A time and photon efficient method of 3D microscopic imaging

A method to obtain three-dimensional (3D) image from a series of two-dimensional (2D) images which are obtained without moving the relative positions of the target, the detector, or the focusing lens. The 2D images consist of one centered image obtained with the aperture at the center of optical system, and at least two directional images obtained with apertures at off-axis locations. The images can be obtained simultaneously or sequentially. The 2D images are sectioned into different depths by computational method resulting in a set of decoupled 2D layers of the 3D object at different axial planes. The 3D image is displayed by color-coding. This technique provides fast data acquisition and fast image reconstruction and eliminates problems associated with motion, phototoxicity and photobleaching.