CPLC Station 1

The CPLC Station 1 is a hybrid instrument combining ultrahigh resolution optical tweezers and single molecule fluorescence confocal microscope. The instrument, jointly developed by the laboratories of Yann Chemla and Taekjip Ha, can measure mechanically movements of DNA polymerase, RNA polymerase, helicases and ribosomes with single base pair resolution and at the same time detect fluorescence signals in multiple colors with single fluorophore sensitivity. The initial development and proof-of-principle demonstrations have been published in Nature Methods (Comstock et al, Nature Methods, 2011). Currently, we are measuring the conformational changes of UvrD helicase while at the same time measuring DNA unwinding.

CPLC Station 2

The CPLC Station 2 is a specially designed single molecule fluorescence microscope. It is centered around an inverted Nikon TE2000 microscope. It has an ASI Imaging stage with < 5 nm resolution in the x-y plane and about 40 nm in z. In addition, fiduciary markers can be put on the coverslip to know where the stage is to < 1 nm in x-y and < 17 nm in z. It is also enclosed for temperature and chemical (CO2) stability. We have an Agilent laser launch, with 405nm (20mW), 488nm (90mW), 561nm (45mW), and 640nm (45mW), along with a 532 nm laser. An Andor EMCCD is used for detection, with a Photometric Dual-View or Quad-View, or Cairn Research OptoSplit II for multi-color detection. It is controlled with Nikon Elements software. There are also automated injection needles, used primarily for neuroscience research. Some molecular motor research, and DNA-mutation research is also done.

CPLC Station 3

The CPLC Station 3 is optimized to study live cells using optical traps. Optical traps are formed from a 5-W, 1064-nm laser beam that is time-shared using an acousto-optic deflector (AOD). Samples are mounted on a three-axis stage and visualized via bright-field microscopy. Widefield fluorescence excitation of the sample can be achieved using either a 30-mW, 532-nm laser source, or a 1-W, 488-nm laser source, interlaced with the optical trapping beam to minimize photo-induced damage of the sample. Emitted fluorescence is imaged using an EMCCD camera equipped with a 128x128 pixel array. The small imaging array enables fast imaging rates, up to 500 Hz. Measurements currently underway using this instrument include simultaneous measurement of adaptive swimming behavior and internal protein dynamics in single E. coli cells, as well as visualization of flagella dynamics in swimming E. coli.