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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.
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.
The ability to visualize protein-DNA interactions at the single-molecule level and in real-time has provided significant insight into genome stability1,2. In addition to working with single molecules of DNA one at a time, the ability to view transactions between individual molecules nearby provides additional insight3,4,5. The manipulation of additional DNA molecules requires both additional optical traps as well as high-quality, multi-channel, microfluidic flow cells6.
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.
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.......
The careful assembly of the flow system is critical to the successful outcome of experiments4,6. 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.......
Research in the Bianco laboratory is supported by NIH grants GM100156 and GM144414 to P.R.B.
....Name | Company | Catalog Number | Comments |
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. | ||
Cy5Â 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; BHM1Â | 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 |
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