Holographic interferometric microscopy systems for the application on biological samples

Abstract

Holographic interferometric metrology allows a fast, non destructive and quantitative high resolution full field detection of optical path length changes. Furthermore, by utilization of modern CCD sensor technology and digital image processing algorithms an on-line application of these methods even on biological specimens is possible. In combination with a microscopic resolution this offers new possibilities for the detection of variations of shape, micro movements or refractive index changes e. g. for the marker free analysis of cellular samples. Three holographic interferometric systems for microscopy applications based on digital holography, (speckle) interferometry and photorefractive crystals as holographic recording medium are introduced. Results of investigations on test charts and biological samples to characterize and optimize the lateral resolution as well as the resolution of the detected phase difference changes are presented and discussed. Finally, the applicability of the developed measurement techniques on living cells is demonstrated. INTRODUCTIONHolographic and speckle interferometry are well-established and frequently applied tools for non destructive testing (NDT) and the analysis of displacements, movements and vibrations. With a measurement accuracy in sub-micrometer range this methods have already been applied in many fields of industry [1] as well as for biomedical applications [2]. Furthermore, by utilization of modern CCD sensor technology and fast digital image processing an on-line application of these methods even in vivo [3] is possible. Holographic interferometric metrology allows a fast, non-contact and quantitative high resolution full field detection of optical path length changes. This offers in combination with a microscopic resolution new possibilities for the analysis of cellular tissues and cells. In the upcoming fields of life sciences and biophotonics there is a requirement for an optical instrumentation of timely and spatially high resolution analysis, measurement and documentation in microscopic dimensions. Here, applications are of particular interest to get enhanced information about cellular tissues and single cells e. g. the detection of shape variations and refractive index changes as well as the full field analysis of parameters like micro movements or the local distribution of elasticity. Up to the present, such parameters are in general not considered in cellular and sub cellular dimensions although they are already routinely used e. g. for defect detection and tumor diagnostics in the macroscopic scale. In that view, holographic interferometric microscopy tools open up new facilities for a marker free monitoring of life processes and cell manipulation as well as for an automated cell differentiation and feedback systems for growing processes in tissue engineering.For such applications of holographic interferometric metrology, the specific requirements for the analysis of cellular tissues and living cells have to be considered and a lateral resolution in microscop...

High-resolution digital holography for shape measurement of microscopic object

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Reconstruction Method of Wavefronts in Digital Micro-Holography

“…Therefore, digital holography has been paid a wide attention in recent years. The main applications includes observation of biological samples [1,2], living cells analysis [3][4][5][6], 3D pattern recognition [7,8], vibration analysis [9], measurement of MEMS and MOEMS [10] and so on.…”

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