a2960_1.pdf CTuV2.pdf
Quantitative Phase Microscopy with Asynchronous Digital Holography System Kevin Chalut, William Brown, Neil Terry, and Adam Wax Biomedical Engineering, Fitzpatrick Institute for Photonics, Duke University 136 Hudson Hall, Durham, NC 27708 email:
[email protected] / t: 919-660-5588 / f: 919-613-9144
Abstract: We demonstrate a new method of measuring quantitative phase in biological materials. The method utilizes asynchronous digital holography, which uses a moving fringe created by acousto-optic modulators. Results are demonstrated on live cell samples. OCIS codes:(090.2880, 110.1650) Holographic interferometry, coherence imaging. Cells are dynamic entities, constantly adjusting their biophysical properties to stay in equilibrium with their environment. An understanding of the cell features, including the behavior of the cytoskeleton, can lead to a better understanding of the feedback mechanisms which control these dynamics. The method we propose, which performs quantitative phase microscopy using an asynchronous digital holography system (ADHS), can measure subtle dynamic changes in the cell volume of normal and diseased cells. Coherent imaging, which is used for measurement of the optical phase, has been of considerable interest lately (1). Measurement of the optical phase, as opposed to the intensity, allows for imaging with axial resolution on a nanometer scale. Phase measurements are also less sensitive to noise than intensity measurements. Zhang and Yamaguchi (2) used a phase-shifting digital holography scheme that combines four holograms to yield phase information. This does not work well with samples that change on a millisecond time scale however, as there could be considerable change in a sample in the time it takes to record four holograms with a CCD. Cuche (3) proposed an inline digital holography system that uses only one hologram to reconstruct the phase information. Feld (4) proposes a system that is off-axis but also uses only one image of the wavefront to reconstruct the phase information. Both of these methods can image dynamic biological samples on a millisecond time scale. Our method provides an alternative for performing quantitative phase measurements on cells using asynchronous digital holography in an off-axis geometry. This is accomplished with optical phase-shifting using acousto-optic modulators (AOMs), as proposed by Li (5). Our method utilizes the line transfer capabilities of a CCD to record two wavefront signals on a microsecond time scale, and then combining them using the knowledge imparted to us by analysis of the rolling fringes created by the AOMs. The CCD is then cleared and ready for another series of two wavefront signals. In this way, we can record quantitative phase measurements on a millisecond time scale with the phase stability and low phase noise associated with phase-shifting interferometry. The integration of the AOMs into a digital holography system opens up new doors for doing experiments that require the high-speed dynamic beam control that AOMs provide. Our experimental setup is similar to the traditional off-axis digital holography microscopy setup (1) with two exceptions. The first is that it is set up in a 4f optical configuration, so that the CCD is in the image plane, the second is that an AOM is inserted into each arm of the interferometer, as shown in figure 1. For our experiments, the AOMs were set to 80 MHz with a 100 Hz beat frequency between them.
Beam splitter laser
mirror
AOM1
AOM2 L1 (f=7.5 mm) 40x MO
mirror sampl
CCD Beam L2 (f=150 mm)
a2960_1.pdf CTuV2.pdf
Figure 1: Setup for ADHS. The optical phase is tightly controlled with the AOMs (acousto-optical modulators). The MO and L1 form a 4f imaging system with L2, so that the CCD is in the image plane. Theory: It is possible to recover the complex analytical signal from a single interferogram via the Hilbert transform. The CCD records the wavefront interference between the reference and sample arms, a purely real signal. The Hilbert transform recovers the complex analytic signal, converting the real signal into a phasor in the complex plane. The AOMs impart a phase shift from one image to the next, thereby rotating the phasor in the complex plane. The fringe (which is linear due to the inherent wavefront matching in our configuration) is fit to a sine wave and analyzed to find both the phase shift Δ and the spatial frequency q. Using this information, the two pictures are combined to cancel the DC and common noise elements, and the phase can be found unambiguously:
I1 I2 G
A( x, y ) B( x, y ) exp ⊥ i> qx
Ι x, y ≅ cc; A( x, y ) B( x, y ) exp ⊥ i> qx Ι x, y Δ ≅ cc; HT ⊥ I 1 I 2 exp iqx /(1 exp i Δ ;
Ι x, y
tan
1
♣Im G ♦ ♥Re G
•. ÷ ≠
Results: We show below a quantitative phase image of a macrophage cell. The phase noise for this measurement was 2.3 nm (as measured by calculating the standard deviation over an area that corresponds to the diffraction-limited spot size, in this case, 8 x 8 pixels). We will show that this is an improvement over the case where only one hologram is used to reconstruct the quantitative phase topography.
Figure 2: A quantitative phase image of macrophage cells. Expected results: We will demonstrate that the ADH system can take real-time quantitative phase measurements on a millisecond time scale. We will demonstrate this with several cell lines, including T84s, macrophage cells, smooth muscles cells, and metachysmal stem cells. 1. Schnars, U., and Jueptner, W. Digital Holography : Digital Hologram Recording, Numerical Reconstruction, and Related Techniques. Berlin: Springer, 2005. 2. Zhang, T., and Yamaguchi, I. Three-dimensional microscopy with phase-shifting digital holography. Optics Letters, 23: 1221-1223, 1998. 3. Cuche, E., Bevilacqua, F., and Depeursinge, C. Digital holography for quantitative phase-contrast imaging. Optics Letters, 24: 291-293, 1999. 4. Ikeda, T., Popescu, G., Dasari, R. R., and Feld, M. S. Hilbert phase microscopy for investigating fast dynamics in transparent systems. Optics Letters, 30: 1165-1167, 2005. 5. Li, E. B., Yao, J. Q., Yu, D. Y., Xi, J. T., and Chicharo, J. Optical phase shifting with acoustooptic devices. Optics Letters, 30: 189-191, 2005.
