Off-axis digital holography (DH), which is based on a Mach-Zehnder interferometer asshown in Fig. 1, is used to acquire digital holograms for 3D image reconstruction. In the offaxis configuration, the coherent laser source is divided into an object (O) and a reference waves (R) using the beam splitter. The object wave illuminates the sample such as red blood cells and creates object wave front. The microscope objective (MO) collects and magnifies the object wave front. A detector such as a CCD camera records the hologram generated by the interference of the object wave and the reference wave, which is incident at a small angle (θ) with respect to the object wave, as shown in the inset of Fig. 1. The recorded holograms are sent to the PC for filtering, encrypting and multiplexing, and reconstruction of the phase contrast image.When the 3D image is reconstructed numerically on computer from the recorded digitalhologram, the reconstructed image includes zero-order noise of diffraction (the first two terms in Eq. (1)) and the virtual image (or twin image) and the real image, which correspond to the third and fourth terms in Eq. (1), respectively [19,34].We need to suppress the undesired data, i.e. zero-order noise and virtual image, byapplying a digitally defined filter mask to a Fourier transform of the hologram in the spatial spectrum domain. This is shown in Fig. 2(c) and results in the filtered hologram, which is shown in Fig. 2(d) and represented bywhere FT and FT −1 are the Fourier and inverse Fourier transforms, respectively, and SF denotes spatial filtering in the Fourier domain. A non-circular shaped SF, as shown in Fig.2(c), is used to filter only the first-order spatial spectral component of a hologram while removing the second-order spectral component at the top right corner of Fig. 2(b). The center of the SF is not intentionally centered on the RO* in order to filter the first-order spectrum as much as possible while minimizing the overlap of the first-order spectrum with the secondorder spectrum and the zero-order noise.The reconstruction of a hologram in the hologram plane is achieved by illuminating the hologram with a replica of the reference wave. The wave front of the reconstructed image is propagated toward the observation plane, in which the 3D image of the object can be observed. The digitally reconstructed image in the observation plane is computed by a numerical calculation of scalar diffraction in the Fresnel approximation, which is expressed as [19,34]where A = exp(i2πd/λ)/(iλd) is a constant, d is the distance between both planes, λ is the wavelength of illumination light, m, n, k, and l are integers (−N/2 ≤ m, n, k, l ≤ N/2), and N × N is the number of pixels on the CCD camera. Δx and Δy are the sampling intervals in the hologram plane, Δξ = λd/(NΔx) and Δη = λd/(NΔy) are the sampling intervals in the observation plane, and RD is the digital reference wave: