The Use of Multiphoton Microscopy for Deep Tissue Imaging

Multiphoton Microscopy is the method of choice for non-invasive deep-penetration fluorescence microscopy of thick biological samples. It is optimal for a diversity of fixed and living biological samples. Specimens can range from thick organ sections, intact organs and whole embryos to entire animals. As compared to the multiphoton excitation microscopy, fluorescence microscopy comes to its limits when imaging thick samples. Visible light is strongly scattered in biological tissues and fluorescence imaging is therefore restricted to an imaging depth of around 100 ┬Ám. The former uses excitation wavelengths in the infrared taking advantage of the reduced scattering of longer wavelengths. This makes multiphoton imaging the perfect tool for deep tissue imaging in thick sections and living animals. Applications range from the visualization of the complex architecture of the whole brain to the study of tumor development and metastasis or the responses of the immune system in living animals. To image several hundreds of micrometers deep into biological samples this process takes place: Biological tissues scatter light which limits the penetration depth of microscopy using visible light Multiphoton microscopy uses red-shifted light, i.e. light that is scattered less Deeper tissue sections may be imaged For multiphoton excitation, pulsed infrared lasers with wavelengths of up to 1300 nm in case of the OPO or optical-parametric oscillator. Deep tissue imaging of thick specimens or in whole animals allows observing cellular processes in their natural context. Especially intravital imaging with multiphoton excitation plays a growing role in many biomedical research areas. How deep one can image sample with the multiphoton microscope depends on the tissue type, age of the tissue, quality of the staining but also on the excitation efficiency which is correlated to the peak power of the laser pulses and how well this peak power can be maintained in the imaging system. The key benefits of this multiphoton excitation process are: Lower scattering, therefore greater penetration depth Spatial confinement of excitation due to low probability of two photons reaching the fluorophore near simultaneously, therefore reduced phototoxicity and bleaching restricted to focal spot Optical sectioning without the need for a detection pinhole allows for the use of non-descanneddetectors; minimize loss of emitted photons, short light path. Along with this, it also provides bleaching restricted to focal plane, no volume bleaching and intrinsical optical sectioning properties.