Even though holography is a powerful technique for three-dimensional (3D) imaging, it could not be applied easily to an incoherent imaging system, for instance fluorescence microscopy. 3D imaging capability in fluorescence microscopy becomes more and more important lately because of great research demand on cell behavior changes in 3D environmental conditions triggered by recently developed 3D cell culture techniques. Biological research on contact-based cell-to-cell or membrane-to-membrane interactions need 3D imaging as well. Lately, several incoherent holographic imaging methods such as scanning holography, self-interference incoherent digital holography (SIDH), and Fresnel incoherent correlation holography (FINCH) have been proposed for 3D holographic imaging with incoherent light. Most of these methods commonly use an in-line holographic microscopy configuration, which has fundamental image quality degradation problems associated with the two DC terms and the twin image term in a measured hologram. It can be removed by taking multiple holograms with different phase delays in measurement or by iterative methods in numerical reconstruction algorithms. Even though off-axis holography is a straightforward method to remove the DC terms and the twin image, it is difficult to be used with incoherent light due to large path length difference between two beams in an off-axis holographic microscopy configuration. In this study we propose a new off-axis FINCH system which can overcome the path length mismatch problem by using a wedge plate. The combination of a spatial light modulator and a wedge plate in our proposed off-axis FINCH system makes the path lengths of two beams almost identical.
We propose a simple non-iterative in-line holographic measurement method which can effectively eliminate a twin image in digital holographic 3D imaging. It is shown that a twin image can be effectively eliminated with only two measured holograms by using a simple numerical propagation algorithm and arithmetic calculations.
We have investigated distortions in the axial position calculations of a sample in lens-free digital inline holography (LDIH). Three-dimensional structure of a sample can be accurately obtained through a series of processes in LDIH, Fourier-domain digital filtering, and numerical focusing. The axial information of a sample is calculated through numerical beam propagation using diffraction theory and can be easily distorted because of approximations and assumptions used in the diffraction formula and the numerical beam propagation analysis used in LDIH. Since the reference light in LDIH is normally a diverging spherical beam from a point source, axial information of a sample calculated by a numerical focusing algorithm with a plane reference beam is off from the real axial position of a sample. We propose an algorithm that can correct this distortion in LDIH.
3D imaging is demanding technology required in fluorescence microscopy. Even though holography is a powerful technique, it could not be used easily in fluorescence microscopy because of low coherence of fluorescence light. Lately, several incoherent holographic methods such as scanning holography, Fresnel in coherent correlation holography (FINCH), and self-interference incoherent digital holography (SIDH) have been proposed. However, these methods have many problems to be overcome for practical applications. For example, DC term removal, twin image ambiguity, and phase unwrapping problems need to be resolved. Off-axis holography is a straightforward solution which can solve most of these problems. We built an off-axis SIDH system for fluorescence imaging, and investigated various conditions and requirements for practical holographic fluorescence microscopy. Our system is based on a modified Michelson interferometer with a flat mirror at one arm and a curved mirror at the other arm of the interferometer. We made a phantom 3D fluorescence object made of 2 single-mode fibers coupled to a single red LED source to mimic 2 fluorescence point sources distributed by a few tens of micrometers apart. A cooled EM-CCD was used to take holograms of these fiber ends which emit only around 180 nW power.
An effective volumetric measurement method for determination of the 3D position of a single particle based on off-axis digital holographic microscopy is presented in this paper. 3 μm polystyrene bead suspended water is used as a particle to be traced in our experiment. We have demonstrated the feasibility of our method by observing the bead undergoing Brownian motion in water by implemented setup. For fast determination, a series of transverse intensity images are numerically reconstructed from a single hologram of the bead, then centroid technique is applied to the reconstructed images for enhancing the position resolution. Nano-meter scale resolution with a frame rate of 30 is achieved in this experiment in both lateral and transverse direction.
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