Deep <i>in vivo</i> Imaging With New Multiphoton Excitation (MPE) Microscope Range from Olympus

Olympus Life and Material Science Europa GmbH, has introduced a Multiphoton* deep fluorescent imaging system. Based on the unique Olympus FluoView FV1000 confocal Laser Scanning Microscope (cLSM), the MPE system is available in three models providing the highest penetration depths on the market.

Each model incorporates Olympus’s market leading components, such as the UIS2 optics including among others well-known long working distance water-immersion objectives. The systems are fully configured to ensure best excitation efficiency and thus imaging hundreds of microns deep into a specimen becomes possible.

Each of the three models in the Olympus FV1000MPE range uses IR femtosecond pulsed lasers and non-descanned detectors to cover every multiphoton excitation imaging requirement. The entry level model is designed to achieve maximum penetration depth with minimal complexity. The advanced models offer IR laser attenuation via an acousto-optical modulator (AOM). This enables the use of all the sophisticated scanning and laser control modes of the FV1000, such as ‘region of interest’ imaging and fly back beam blanking. The carefully designed laser introduction path with AOM laser control provides negative-chirp laser beam conditioning for the optimum fluorochrome excitation efficiency in MPE applications. This produces a pinpoint photon density precisely at the focal plane in the specimen with minimal damage or photobleaching to living cells.

The third model incorporates the unique SIM scanner, available for the Olympus FluoView FV1000. This system provides an independantly controllable, fully integrated second IR laser for simultaneous multiphoton imaging and multiphoton laser manipulation. A solution, which is essential e.g. in uncaging applications deep within the tissue. This provides a unique system for the ultimate in multiphoton microscopy.

In multiphoton microscopy, fluorescence molecules and proteins absorb the energy from multiple photons, carefully targeted to arrive simultaneously at the focal layer in the focus point of the objective. As a result the specimen emits a fluorescence signal as if it had been excited by a single photon of double (or triple) the photon energy, but only in the excited focal layer. One of the main advantages of the Olympus FV1000MPE systems is the design of the complete optical light path ensuring best excitation efficiency with pulsed IR laser light and sensitive signal detection. Non-descanned detectors are used to acquire the maximum amount of the fluorescence emission. The detector module is positioned close to the objective, maximising the detection efficiency, since there is no need for the signal emitted from the excited focal layer to pass through a pin-hole. As a result, Olympus multiphoton systems enable researchers to achieve in vivo optical sectioning deep into a sample without causing significant photobleaching or phototoxicity.

*The use of lasers with sub-picosecond pulses for two-photon microscopy is protected by U.S. Patent No 5.034.613 This technology was integrated in Olympus laser scanning microscopes, Models FV1000MPE, under a license from Carl Zeiss MicroImaging GmbH and Cornell Research Foundation Inc.