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Visualizing Cellular Networks

Dr. Andrea Pfeifer, Leica Microsystems

A common cause of the most life-threatening diseases is the disruption of complex biological networks. The importance of intercellular connections is an emerging feature of cancer, neurological and immunological diseases. Therefore, a growing number of research project focus on intact cellular networks. Specimens used for these studies are thick tissue sections or whole animals. Imaging cellular networks at high resolution deep within biological tissues is a demanding task. Visible light is strongly scattered by the specimens and cannot excite fluorophores in deep tissue sections. Here, multiphoton microscopy is the perfect tool as it uses infrared light for excitation which is less prone to scattering in tissues. The new Leica TCS MP5 is dedicated to the needs of multiphoton imaging. Optimal transmission up to 1300 nm and an integrated Optical Parametric Oscillator (OPO) solution allow researchers to reach even deeper into samples.

Intravital imaging of soft tissues

Gaining access to the tissue of interest without interrupting the studied process is crucial for the success of intravital imaging. In neurosciences, connections and interactions between neurons and other cell types of the nervous system are regularly studied in living animals by multiphoton microscopy.

More recently, intravital imaging of soft tissues provides exciting new insights into the biology of cancer. Repeated observation of tumor metastases and blood vessel formation over several days is possible with imaging windows implanted in the skin of the animal.

Multiphoton imaging – individually configured

As a dedicated multiphoton microscope, the Leica TCS MP5 is the perfect platform for intravital imaging. In combination with the Leica DM6000 CFS fixed stage microscope it offers the highest mechanical and electronical stability for demanding applications like electrophysiology. The Leica TCS MP5 equipped with the inverted microscope Leica DMI6000 B is optimal for observation through ventral
imaging windows.

Many processes in living organisms are highly dynamic and need to be monitored with high temporal resolution. The Leica Tandem Scanner combines two scanners to offer the full range of scan speeds in one Leica TCS MP5 system. The resonant scanner allows recording of fast dynamics and faster acquisition of detailed z-stacks by high-speed scanning. For optimal spatial resolution the conventional scanner can be used with an exceptionally large field of view of 22 mm.

Explore the full range of fluorescent proteins

Interactions of proteins and different cell types are monitored by using specific fluorescent markers for each species. The Leica TCS MP5 can accommodate up to seven external detectors for simultaneous multichannel acquisition. This includes non-descanned detectors (NDDs) for reflected and transmitted light detection of fluorescent signals and a high-contrast brightfield detector to give researchers the full picture of their sample.

Excitation up to 1300 nm with an optical parametric oscillator (OPO) allows researchers to explore the full range of fluorescent proteins and dyes by multiphoton microscopy. With Leica’s OPO solution simultaneous multiphoton excitation of green and red fluorophores is possible. The longer wavelengths of the OPO can reach deeper into tissue and are less harmful to samples. Leica Microsystems offers an OPO solution that is fully integrated in the software interface of LAS AF for easy tuning and setup of laser wavelengths. High transmission up to 1300 nm of the Leica TCS MP5 scan optics brings all the power from the OPO to the sample (see Research reSOLUTION No.9, p. 8).

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