Multiphoton microscopy, also known as non-linear or two-photon microscopy, is an alternative to laser scanning (single photon) or deconvolution microscopy that provides distinct and clear advantages for three-dimensional imaging. Specifically, multiphoton excitation is superior for imaging living cells that reside within intact tissues such as brain slices, embryos, whole organs, and even entire animals. The technique provides optical sectioning with reduced absorption of excitation light in specimen regions removed from the objective focal plane, thus minimizing photobleaching and phototoxicity. The longer excitation wavelengths also enable increased depth penetration over confocal microscopy and are therefore more useful to deep imaging. (For more detailed information click here).
Figure 1: This upright system is optimized for live tissue and whole animal experiments as well as large volume reconstruction of both fluorescent labels and second harmonic generation signals.
Figure 2: Silicone immersion objective and Microprobe objective.
Figure 3: Confocal microscope image of a ZO-1 labeled monolayer of bEnd.3 endothelial cells. The dark patch is an example of photobleaching that is more likely to occur in confocal microscope. Use of a multiphoton microscope can prevent photobleaching by decreasing the intensity and exposure of light to a specific area, making this approach and excellent choice for imaging of living tissues.
Figure 4: Muscle surrounded by adipose tissue. Optical sectioning allows for capture of unique 2D images at multiple planes (different depths) through the tissue, called a z-stack. The z-stack of images can be rendered into a 3D model or compressed (projected through the z-axis) into a single image as shown here. For this image, 56 unique images were taken at 3 μm intervals in the z-axis and compressed into a single 2D image.