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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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.

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.

  1. HeNe Beam alignment
    1. 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 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.......

Acknowledgements

Research in the Bianco laboratory is supported by NIH grants GM100156 and GM144414 to P.R.B.

....

Materials

NameCompanyCatalog NumberComments
100x objectiveLeica506318 or 506038Oil immersion lenses; Imaging and optical trapping only; Plan APO objectives optimized for fluorescence imaging
10X ObjectiveLeica506263Used to locate laser beams spots during alignment; to find focus and X-Y position in flow cell
1 mm fluorescent beadsBangs LabsFSDG004Used for tap performance, focal position determination
1 mm polystyrene beadsBangs LabsCPO1004Used for trap performance evaluation and binding to biotinylated molecules
63x objectiveLeica506081Used 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 laserLumentum1100 series10mW HeNe laser that is visible to the naked eye that is used to position optics
Beam alignment cameraAmscopeMU303A simple, inexpensive and software controlled camera for imaging of the beam position
Camera control and Image capture softwareHamamatsuHCImageCoordinates activities of the Lambda DG4 with the camera to facilitate rapid wavelength switching
Camera; Orca flash 4Hamamatsuc13440-20cuCCD camera for imaging of single-molecule experiments
C-mount for the beam alignment cameraSpot imaging solutionsDE50CMTProvides optimal positioning of the camera for imaging of laser beams during alignment
C-mount for the Orca Flash 4 cameraHas 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 cubeSemrockcy5-404a-lsc-zeroUsed in conjunction with Lambda DG4 to image Cy5 only
Fitc-Txred  fluorescence filter cubeSemrockfitc/txred-2x-b-000Used in conjunction with Lambda DG4 to image FITC and TXRed
Fluidics tubingGrace Bio46004PTFE 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 cubeSemrockgfp-3035b-lsc-zeroUsed in conjunction with Lambda DG4 to image GFP only
Glass flow cellsTranslumeCustomClear flow channels for imaging (Fig. 2E)
Glass glueLoctite233841Securely and easily bonds Nanoport assemblies to glass flow cells
Glass/PDMS sandwich flow cellsCIDRA Precision servicesCustom designFlow cells built according to your specifications; imaging channels are clear (Fig. 2C)
Hamilton Cleaning solutionHamilton18311Gentle but efficient cleaning solution for glass flow cells; does not bubble when used carefully
Illumination systemSutter InstrumentLamda DG4Discontinued so recommend Lambda 721
Illumination systemSutter InstrumentLamda DG4Discontinued so recommend Lambda 721
Image analysis softwareMedia cyberneticsImage Pro PremiereAnalysis of images and single molecule tracking
Image analysis softwareFiji/NIH Image/Image JSharewareAnalysis of images and single molecule tracking
Image display cardMelles Griot06 DLA 001Alternate product from Thorlabs: VRC5
Immersion oilZeiss444960Immersol 518 F fluorescence free
Laser beam alignment toolsThor labsFMP05/M; dgo5-1500-h1; BHM1 Used to ensure beams are horizontal and at the correct height
Laser beam viewerCanadian Photonics labsIR 3150Used to image IR beam spots on mirrors and  targets
Laser power meterThor labsMeasurement of laser output as well as trap strength
Laser safety glasses (HeNe)Thor labsLG7 or 8Blocks >3 OD units of light of wavelengths >600 nm
Laser safety glasses (IR)Thor labsLG11Blocks >7 OD units of light of wavelengths ³1000 nm
Mcherry  fluorescence filter cubeSemrockmcherry-a-lsc-zeroUsed in conjunction with Lambda DG4 to image mcherry only
MicroscopeLeicaDMIRE2DIC port removed to accommodate Dichroic trapping/alignment mirror
Microscope control software UCSF/sharewareuManagerControls the microscope, permits focal alignment of objectives as well as stage control
Nanoport assemblyIDEXN333Connectors that are bonded to flow cells
Optical table supportThor LabsPA52502Active isolation table support
Optics and lensesSolar TIIVariousInterference mirrors, telescopes and lenses custom designed for the system
PDMS flow cellsufluidixCustomFlow cells built according to your specifications; imaging channels are clear (Figs. 2B and D)
PEEK tubingIDEX1532Provides excellent connection to flow cells and switching valves
Pinkel fluorescence filter cubeSemrocklf488/543/635-3x-a-000Used in conjunction with Lambda DG4 to image multiple fluorophores rapidly
Press fit tubing connectorsGraceBio46003Clear silicone connector with adhesive that binds well to glass
Scanning mirrorsGSI LumonicsVM500Used to provide control of the second optical trap. GSI Lumonics no longer exists. Similar mirrors can be purchased from Cambridge Scientific
StageLeica
Stage micrometerElectron Microscopy Sciences68042-08Provides on screen ruler for positioning of the beam and system calibration
Switching valvesIDEXV-101TControl direction of fluid flow and eliminate introduction of bubbles into flow cells
Syringe and valve manifoldMachine shopNoneCustom built
Syringe pumpHarvard ApparatusPHD 2000Controls fluid flow through flow cells
Syringe pump softwareHarvard Apparatus70-6000Flow control provides seamless, programmable control of fluid flow
SyringesHamilton81320Gas-tight, PTFE Luer Lock, glass barrels with Teflon-coated plungers
Table topThor LabsT36HOptical table top or breadboard
Trapping laserNewport/Spectra PhysicsJ-series; BL106CNd:YAG laser; 1064 nm; 5W laser

References

  1. Bianco, P. R., Lu, Y. Single-molecule insight into stalled replication fork rescue in Escherichia coli. Nucleic Acids Research. 49 (8), 4220-4238 (2021).
  2. Kaur, G., Lewis, J. S., van Oijen, A. M. Shining ....

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