A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force-Confocal Fluorescence Microscopy

Abstract

Integrated single-molecule force-fluorescence spectroscopy setups allow for simultaneous fluorescence imaging and mechanical force manipulation and measurements on individual molecules, providing comprehensive dynamic and spatiotemporal information. Dual-beam optical tweezers (OT) combined with a confocal scanning microscope form a force-fluorescence spectroscopy apparatus broadly used to investigate various biological processes, in particular, protein:DNA interactions. Such experiments typically involve imaging of fluorescently labeled proteins bound to DNA and force spectroscopy measurements of trapped individual DNA molecules. Here, we present a versatile state-of-the-art toolbox including the preparation of protein:DNA complex samples, design of a microfluidic flow cell incorporated with OT, automation of OT-confocal scanning measurements, and the development and implementation of a streamlined data analysis package for force and fluorescence spectroscopy data processing. Its components can be adapted to any commercialized or home-built dual-beam OT setup equipped with a confocal scanning microscope, which will facilitate single-molecule force-fluorescence spectroscopy studies on a large variety of biological systems.

Figure 1

Dual-beam OT setup for force–fluorescence spectroscopy measurements. An individual DNA molecule (purple) is tethered between two micrometer-sized beads (gray spheres) that are held in optical traps (red) to act as a substrate for a fluorescently labeled molecule of interest (orange oval with green dot). A: (left) One of the beads is moved away from the other along the x-axis in order to exert a stretching force F_x on the tethered DNA molecule. (right) One then records force–distance data by plotting this force against the end-to-end distance of the DNA measured along the x-coordinate. B: (left) A confocal scanning laser (green) integrated with a dual-beam OT performs a line scan along the x-axis. (right) A series of offset line scans imaging the x–y plane to form a 2D image, where a fluorescently labeled CMG holo-helicase is shown as a fluorescent spot. This allows one to capture the diffraction-limited spots of fluorescently labeled molecules bound to the tethered DNA molecule and monitor their dynamics.

Figure 2

Strategies to prepare protein:DNA substrates. (A) Protein of interest is bound to a DNA substrate through bulk incubation. The assembled protein:DNA complex can be either directly loaded into the dual-beam OT-confocal scanning microscope setup for single-molecule force–fluorescence measurements or purified in bulk prior to single-molecule measurements. (B) Individual DNA substrate molecule is trapped in the dual-beam OT and incubated with a fluorescently labeled protein of interest to form a protein:DNA complex. The complex can subsequently be steered into a separate buffer reservoir for single-molecule measurements in the absence of background fluorescence.

Figure 3

Microfluidic system design and procedure of automated dual-beam OT force–fluorescence microscopy measurements. (A) Microfluidic chip designed to carry out dual-beam OT force–fluorescence microscopy measurements. The bead, DNA, and buffer channels are separated by laminar flow without a physical barrier. Multiple large buffer reservoirs (0.5 μL) are connected to the buffer channel by thin (0.25 mm wide) and long (1.5 mm long) necks. These necks ensure separation between the reservoir and the main channel in the absence of flow. (B) Flowchart showing the procedure for performing automated force and/or fluorescence spectroscopy measurements on an individual DNA molecule. The procedure starts by moving the two trapping laser foci to the bead channel in order to trap beads. Once beads of sufficient quality are trapped, they are steered to the DNA channel to tether a single DNA molecule. The qualities of beads and tethered DNA are monitored as indicated, and not passing the checkpoints leads to a restart of the protocol. Dashed lines show that the movement to the buffer reservoir and subsequent incubation are optional, depending on the experiment. Force spectroscopy and fluorescence imaging are carried out in the buffer channel or a separate buffer reservoir. When measurement termination criteria, e.g., fluorescence intensity or stretching force, are reached, the procedure is restarted to trap and measure another DNA molecule. This procedure is repeated until the desired replicate number of DNAs is reached, and the experiment is terminated.

Figure 4

Examples of kymograph and 2D full confocal scanning images collected using dual-beam OT-confocal scanning microscope. (A) A kymograph illustrating the diffusive motion of a fluorescently labeled Mcm2-7 helicase on DNA (green trace indicated by the green arrow). The top panel shows a full 2D scan at the start of the measurement that includes the beads and the fluorescent Mcm2-7 helicase. The kymograph is constructed by repeating 1D scans along a given line on the x-axis and stacking the scans (Figure S3). (B) Three 2D confocal scanning images that sample the x–y plane at the time stamps are indicated. The green spot illustrates the unidirectional translocation of a fluorescently labeled CMG holo-helicase on DNA oriented along the x-axis.

Figure 5

Use of site-specific fluorophores to determine the genomic locations of fluorescent spots. Determination of the bead extremity and pixel-to-nanometer conversion in the confocal scanning images is required for determining the locations of DNA ends.(A)The location of the bead extremity in the bright-field image is mapped onto the corresponding location in the confocal scanning image by calibration of the offset between these two sets of images (indicated by the red dashed lines and arrow). To do so, a static fluorophore is bound to the DNA at a known distance away from the DNA center. This fluorophore will appear at two x-locations in the confocal scanning image (green dots, x_dye,confocal and x_{dye,confocal, and mirrored}). The arithmetic mean between these two locations yields the center of the DNA in this image (x_{DNA center, confocal}). Note that the fluorophores are not visible in the bright field image. Therefore, the center of the DNA in the brightfield image is given by arithmetic mean between the bead locations . The offset equals the difference between the DNA center locations in bright field and confocal scanning images. (B) Dividing the distance between the locations of the two fluorophores in the population of traces measured in the confocal scanning images (in pixels) by the length of the DNA measured in the bright-field image (in microns) yields the confocal scanning image pixel size.

Figure 6

A flowchart summarizing the data hierarchy of confocal scanning data analysis pipeline and the output at each level. The analysis performed to move to the next level are shown in blue. The information extracted at each level is shown in red. The input data include the raw image data and the associated metadata. Spot detection, localization, and tracking algorithms are used to identify fluorescent spots in the image (Track) of a certain color, providing information about locations, lifetime, and number of bleaching steps of the fluorophores. Colocalized spots (Tracks) of different colors are combined within one Trace, which provides information about stoichiometry and motion properties. Different traces within one image are stored in the same scan object, providing information about the number of spots and/or fluorophores on one DNA molecule. All the scans collected under one experimental condition are stored in one experiment object, where the spot analysis results are reported in a table.