Multi-color digital holographic microscope (DHM) for biological purposes. Zolt´ anG¨or¨ ocs, M´arton Kiss, Veronika T´ oth, L´ aszl´oOrz´ o, Szabolcs T˝ok´ es Computer and Automation Research Institute (MTA SZTAKI), Kende ut 13., Budapest, Hungary; ABSTRACT Our digital holographic (DH) approach can be used to study tissue structures both in vitro and in vivo. This DHM architecture can produce three color microscopic 3D and 4D (video) images. We record 3 color (RGB) holograms with single exposures, and the perfect compensation of color crosstalk is solved. An in-line holographic setup and reconstruction algorithms are presented with demonstrative simulations and experimentally captured and numerically reconstructed images. Comparing the individually reconstructed color images with each other can provide information both for recognition of different types of cells or microorganisms, and for diagnostic purposes as well. Experimental example is given observing microscopic hydro-biological organisms using a color digital holographic microscope. Keywords: Color Digital Holography, Digital Holographic Microscope, Color crosstalk compensation 1. INTRODUCTION In recent years several journal articles reported the accelerating developments in the field of digital holography. The possibility to obtain volumetric information from a single image using wave field propagation algorithms makes digital holography a promising method to investigate moving biological organisms in fluids. As in ordinary holography, digital holography is based on capturing the interference pattern of the object and the reference beams. The difference here is, that instead of using a high resolution holographic recording medium the digital holographic system uses an image sensor to acquire the holograms. The reconstruction of this digitally recorded hologram is done numerically. Digital holography is frequently used to capture 3D information of objects within a volume 1 . 2 The recorded holograms are inherently monochromatic, however several multi-wavelength approaches are known as well, as the color of the object can sometimes carry relevant information. These approaches usually use time multiplexed recordings, where the illumination of the object is done by only a single wavelength at a given time, and the captured image is later reassembled algorithmically. 3 There are measurements where objects were simultaneously illuminated by several wavelengths 4 and refractive index measurements of phase samples using multi wavelength illumination. 5 To our knowledge there are methods that use only monochromatic, 6 two-color, 7 or sequentially exposed three color cases, 8 where digital holography is used in a digital holographic microscope. In the case of single shot three-color exposure, color-crosstalk can cause noise by false reconstructions. Our research focuses on measuring the biological content of water samples by DHM. The essence and novelty of our method is the way how we can remove the color crosstalk of commercial color cameras, and thus the ability to capture color digital holograms at video rate. The three color holograms are recorded simultaneously, with a single shot. Using in-line digital holography for multiple object recognition requires additional segmentation and twin-image removal tasks. Our applied methods and the first results of this research are presented in this paper. Further author information: (Send correspondence to Sz.T.) Z.G.: E-mail: zoli.gorocs@gmail.com, L.O.: E-mail: orzo@sztaki.hu, Sz.T.: E-mail: tokes@sztaki.hu, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VIII, edited by Daniel L. Farkas, Dan V. Nicolau, Robert C. Leif, Proc. of SPIE Vol. 7568, 75681P · © 2010 SPIE · CCC code: 1605-7422/10/$18 · doi: 10.1117/12.841962 Proc. of SPIE Vol. 7568 75681P-1