Phase Unwrapping Problems in Digital Holographic Microscopy of Biological Cells - Biomedical Optical Phase Microscopy and Nanoscopy - page 124

CHAPTER 7
Phase Unwrapping Problems in Digital
Holographic Microscopy of Biological Cells

Alexander Khmaladze
Department of Chemistry, University of Michigan, Ann Arbor, MI

Editor: Natan T. Shaked

7.1 Introduction

Quantitative phase mapping in digital holographic microscopy carries the three-dimensional
(3D) information about the object. However, phase mapping is ambiguous, as absolute phase
is wrapped in the intervals of 2 . This results in a discontinuous imaging, as the phase signals

from the points with heights that are exactly integer number of wavelengths apart are the
same. The phase unwrapping can be done using a software algorithm that looks for jumps in
the phase image and shifts the image segments up or down depending on the surrounding
topology. However, when it comes to imaging real objects (and especially biological cells),
the typical phase image is noisy and may have areas of low intensity, where the interference
fringes are not visible. Many phase unwrapping software algorithms using different methods
have been developed in the past [1] . Ultimately, however, the software algorithms rely on
surrounding pixels to unwrap the 2

π

discontinuities. As a result, if a certain area within the
phase image proves to be problematic for the unwrapping program, not just the unwrapping
from this area is affected, but the error often propagates to other regions as well.

π

In this chapter, first a method to computationally unwrap the phase is discussed. This
method, instead of heavily relying on the immediate surrounding pixels in a single phase
image, uses a set of multiple phase images obtained using the angular spectrum method
[2,3] , with the reconstruction performed at various axial distances. While this method is
generally applicable, it was designed for imaging cell-like objects on a flat background, in
which case it is especially fast and effective.

Also, the multiple wavelength phase imaging technique, which is based on the comparison
of phase maps, acquired using different wavelengths, is discussed. This dual-wavelength