Nikon A1R-MP Multiphoton Microscope
The A1R-MP+ multiphoton microscope system is equipped with a Coherent Ti:S Chameleon Vision S laser tunable from 690nm to 1050nm. Further information on the laser can be found here. Raster scans can be switched between a Galvo-controlled mechanism or a resonant scanner, the latter allows user to acquire images at a rate of hundreds of frams per second. The multiphoton system should be used primarily for deep specimen penetration for up to 1.5mm (actual penetration depth is sample-dependent). The Ti:S laser will allow users to combine mainly blue, green and red emitting fluorophores.
The system is also equipped with forward and backward detectors for second harmonic wave detection, allowing users to image anisotropic biomaterials such as collagen fibers and myofibrils label-free. Operation of the system is driven by Nikon Elements NIS software.
Principle of Non-linear Multiphoton Microscopy
Multiphoton microscopy relies on red-shifted (longer) excitation light that illuminates the samples in very short pulses, usually in femtoseconds. Two photons of longer wavlength and weaker energy will be absorbed by the fluorescent dye in one quantum event. The multiphoton effect - wherein near-simultaneous absorption of two excitatory photons - is so low that it will only occur at a diffraction limited spot at the focal point of the objective lens. Therefore, multiphoton microscopy obviates the need for the pinhole to reject out-of-focus light, and can employ non-descanned detectors which are significantly more efficient at light collection than standard PMTs used in confocal microscopes. The near-IR laser also minimizes scattering within the specimen. The combined effect is that multiphoton microscopy provides deeper tissue penetration, low background, and efficient light detection, all critical for intravital imaging.
In addition, multiphoton microscope set-up can also take advantage of another non-linear phenomenon, second harmonic generation or frequency doubling. In second harmonic generation, photons interacting with an anisotropic medium (basically structures that are directionally dependent, or highly ordered materials, such as collagen fibers) are essentially "combined" to form new photons with twice the energy, thus half the wavelength of the initial photon. This becomes a very powerful and convenient way to detect certain biomaterials i.e. collagen, myfibrils, without any fluorescent label, as shown in the example below.
Non-linear second harmonic generation to detect anisotropic structures, the A1R-MP is also equipped with backward scattered and forward propagated second harmonic waves to image label-free collagen, as well as myofibrils in situ. As shown above, green channel is fluorescence, and blue channel is second harmonic generation by in situ collagen I in skin.
ThHe Nikon A1R MP was acquired through an S10 shared instrumentation grant (1 S10 OD010398-01) awarded to Dr. Chew.