Optical Tweezers Combined with Single-Molecule Fluorescence Imaging

We are monitoring the structural re-arrangements and conformational changes during the steps of PIC assembly in real-time using a combination of Optical Trapping with Total-Internal-Reflection microscopy. The experimental setup is based around a multi-color single-molecule fluorescence TIR microscope capable of sub-nanometer co-localization of individual dye-molecules. State-of-the-art optical trapping and nano-positioning capabilities in this instrument allow simultaneous imaging and manipulation of surface tethered DNA molecules. This technique allows detecting the real-time dynamics during the PIC assembly.

A current implementation of the setup, built around a commercial Olympus IX-71 is shown above. The setup is comprised of individual functional blocks, shown below.

We excite different dye molecules by three lasers (Block B: 488nm (not shown), 532nm frequency-doubled Nd-YAG, 635nm diode), in objective-type TIR mode (green/red light path). Single-molecule fluorescence (yellow light path) is collected by the same objective and is detected by an Electron-Multiplication CCD Camera (CCD1) for wide field imaging. Dichroic mirrors and band-pass filters are used to separate the fluorescence emission of the different dyes and form images on two separate regions of the EM-CCD (home-built 2-color imaging optics Box, details not shown). Also a video-rate camera (CCD2) and an LED are included for general-purpose imaging/alignment.

A Nd-YAG laser beam (2W maximum at 1064nm) is used to form the optical trap (Block D: pink light path). The position of the trap in the specimen plane is adjusted using a set of Acousto-optic Deflectors(not shown). For position detection, the forward-scattered light off the trapped bead is collected with a condenser lens and the condenser back-focal plane is imaged in a Quadrant-photo-detector (QPD). The QPD output is digitized and stored on a PC for off-line analysis or observed in real-time with a spectrum analyzer. During combined optical trapping/single molecule fluorescence imaging it is known that absorption of a NIR photon during the excited state lifetime of certain cyanine dyes (Cy3 and Alexa-555) can cause irreversible photo-destruction. Since the excited state lifetime of Cy3 is ~nsec while relevant experimental time scales are ~msec or longer a straightforward way to circumvent this problem is alternating the excitation and trapping beams. However, chopping at frequencies close to the viscous relaxation time of the bead can compromise the performance of the trap and increase position noise. In our setup the same RF signal from a Voltage-controlled Oscillator drives both the 532nm and 1064nm Acousto-optic Deflectors, while an RF switch alternates between the two. The signal driving the switch is a square wave from a function generator and the RF signal is amplified to drive the AODs. By also reducing the beam waist (~100um) inside the Acousto-optic crystals our homemade electronics achieve modulation frequencies up to (at least) 1MHz, as verified by a fast photodiode. Chopping the trapping beam at the 100KHz-1MHz frequency range does not result in any adverse effects, other than a 2-fold reduction in the trap stiffness due to the 50% duty cycle. At the same time the enhanced photo-bleaching of the Cy3 is eliminated.

Back-focal-plane detection of a fiducial marks on the sample detects stage and focus drift. The fiducial is a ~1um bead stuck on the coverslip. The beam from a temperature-stabilized and optically isolated diode laser (780nm, orange optical path) forms a bright-field image of the mark on QPD. The beams is coupled to the microscope through a polarization-maintaining single-mode optical fiber for enhanced pointing stability. The signals of the position detector are compared to the desired set-point for the stage coordinates. The error signal drives the control electronics of a 3-axis Nano-Positioning stage to correct drift in each axis (X, Y, Z) independently. This closed-loop feedback-stabilization approach is essential in alleviating drift and allowing precise measurements on the sub-nanometer scale during extended experiments.