Lumicks C-Trap Optical Tweezers with Confocal Fluorescence Microscope

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

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Abstract

Title: Lumicks C-Trap optical tweezers with confocal fluorescence microscope. PI: Bennett Van Houten, PhD Abstract This proposal requests the purchase of a Lumicks C-trap correlative single-molecule and force microscope, which consists of four optical traps, and a 3-color confocal fluorescence platform for Major and Minor user groups at the University of Pittsburgh and Carnegie Mellon University. The C-trap is the only commercially available instrument that fully integrates laser traps with confocal microscopy. Single molecule analysis has helped revolutionize the field of protein-DNA interactions and each of the users have a focused funded project that would be greatly facilitated by access to this instrument. Through a lease agreement we have been using the instrument since Feb of 2021 and several user groups have successfully generated preliminary data on the instrument with up to three different fluorescent proteins. This instrument represents the first placement of this novel technology within the entire Common Wealth of Pennsylvania, and the new four laser trap module is planned to be added June 2022 to keep up with this cutting-edge technology. The C-trap allows investigators to use biophysical approaches to interrogate protein-DNA interactions and follow molecular motors and assembly of protein machines at specific sites on DNA. This instrument combines several unique features that makes single molecule and force measurements accessible to wide group of users that might be relatively new to the single molecule field. The instrument has an automated microfluidic system with a five chamber imaging flow cell, which provides a wide variety of experimental designs. The instrument is fully temperature controlled so kinetics of protein-DNA interactions can be followed at different temperatures. The laser traps have high temporal and spatial control and can measure down to sub pN forces accurately. The force measurements are real-time correlated with the confocal scanning which can scan at speeds up to 5 msec per scan allowing to capture rapid single molecule kinetics. The software that controls the instrument and allows data analysis is easy to use through a Python user interface. Scripting of experimental work-flow as well as data analysis allows highly reproducible conditions to be established. The two user groups consists of members of the Genome Stability and Cancer Virology Programs at the UPMC-Hillman Cancer Center, where the instrument is housed, as well as other minor users throughout the campus. These groups are interested in the role of DNA damage and repair in cancer and neurodegenerative diseases, as well as how specific viruses can cause cancer. Five Major users have been identified for the purpose of this application and they in turn have requested a majority of the time. The five Minor users will work on the instrument in the remaining time. The Lumicks C-trap is being managed as a user facility at the Hillman Cancer Center and will afford the capacity for advanced single molecule and mechanical force measurements and will ensure sustained and efficient use by faculty working in the biomedical sciences in Pittsburgh