Nonlinear microscopy


SHG z-stack from the external surface to the internal surface of an excised salamander eyeball (through sclera). Sample is unlabeled. SHG from external surface is produced by muscle fiber (scaly structures) and collagen (wavy structures). SHG from internal surface is possibly produced by axon bundles. Sample provided by Dr. Chris Passaglia (BU, BME Dept.)

Nonlinear optical microscopy is generally divided into two categories: incoherent or coherent. Incoherent microscopy produces an optical signal whose phase is random and whose power is proportional to the concentration of radiating molecules. Fluorescence is a common example of an incoherent signal. Nonlinear versions of fluorescence microscopes are based on the simultaneous absorption of two or more photons, the most well known being two-photon excited fluorescence (TPEF) microscopy. In TPEF microscopy, two excitation photons from a pulsed laser (typically a Ti:sapphire laser of wavelength 700nm-1000nm) combine to excite a fluorescent molecule. The molecule then releases its excitation energy as a fluorescence photon (typically a visible wavelength). Because the excitation is nonlinear, the fluorescence is confined to the focal center of the laser beam, and fluorescence power decays as 1/z2, where z is the axial distance away from the focus (see figure). TPEF microscopy therefore confers 3D-imaging with out-of-focus background rejection similar to a confocal microscope. The advantage of TPEF microscopy over confocal microscopy is that it can penetrate deeper in thick tissue.

Coherent microscopes produce optical signals whose phase is rigorously prescribed by a variety of factors including the excitation light phase and the geometric distribution of the radiating molecules. Coherent signal power is proportional to the concentration of radiating molecules squared. Nonlinear versions of coherent microscopy are based on the simultaneous scattering of two or more photons. Examples are second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy. SHG signals are highly sensitive to molecular orientation.

We have worked on further developments of TPEF  microscopy and its applications to brain imaging. For example, we have implemented TPEF microscopy with simultaneous autoconfocal microscopy, differential aberration imaging (DAI), and reverberation multiplane imaging. We have also implemented an optical dynamic clamp for calcium uncaging.

TPEF z-stack of glomeruli in the excised olfactory bulb of an OMP-synaptopHluorin mouse. The resting GFP fluorescence indicates presynaptic regions of olfactory receptor neurons. Image width is 350μm. Sample provided by Dr. Matt Wachowiak (BU, Biology Dept.)
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