accommodate up to 10 syringes is used (Figure 3A). This provides flexibility to use single-channel flow cells or flow cells with multiple inlet channels. Here, a three-channel flow cell is used and is mated to syringes held in place on the syringe pump using poly-ether-ether-ketone (PEEK) tubing (Figure 3A–C). The flow of fluids is controlled by four-way switching valves and thus serve to minimize the introduction of bubbles into the flow cell (Figure 3A,D). In addition, Hamilton gastight syringes which have stiff glass walls and polytetrafluoroethylene (PTFE)-coated plungers, are recommended as they provide exceptionally smooth plunger motion that is essential to obtaining a smooth flow^14,27.

In the experimental system described, flow cells with two to five inlet channels have been used. The number of inlet channels is dictated by the experiment being done. For the study of RecBCD and Hop2-Mnd1, two stream channels were sufficient^14,28. For the helicase, the enzyme was bound to the free end of the DNA and translated into a stream containing magnesium and ATP to initiate translocation and unwinding. For Hop2-Mnd1, optically trapped DNA was translated into the adjacent fluid stream containing proteins and buffer ± divalent metal ions. The use of three-channel flow cells enables one to trap DNA in stream 1, translate the DNA into stream 2 to allow protein binding to occur, and then to stream 3 where ATP is present, for example, to initiate reactions. A variation on the above is to use fluorescent-tagged protein in channel 2, which results in the fluid stream being completely white and precludes visualization of the DNA. When this molecule is translated into stream 3, both proteins and DNA are now visible when reactions are initiated.

The use of four-way switching valves to control fluid flow is a critical component of the system to eliminate bubbles in the flow cells. Bubbles are detrimental to stable fluid flow as they contract and expand in unpredictable ways resulting in rapid changes in flow velocity and the introduction of turbulence. When the valves are positioned between syringes and inlet tubing, the flow path is disconnected by switching the valve position when syringes are changed out. When the new syringe is put in place, the plunger can be manually depressed so that >6 μL is ejected (the dead volume of the valve) and this eliminates bubbles almost entirely.

The attachment of connectors to flow cells is frequently the rate-limiting step in flow cell use. We describe the use of two types of connectors: removable known as press-fit and permanent ones (nanoport assemblies). The removable connectors are simple to adhere to a flow cell and different types of flexible tubing in addition to the recommended PTFE can be tested with these connectors. This is a rapid and cost-effective way to test tubing and connectors without sacrificing more expensive glass flow cells. In contrast, nanoport assemblies are attached permanently, withstand pressures up to 1,000 psi, and, in our hands, their use is restricted to PEEK tubing of different diameters. This is not a disadvantage as PEEK tubing is preferably used. A single glass flow cell with permanent assemblies attached can be reused for more than 1 year with careful use.

Protocol

1. Laser trap alignment and testing with polystyrene beads NOTE: For the setup, refer to Figure 1A,B. CAUTION: The experimentalist should wear appropriate protective eyewear or laser safety glasses during laser beam alignment. As the optical tweezer system described herein uses both HeNe and IR beams, two separate sets of laser safety glassware are required. HeNe Beam alignment P…

Representative Results

The initial testing of the trap alignment and strength is done with 1 μm, non-fluorescent polystyrene beads. Since most of the research done in the laboratory uses fluorescence, we further test trap strength using 1 μm, Dragon green polystyrene beads (Figure 1D,E). Thereafter work changes to optical trapping of DNA-bead complexes where the DNA is stained with the bis-intercalating dye YOYO-114,29. When thes…

Discussion

The careful assembly of the flow system is critical to the successful outcome of experiments^4,6. One of the most challenging aspects of the protocol is the attachment of connectors to the glass surface. For this, we use the following two approaches: press-fit fit tubing connectors and nanoport assemblies. Press-fit connectors adhere easily to glass followed by pushing of PTFE tubing into the preformed holes using forceps. When a more stable attachment is required…

