Multiphoton microscopy has been adopted to perform imaging c

Multiphoton microscopy has been adopted to perform imaging cells in a living animal. - Describe the role of pulse lasers in performing multiphoton microscope. - Typical multiphoton microscope systems uses laser scanning technologies to capture fluoresent image. Please describe the principles of image formation using laser scanning.

Solution

Multiphoton microscopy (MPM) is regarded as the method of choice for imaging of living, intact biological tissues on length scales from the molecular level through the whole organism.

Two-photon excitation microscopy (a type of nonlinear microscopy, also known as multi-photon microscopy is an alternative to confocal microscopy that provides clear advantages for three-dimensional imaging. In particular, two-photon excitation microscopy excels at high-resolution imaging in intact thick tissues such as brain slices, embryos, whole organs, and live animals.

Two-photon excitation depends on the simultaneous absorption of two photons, so the resulting fluorescence emission intensity depends on the square of the excitation intensity. This quadratic dependence gives rise to the enhanced excitation provided by a pulsed laser source, as well as the intrinsic optical section capability of two-photon excitation microscopy .

In a pulsed laser source, all of the photons are concentrated into discrete pulses such that the peak power of the pulse is enhanced relative to the time-averaged power—there is a much greater probability of two photons being incident at a fluorophore at the same time. The shorter the laser pulse, the greater the concentration of photons in time and thus the higher the peak power relative to the average power. In this way, by using an ultra-short pulsed laser source, there can be a significant probability of two-photon absorption occurring while still maintaining low incident power.

High speed scanning with a pulsed laser source has many natural advantages in fluorescence microscopy. It is a common practice in MPM to interrogate samples labelled with several fluorescent probes. The cost of femtosecond lasers often limits a multiphoton imaging system to a single laser. Imaging a sample with several fluorophores often requires tuning of the laser to a compromising wavelength to excite the fluorophores simultaneously, though less efficiently. Alternatively, at the expense of imaging speed and photodamage, the laser can be tuned to the optimal excitation wavelength for each fluorophore and the entire specimen rescanned sequentially. To reduce the damaging effects of rescanning the sample or exciting at a compromising wavelength with higher power, a laser with a shorter pulse width and, therefore, broader bandwidth can be employed.

The total fluorescence signal generation is proportional to the overlap of the two-photon excitation spectrum of the fluorophore and the two-photon excitation spectrum of the broadband laser. Broadband laser pulses, therefore, increase the signal generated by the fluorophores without the necessity to have the laser tuned exactly at the excitation peak. It should also be noted that the two photons required to generate the nonlinear signal do not have to be equal in wavelength, allowing the entire laser pulse spectrum to contribute to the excitation of the fluorophore.

Examples of multiphoton microscopy techniques include second harmonic generation (SHG), third harmonic generation (THG), coherent anti-Stokes Raman spectroscopy (CARS) and stimulated-emission-depletion (STED) microscopy. Because each of these techniques utilized pulsed lasers, it is important to choose optical components that minimize pulse dispersion, and laser-reflecting dichroics should have low GDD characteristics.