Applications of Optical Techniques for Imaging and Manipulating Live Cells

 

Chi-Hung Lin, M.D., Ph.D.

 

Institute of Microbiology and Immunology,

National Yang-Ming University, Taipei, Taiwan

 

 

In the past, investigators wishing to address basic cellular structure-function questions have been limited to work on chemically fixed tissue.  Recently, however, powerful electronic imaging techniques have been applied to the study of live cells.  Although the physical laws of optics limit the resolution of light level measurements to about 200 nm, it is now possible to accurately detect and image objects on the order of tens of nm using digital-enhanced differential interference contrast (DIC) microscopy.  Along with low light level fluorescence imaging and live cell manipulation, modern techniques have brought physiological approaches to structure-function questions to the level of macromolecular resolution. 

My lab has been using the optical tweezers for manipulating biological samples and measuring biological forces.  We have successfully integrated a forward scatter analysis (FORSA) module onto the "single-beam gradient force optical tweezers".  The entire set-up could then be incorporated onto an inverted microscope.  In the FORSA module, a Helium-Neon probing laser was spotted (at a slightly out-of-focus way) onto the object being trapped by the infrared laser-based tweezers and generated a diffraction pattern.  The image of the diffraction pattern was captured by a charge-coupled device (CCD), and digitized and processed by a computer.  We demonstrated that tracking the “amplified” scattering pattern was much more precise to determine the movement of the object within the trap than analyzing the minute motion of the object itself.   The displacement of the object could then be translated into the force being applied to the trapped object.  Also, using an algorithm developed in the lab, we were able to follow the movement of the scattering pattern at a temporal resolution close to video rate.  We have used this system to investigate the binding force associated with cell-cell and molecular interactions, especially during the immediate early phase, i.e., seconds to tens of seconds after the initial contact, with great convenience and accuracy. 

Our results demonstrate that combined optical tools could provide us with effective and convenient ways for monitoring biological activities in situ and measuring changes of biological forces with precision.  Integrating such methodology with powerful biochemistry, genetics and molecular biology tools has made possible a multi-disciplinary approach that offers new understanding and helps raise novel questions to facilitate future life science research.

 

Keywords: microscopy, optical tweezers, forward scatter, DIC