Polarization and Spectral Interferometric Techniques for Quantitative Phase Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 275

(A) (B) (C)

T=0ms

5 μ m

(D)

(E)

Biased
optical
path
length

5

Without sample
σ = 0.67 nm

5
4
3
2
1
0

0

Outside cell
σ = 0.87 nm

Cell edge
σ = 1.83 nm

Cell center
σ = 1.31 nm -5

0 100 200 300 400 500

Figure 14.2
MDA-MB-468 human breast cancer cell. (A) Transmission brightfield microscopic imaging
through the system; (B) the two phase-shifted interferograms in a single camera exposure; (C)
final unwrapped phase profile; (D) surface plot of the phase profile shown in (C); (E) temporal
phase stability without the sample and with the sample in the three points marked in (C). Source:
Adapted from Ref. [13] .

(5 mW, 633 nm) as the light source. In both interferometer arms, 40 3 , 0.65 NA achromatic

objective lenses were used, each creating 33 magnification when paired with lens L 2

(15 cm focal length). The resulting complex amplitude was spatially restricted by a
3.2 mm

3

2.3 mm aperture. The 4 f image-splitting system, as shown in Figure 14.1B ,
consists of two lenses L 3 and L 4 of similar type, each with a focal length of 7.5 cm.
Between these lenses, the Wollaston prism (crystal quartz, 2 of angular separation) splits
the pattern from the aperture plane so that the charge-coupled device (CCD) camera (AVT
Pike F032-B, 640

3

480 pixels) captures the two phase-shifted interferograms I 1 and I 2
shown in Figure 14.2B in one frame but with a spatial separation of about 2.4 mm. To
determine the coregistration between the two phase-shifted interferograms recorded in the
single CCD image, images of a test target (United State Air Force (USAF) resolution chart)
were used as a calibration standard. The frequency q and the actual phase shift α of the
interferometric fringes were extracted by fitting each of the interferograms to a sine wave.
The background interference fringes are oriented vertically in each of the interferograms,
yielding

3

φ C 5qx , where x is the horizontal coordinate in each interferogram frame.
Figure 14.2C and D shows the final unwrapped phase profile of the sample, obtained by
applying Eq. (14.2) , and a phase unwrapping algorithm. The same cell was imaged every
8.3 ms (120 frames per second (fps)) for a duration of 500 ms.