Third Harmonic Microscopy: Dynamic, High-Resolution, Three-Dimensional Imaging Without Bleaching

University Of California-San Diego, La Jolla CA

Investigators

Abstract

A high-resolution, real-time, microscope that is based on third-harmonic generation and provides unique possibilities to visualize interfaces in refractive index or third-order nonlinear susceptibility in optically transparent media, and biological systems is being developed. The thin optical sections produced in the third harmonic microscope can be used in conjunction with standard surface rendering techniques to produce three-dimensional images. No fluorophore is necessary to label the specimen as is used in traditional laser fluoresence microscopy, as the signal is generated by endogenous interfaces. Thus, in third harmonic microscopy the images do not fade due to bleaching, and can be viewed for extended periods without loss of intensity or clarity. This imaging technique is therefore particular useful for imaging three-dimensional dynamics in microscopic systems. For dynamic measurements, imaging volumes must be repeatedly addressed in order to develop a reliable time series of transient phenomena. In the past, the effectiveness over which dynamic systems could be measured was limited by the bleaching characteristics of an exogenous label. Third harmonic imaging relies on naturally occurring boundaries within the sample to generate image contrast, and therefore does not fade, and is highly suitable for visualizing dynamic three-dimensional systems. A diode-pumped, femtosecond Nd:glass oscillator optimized for microscopy will be constructed. The laser will produce third harmonic signals that are 16-21 times greater than can be achieved with commercially available lasers. The laser will also be beneficial for 2-photon imaging. Operating at a wavelength of 1.06 micro meter, this light penetrates tissue ~20% deeper than present femtosecond lasers that commonly operate at 800 nm. A novel microscope design will be constructed that can simultaneously capture fluoresence images and third harmonic images with perfect registration. This is important for fully quantifying the image contrast mechanisms in third harmonic microscopy, and enables a quantitative analysis of this new imaging process. The third harmonic microscope will be used in conjunction with an image correlation spectroscopy technique developed specifically to allow rapid measurements of macromolecular aggregation within an intact cellular environment. By combining temporal and spatial autocorrelation analysis of an image time series, it is possible to obtain information on the molecular dynamics and transport properties as well as measuring the state of aggregation of the molecules as a function of time. The combined information can provide insight into the molecular mechanisms that govern many biochemical reactions in living cells. Measurement of molecular interactions and dynamics is integral for a full understanding of how cells build functional macromolecular assemblies as well as how they transduce signals across the plasma membrane following binding of external ligands to receptors localized to the cell surface.