ABSTRACT
As was the case with the introduction of confocal fluorescence microscopy in life sciences, the application of second harmonic and multiphoton imaging was another milestone in biological imaging, since it enabled researchers to move from cultured cells deep into tissues and whole organs, even in live animal models. A well-aligned confocal microscope provides faithful representation of the 3D distribution of the fluorophores without image-processing artifacts. Deep tissue imaging, however important in life sciences, is very difficult and faces many limitations and challenges. Methods based on x-ray absorption, nuclear magnetic resonance or emission from radioactive markers, as mere examples, do allow whole body imaging, but lack the resolution and specificity of light microscopy. The high power of incident light required for appreciable 2P absorption in fluorescently labeled samples necessitates the use of lasers as excitation sources. With these lasers, multiphoton imaging of some fluorescent proteins and second harmonic generation imaging are readily achievable.
