N. Tavernarakis, Director IMBB

Institute of Molecular Biology and Biotechnology, FORTH

Non-linear microscopy and optical projection tomography in Biology

Abstract

 

Imaging of cellular and molecular processes in vivo, as they transpire over time and in the context of the whole organism is becoming increasingly important in biomedical research. This capacity is of considerable relevance to studies of complex biological phenomena such as development and ageing. Currently available methodologies for imaging live small model organisms, such as Drosophila or Caenorhabditis elegans are based mainly on differential interference contrast (DIC) and confocal epi-fluorescence microscopy. While these approaches allow detailed two-dimensional (2D) visualization of cellular and sub-cellular structures, they are inefficient for obtaining high-resolution, three-dimensional (3D) reconstructions of whole animals over extended periods of time, during post embryonic development and ageing. We have developed a versatile optical projection tomography system for rapid three-dimensional imaging of microscopic specimens in vivo. The tomographic setup eliminates the in xy and z strongly asymmetric resolution, resulting from optical sectioning in conventional confocal microscopy. It allows for robust, high resolution fluorescence as well as absorption imaging of live transparent invertebrate animals such as C. elegans. This system offers considerable advantages over currently available methods when imaging dynamic developmental processes and animal ageing; it permits monitoring of spatio-temporal gene expression and anatomical alterations with single-cell resolution, it utilizes both fluorescence and absorption as a source of contrast, and is easily adaptable for a range of small model organisms. Two-photon excitation fluorescence (TPEF), Second-harmonic generation (SHG) and Third Harmonic Generation (THG) are relatively new and promising tools for detailed imaging of biological samples and processes at the microscopic level. Due to their inherent advantages in comparison with conventional microscopy (increased resolution, ability to section deep within tissues, minimization of photodamage and photobleaching effects), these non-linear microscopy techniques comprise an extremely powerful tool for the extraction of valuable and unique information from biological samples. We developed a compact, reliable, inexpensive non-linear imaging system, utilizing femtosecond laser pulses (1028nm) for excitation of biological samples. The use of 1028nm wavelength as excitation source minimizes photodamage effects and unwanted heating (due to the water absorption) of biological specimens. The emitted THG signal lies in the near UV part of the spectrum (343nm). We achieved high-resolution imaging and mapping of Caenorhabditis elegans (C. elegans) neurons and muscular structures of the pharynx, at the microscopic level by performing SHG and TPEF measurements. Detailed and specific structural and anatomical features can be visualized, by recording THG signals. Thus, the combination of three image-contrast modes (TPEF-SHG-THG) in a single instrument has the potential to provide unique and complementary information about the structure and function of tissues and individual cells of live biological specimens.


Date: 9/4/2014
Time:12:00 (coffee & cookies will be served at 11:45)
Place:FORTH Seminar Room 1