Influence of defocus on quantitative analysis of microscopic objects and individual cells with digital holography

Digital holography offers a unique method for studying microscopic objects using quantitative measurements of the optical phase delays of transmitted light. The optical phase may be integrated across the object to produce an optical volume measurement, a parameter related to dry mass by a simple scaling factor. While digital holography is useful for comparing the properties of microscopic objects, especially cells, we show here that quantitative comparisons of optical phase can be influenced by the focal plane of the measurement. Although holographic images can be refocused digitally using Fresnel propagation, ambiguity can result if this aspect is not carefully controlled. We demonstrate that microscopic objects can be accurately profiled by employing a digital refocusing method to analyze phase profiles of polystyrene microspheres and red blood cells.