The Optical Imaging Facility (established in 2002) is the optical microscopy facility of the Institute of Neuroscience (ION), situated in the campus of the Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS). At present, the laboratory has five technical staff members and runs under the supervision of Dr. Qian Hu. There are five large-scale optical microscopes in the laboratory: a ZEISS LSM 510 META confocal microscope, a ZEISS LSM 510 inverted confocal microscope,a NIKON A1R inverted confocal microscope, a Prairie in vivo two photon microscope and a live cell imaging system based around a NIKON TE-2000E inverted microscope. The laboratory is also equipped with some electrophysiology instruments (including a Hamamatsu infrared CCD, two Heka EPC-10 patch-clamp amplifiers, and an AMPI programmable MASTER-8 multi-channel stimulator) for simultaneous optical and electrophysiological recording. Open 13 hours a day from Monday to Friday and 8 hours a day on Saturday and Sunday, the two-photon microscopy laboratory is known for its advanced technology and high rate of equipment usage.
Laser Scanning Confocal Microscopy (LSCM) is proving to be a most important technique in morphological analyses, molecular and cellular biology, neuroscience and pharmacology. Using lasers as polarized light sources of specified wavelengths, combined with scanning devices, conjugate focusing devices, and computer-controlled image digitizing and analyzing softwares, LSCM enables researchers to collect fluorescent signals from the interior of cells or organisms with improved depth discrimination and resolution. With LSCM, researchers can: 1) observe very small structures within a cell or an organism and follow their morphological changes; 2) optically slice a sample and then reconstruct and analyze its three-dimensional structure; 3) record concentration change of important ions or pH changes at the sub-cellular level, and 4) in combination with electrophysiological methods, study the physiological activity of cells.
The ZEISS LSM510 META can collect fluorescence signals of different wavelengths simultaneously through its META channel, which consists of a light diffracting device and 32 highly sensitive photomultiplier tubes. With the integrated Emission Fingerprinting function, widely overlapping emission spectra can be precisely separated, fluorescence bleed-through can be mostly eliminated, and loss of fluorescence signal can be reduced to a minimum. The LSM510 META provides a flexible means for life science research, and is suitable for newly emergent fluorescence applications such as simultaneous intracellular tracking of multiple fluorescent proteins, as well as FRAP and FRET experiments.
Two-Photon Fluorescence Microscopy is a powerful research tool that combines the advanced optical techniques of laser scanning confocal microscopy with the long wavelengths of two-photon excitation. It is based on the phenomenon that at high photon density, two photons can be simultaneously absorbed (combining their energies) to provoke the electronic transition of a fluorophore to the excited state. Because the energy of a photon is inversely proportional to its wavelength, the two photons have approximately twice the wavelengths of light required for single-photon excitation. In two-photon excitation high photon density is necessary to ensure a sufficient level of fluorophore excitation. To prevent photo-damage, the two-photon microscope utilizes high-power mode-locked pulsed laser, which generates a significant amount of power during pulse peaks, but has an average power that is sufficiently low as not to damage the specimen. A typical pulsed laser configuration employs short duty cycles of around 100 femtoseconds with a repetition rate of 80 to 100 megahertz. Since with high numerical aperture objectives two-photon excitation occurs only at the focal point of a diffraction-limited microscope, a detecting pinhole is not need in two-photon microscope, thereby increasing detecting efficiency. Two-photon microscopy has many advantages over confocal microscopy: 1) long wavelength infrared light scatters less than light of shorter wavelengths and penetrates through specimen more easily; 2) lack of absorption from fluorophores positioned outside the focal plane allows more of the excitation light to penetrate through the specimen and reach the plane of focus; 3) long wavelength infrared laser is less phototoxic than lasers of shorter wavelengths; 4) photobleaching and phototoxicity occurs only at the focal point. For these reasons, two-photon microscopy is particularly suitable for experiments including long term live cell imaging, imaging in deep tissues and photobleaching or other manipulations in small localized regions.
The Optical Imaging Facility provides service to investigators of ION as well as those from other SIBS affiliated institutions on an hourly-fee basis or a fee-for-service basis. Outside users from surrounding universities and local biotechnology industries are also accommodated schedule permitting.