Materials

100x objective	Leica	506318 or 506038	Oil immersion lenses; Imaging and optical trapping only; Plan APO objectives optimized for fluorescence imaging
10X Objective	Leica	506263	Used to locate laser beams spots during alignment; to find focus and X-Y position in flow cell
1 mm fluorescent beads	Bangs Labs	FSDG004	Used for tap performance, focal position determination
1 mm polystyrene beads	Bangs Labs	CPO1004	Used for trap performance evaluation and binding to biotinylated molecules
63x objective	Leica	506081	Used to locate laser beams spots during alignment and to find focus and X-y position in flow cell; can be used for optical trapping as it has an identical back aperture diameter to the 100X; oil immersion lens
Alignment laser	Lumentum	1100 series	10mW HeNe laser that is visible to the naked eye that is used to position optics
Beam alignment camera	Amscope	MU303	A simple, inexpensive and software controlled camera for imaging of the beam position
Camera control and Image capture software	Hamamatsu	HCImage	Coordinates activities of the Lambda DG4 with the camera to facilitate rapid wavelength switching
Camera; Orca flash 4	Hamamatsu	c13440-20cu	CCD camera for imaging of single-molecule experiments
C-mount for the beam alignment camera	Spot imaging solutions	DE50CMT	Provides optimal positioning of the camera for imaging of laser beams during alignment
C-mount for the Orca Flash 4 camera			Has a retainer ring to hold an IR blocking filter in place. This eliminates reflected IR beam from the optical traps and facilitates clearer imaging of trapped objects.
Cy5  fluorescence filter cube	Semrock	cy5-404a-lsc-zero	Used in conjunction with Lambda DG4 to image Cy5 only
Fitc-Txred  fluorescence filter cube	Semrock	fitc/txred-2x-b-000	Used in conjunction with Lambda DG4 to image FITC and TXRed
Fluidics tubing	Grace Bio	46004	PTFE tubing as an alternate to PEEK; works well on some flow cells. Can be used with PDMS flow cells or glass flow cells when Grace Bio fit tubing connectors are used
GFP fluorescence filter cube	Semrock	gfp-3035b-lsc-zero	Used in conjunction with Lambda DG4 to image GFP only
Glass flow cells	Translume	Custom	Clear flow channels for imaging (Fig. 2E)
Glass glue	Loctite	233841	Securely and easily bonds Nanoport assemblies to glass flow cells
Glass/PDMS sandwich flow cells	CIDRA Precision services	Custom design	Flow cells built according to your specifications; imaging channels are clear (Fig. 2C)
Hamilton Cleaning solution	Hamilton	18311	Gentle but efficient cleaning solution for glass flow cells; does not bubble when used carefully
Illumination system	Sutter Instrument	Lamda DG4	Discontinued so recommend Lambda 721
Illumination system	Sutter Instrument	Lamda DG4	Discontinued so recommend Lambda 721
Image analysis software	Media cybernetics	Image Pro Premiere	Analysis of images and single molecule tracking
Image analysis software	Fiji/NIH Image/Image J	Shareware	Analysis of images and single molecule tracking
Image display card	Melles Griot	06 DLA 001	Alternate product from Thorlabs: VRC5
Immersion oil	Zeiss	444960	Immersol 518 F fluorescence free
Laser beam alignment tools	Thor labs	FMP05/M; dgo5-1500-h1; BHM1	Used to ensure beams are horizontal and at the correct height
Laser beam viewer	Canadian Photonics labs	IR 3150	Used to image IR beam spots on mirrors and  targets
Laser power meter	Thor labs		Measurement of laser output as well as trap strength
Laser safety glasses (HeNe)	Thor labs	LG7 or 8	Blocks >3 OD units of light of wavelengths >600 nm
Laser safety glasses (IR)	Thor labs	LG11	Blocks >7 OD units of light of wavelengths ³1000 nm
Mcherry  fluorescence filter cube	Semrock	mcherry-a-lsc-zero	Used in conjunction with Lambda DG4 to image mcherry only
Microscope	Leica	DMIRE2	DIC port removed to accommodate Dichroic trapping/alignment mirror
Microscope control software	UCSF/shareware	uManager	Controls the microscope, permits focal alignment of objectives as well as stage control
Nanoport assembly	IDEX	N333	Connectors that are bonded to flow cells
Optical table support	Thor Labs	PA52502	Active isolation table support
Optics and lenses	Solar TII	Various	Interference mirrors, telescopes and lenses custom designed for the system
PDMS flow cells	ufluidix	Custom	Flow cells built according to your specifications; imaging channels are clear (Figs. 2B and D)
PEEK tubing	IDEX	1532	Provides excellent connection to flow cells and switching valves
Pinkel fluorescence filter cube	Semrock	lf488/543/635-3x-a-000	Used in conjunction with Lambda DG4 to image multiple fluorophores rapidly
Press fit tubing connectors	GraceBio	46003	Clear silicone connector with adhesive that binds well to glass
Scanning mirrors	GSI Lumonics	VM500	Used to provide control of the second optical trap. GSI Lumonics no longer exists. Similar mirrors can be purchased from Cambridge Scientific
Stage	Leica
Stage micrometer	Electron Microscopy Sciences	68042-08	Provides on screen ruler for positioning of the beam and system calibration
Switching valves	IDEX	V-101T	Control direction of fluid flow and eliminate introduction of bubbles into flow cells
Syringe and valve manifold	Machine shop	None	Custom built
Syringe pump	Harvard Apparatus	PHD 2000	Controls fluid flow through flow cells
Syringe pump software	Harvard Apparatus	70-6000	Flow control provides seamless, programmable control of fluid flow
Syringes	Hamilton	81320	Gas-tight, PTFE Luer Lock, glass barrels with Teflon-coated plungers
Table top	Thor Labs	T36H	Optical table top or breadboard
Trapping laser	Newport/Spectra Physics	J-series; BL106C	Nd:YAG laser; 1064 nm; 5W laser