Volumetric imaging with two-photon stereomicroscopy

The projection of a 3D image stack is commonly information richer than a single 2D slice of the stack, especially in laser scanning fluorescence microscopy. Similarly, extended depth-of-field (EDOF) microscopy, which acquires the projection of the sample volume, contains more information than an ordinary 2D wide-field image or a single optical section image. However, one inevitable problem of EDOF is the loss of depth information. If isotropic resolution is not a necessity, two projections with a small parallax angle in between can regain some depth information through the principle of stereo vision. Unfortunately, stereo vision in microscopy always involves two separate optical paths, which is not easy to implement in laser scanning microscopy. Even worse, if the two projections are acquired frame by frame sequentially, the stereoscopic approach is meaningless for fast dynamic process, because the frame rate may be too low for capturing the same feature in the same position at one time point for two views. In fact, the parallax angle of EDOF(Bessel beam[2] created by axicon) was previously generated by shifting either the axicon or the objective lens laterally. Obviously, mechanically moving the high inertia optical elements cannot meet the requirement of simultaneity of two view channels.