White Paper: Imaging

How Mode-Locked Lasers Affect the Past, Present, and Future of Two-Photon Microscopy


For more than 100 years, fluorescence microscopy has been utilized in biological sciences as a means for identifying the spatial distribution of molecules of interest in complex heterogeneous samples. The first fluorescence microscopes relied on ultraviolet light to induce autofluorescence. Shortly after, it was shown that fluorescent dyes could be bound to living cells, allowing the excitation and emission wavelengths to be engineered independently of the sample’s native properties.

These fluorescent tags, as they later became known, quickly became standard practice in fluorescence microscopy for many years because they allowed the excitation energy required to induce fluorescence to be greatly reduced. This, in turn, reduced photochemical degradation of the sample by moving from ultraviolet to visible excitation wavelengths, which was extremely advantageous for biological studies, allowing for sample integrity to maintained especially for live samples.

As a result, fluorescence microscopy has become one of the most widely utilized techniques in biological sciences. For many years, the advantages of fluorescence tags outweighed the disadvantages of the sample preparation requirements but with the emergence of two-photon fluorescence microscopy in the 1990s, many of these same advantages can now be achieved without any sample pretreatment. As a result, two-photon microscopy, also referred to as multiphoton microscopy, has once again revolutionized the field of biological imaging, allowing for the creation of high-resolution images of living cells. Even today, new uses are constantly being discovered.

This white paper explores how two-photon microscopy has evolved over the years and the integral role that the mode-locked laser has played in its evolution. The development of new mode-locked fiber laser technologies is allowing for the expanded adoption of the technique in biological science labs all around the world. This, in turn, is opening up new application areas, as more and more people are gaining access to the technology.

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