An instrument to acquire and methods to obtain three-dimensional (3D) images 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 is disclosed. 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 blurred 2D images are sectioned by computational method using point spread function of the optical system resulting in a set of decoupled 2D layers of the 3D object. The layered images are then sharpened by deconvolution using point spread function. The 3D reconstructed image is displayed. This technique provides fast data acquisition and fast image reconstruction and eliminates problems associated with motion, phototoxicity and photobleaching.