Multiphoton Laser Scanning Microscope for Live Cell Imaging

Zeiss has overcome the challenge of imaging deep inside living tissues with the launch of the LSM 5 MP multiphoton laser scanning microscope. The instrument uses longer-wavelength, femtosecond lasers to create and detect fluorescence signals at a depth of up to 500 microns, illuminating cellular structures in great detail whilst eliminating phototoxic cellular damage caused by high intensity light.

The LSM 5 MP is based on the Axio Observer inverted research microscope platform and takes advantage of its optical features. The coatings on the optics ensure minimal power losses of the directly coupled femtosecond lasers while advanced filter technology guarantees suppression of stray and excitation light and sensitive detection of fluorescent signals.

The optical standards are set by the W Plan-Apochromat objective lens, which boasts 20x agnification at a numerical aperture of 1.0 and a working distance of 2mm. It is ideally suited to multiphoton electrophysiology experimentation, allowing larger areas to be imaged with unsurpassed resolution and brilliance.

The specially designed objective lenses feature optical correction in the near infra red (NIR) 700-1100nm spectral range to reduce absorption and scatter. This allows the NIR laser to penetrate tissue to greater depths working in ultrashort pulses of up to 170kW at a frequency of 76 to 90 MHz.

The multiphoton LSM 5 MP restricts fluorescence excitation to the focal spot. The laser beam only excites fluorescence if at least two photons are absorbed by a fluorochrome molecule within less than a femtosecond (10 -15 seconds) and the whole of the emitted light signal can be used for imaging.

The LSM 5 MP is suited to the study of live biological tissue, including intact animals, where its ability to image fluorescence-labelled cellular structures at depths up to 500 microns without damage is a key advantage.