Top view and side view of complete assembly for holding 0.2 × 2.0 × 100 mm microslide (VitroCom, Cat. # 3520-100). See companion photographs in Fig. 1n and 1o.

Magnetic tweezers (MT).
(a) Photograph of MT system on an inverted microscope with belt driven MT. (b–g) Microscope video images of one bead rotating around a magnetic bead due to the rotation of the external magnets of the belt driven MT in (a). Sequential order of images is b, c, d, e, f and g. (h) Schematic example of first generation flow cell design using parafilm and photograph of assembled flow cell. (i) Schematic of MT operation on an upright microscope with inset of individual DNA molecule suspended between a superparamagnetic bead and the surface. Photograph of same area containing 2.8 μm Dynal superparamagnetic beads attached to the glass slide illuminated by white light (j) or by scatter of a 532 nm laser (k). (l) Photograph of the ring around the objective. This large diameter ring can rotate external magnet(s) clockwise and counter-clockwise around the objective and can be used for the MT setup above the microscope slide. (m) Photograph of the external ring setup portion of MT. Counter, motor, measurement of number of clockwise (cw) and counter cw (c-cw) rotations, and ball bearing ring are indicated. On opposite side of ball bearing is the place for magnets as in (l). (n) Photograph of assembly for holding 0.2 × 2.0 × 100 mm microslide (VitroCom, Cat. # 3520-100) with two buffer reservoirs for attachment to the ends of this rectangular cuvette. (o) Photograph of cuvette holder and MT on an upright microscope. (p) Photograph of second generation flow cell design.

Top view, side view and expanded detailed view of complete assembly for holding microslide with measurements in mm. See companion photographs in Fig. 1n and 1o. The Detail A-A section has an expanded view of the two slots in the two prongs that hold the 0.1 mm × 2 mm × 100 mm rectangular glass microslide parallel to the surface of the microscope stage for flat imaging by the upright objective.

DNA attachment to a surface chemistry schematics. (a) Schematic of DNA construct. (b) Schematic of surface modification chemistry. (c) Schematic of microsphere modification.

The pressure-driven microvortex design for application of high radial acceleration on individual DNA molecules. (a) Schematic of the microfluidic configuration used to create the microvortex. The surface of the glass substrate at the bottom of this microvortex system was treated with anti-digoxigenin for at least one hour to allow the attachment of DNA molecules to the surface of the glass. (b) The process of the soft lithography for fabricating this microvortex system with poly(dimethylsiloxane). (c) An optical image of the poly(dimethylsiloxane) microfluidic device. Fluorescent images of YOYO-1 labeled DNA molecules (d) without and (e) with pressure applied to microvortex system. Scale bar=4 μm.

Observation of pressure-driven DNA transport in microfluidic and nanofluidic channels. Schematic of a rectangular, 50-μm-wide, 4-mm-long silica fluidic channel (a) and the channel cross-section over which an applied pressure gradient generated a parabolic fluid velocity profile (b). (c) Imaging a fluorescently labeled 48.5-kbp DNA molecule as it was transported through a 250 nm high channel using an applied pressure gradient of 1.44×10⁵ Pa/m. The red dots indicate the center-of-mass positions, which were recorded at a rate of 5 Hz [21].

Probing fundamental DNA relationships with nanochannels. (a) When a DNA polymer is confined to a nanochannel of diameter D, the polymer must elongate to with a constrained end-to-end distance. (b) Using this approach, the observed standard deviation was determined based on the length of the molecule in a confined channel with a width of 100 nm [22].

Magnetic tweezers used to follow single molecule transcription. (a) Schematic of setup. A double-stranded DNA molecule is tethered between a surface attached molecule of T7 RNA polymerase and a 2.8 μm superparamagnetic bead that is decorated with a 1 μm marker bead. (b) Video image of the 2.8 μm superparamagnetic bead decorated with a 1 μm marker bead. (c) Time lapse images from a movie demonstrating one complete rotation of the marker bead around the superparamagnetic bead. Figure adapted from Pomerantz et al. [24].

Magnetic tweezers using to follow chromatin fiber assembly and disassembly. (a) Schematic of an image of video of the attachment of a single linear DNA molecule tethered between the inner surface of a glass cuvette and a superparamagnetic bead. In the actual video, only the tethered superparamagnetic bead is observed. (b) Schematic of an image of video after flow is introduced. The superparamagnetic bead that was in the upper left hand portion of the image in (a) has now been forced by the left to right flow to be in the upper right hand corner of this image. Additionally, the flow introduces a histone chaperones and core histone octamers, which begin to assemble on the naked DNA molecule and subsequently reduce its overall tether length resulting in the travel of the superparamagnetic bead across the screen and against the direction of the flow. (c) Upon observation that the superparamagnetic bead is traveling across the screen, the flow is stopped and only the external magnet exerts force on the tethered DNA molecule. (d) The flow is off and the observed travel of the bead is only due to assembly caused reduction in the tether length of the DNA molecule as a function of the force exerted by the magnetic field of the external magnet on the superparamagnetic bead. (e) Graph of tracking the center of the superparamagnetic bead over time. After observing the bead traveling across the video screen, we turned off the flow, which results in a precipitous drop in tether length as marked on the graph. Video frame rate 1 per sec. (f) Graph of travel of the bead across the screen as a function of the force on the superparamagnetic bead exerted by the external magnet. Having the external magnet at 2 mm away results in a 10 pN force, which prevents changes in the DNA, tether length and chromatin assembly. Moving the external magnet away using a manual linear caliper weakens the magnetic field exerted on the superparamagnetic bead and results in faster and faster chromatin assembly. Adapted from Leuba et al. [8].

Chromatin assembly by flow in a microfluidic chamber. (a) Schematic of a PDMS-fabricated flow chamber. λ-DNA is tethered to a surface and a syringe pump creates flows containing a Xenopus egg extract to assemble the DNA molecule into chromatin. (b) Video images of a single fluorescently labeled λ-DNA molecule undergoing assembly at shear rates of 26 s⁻¹ (left panel) and 1050 s⁻¹ (right panel). (c) Chromatin assembly at a constant shear rate of 26 s⁻¹ as a function egg extract dilution ranging from 1:2.5 (far left orange curve) to a much diluted 1:400 (far right yellow curve). (d) Assembly of chromatin in the presence of the 1:100-diluted egg extract as a function of shear rates ranging from 26 s⁻¹ (lowest blue curve) to 1050 s⁻¹ (highest black curve). The furthest left green curve is chromatin assembly at shear rate with extracts with a ten-fold addition of H2A/H2B. Data from Ladoux et al. [26].