Quantitative phase measurements have merited great interest lately for measuring cellular dynamics [1
]. One natural modality for studying these dynamics is digital holographic interferometry (DHI) [3
], a simple interferometric configuration that can be used to assess subwavelength changes in an object by measuring the optical path length difference between each imaged point at two distinct times. There are complications, however, in utilizing DHI at a high enough speed to visualize cell dynamics, usually considered to be ~10 ms. Primarily, unambiguous phase extraction requires separating the phase information from the rest of the signal in the interferogram. In this paper, we present a system designed to overcome these complications. The system uses asynchronous digital holography (ADH), a phase referencing method based on acousto-optically generated moving fringes, to perform quantitative phase microscopy. We show that ADH obtains quantitative phase images on the 10 ms time scale, and demonstrate its application to several biological systems.
The goal of holography-based phase imaging is to extract the phase from a holographic image. To do this, the method may take one of three approaches: First, multiple images can be recorded to gain enough information to yield the phase term, second, the background can be removed via optical processing, or third, the background can be removed through digital image processing. Zhang and Yamaguchi [4
] use the first approach, implementing a phase-shifting DHI scheme that combines four interferograms to yield the phase term. This works well with stationary samples, but does not work well with samples that change on millisecond time scales, as considerable changes can occur in the time it takes to record four interferograms with a CCD. Additionally, the longer the recording time, the greater influence phase perturbations due to environmental disturbances have on phase image quality. Depeursinge and coworkers take the second approach, employing an off-axis digital holography system [5
] that uses only one interferogram to reconstruct the phase information, thereby making it an imaging technique applicable to visualizing living cells [1
]. In this method, the reference beam is tilted to induce spatial separation of the background from the desired signal. The tilt of the reference beam can limit the field of view and spatial resolution of the phase image [6
]. Feld and coworkers take the third approach; they created a system that uses only one image, isolating the phase term via image processing [7
], similar to the Fourier transform evaluation method [8
]. This system can be used for wide-field imaging and has no limitations on the spatial resolution; it has been successfully used to quantify cell structure and dynamics [2
]. One possible disadvantage of this approach, among others [3
], is that in using image processing to eliminate unwanted background, some desired signal may be eliminated as well.
We now propose an alternative quantitative phase microscopy method, asynchronous digital hologray (ADH) that integrates two new features into a holographic imaging setup. The first is the use of phase referenced interferometry. Phase shifting is achieved using acousto-optic modulators (AOMs), as proposed by Li [10
]. Here the phase shift is measured, as opposed to tightly controlling it, to greatly simplify the experimental setup by eliminating the need for control electronics. The phase reference is obtained by tilting the reference beam slightly off-axis to observe several fringes across the image plane. These fringes, which move due to a frequency offset between the two AOMs, are analyzed to measure the phase-shift between successive images. The second feature is that only two images, which can be recorded in a near-simultaneous manner (as described in section 2), are used to find the phase distribution. This not only avoids the disadvantages of phase-shifting interferometry described above, but also permits us to eliminate the background and extract the phase unambiguously, without using image processing or introducing a large angular tilt in the reference arm. In the following sections, we will describe the ADH setup and processing algorithm and present quantitative phase microscopy images of water and living cells to demonstrate quantitative phase imaging of biological samples.