Simultaneous two-wavelength transmission quantitative phase microscopy with a color camera

Article (PDF Available)inOptics Letters 35(15):2612-4 · August 2010with28 Reads
DOI: 10.1364/OL.35.002612 · Source: PubMed
We present a quantitative phase microscopy method that uses a Bayer mosaic color camera to simultaneously acquire off-axis interferograms in transmission mode at two distinct wavelengths. Wrapped phase information is processed using a two-wavelength algorithm to extend the range of the optical path delay measurements that can be detected using a single temporal acquisition. We experimentally demonstrate this technique by acquiring the phase profiles of optically clear microstructures without 2pi ambiguities. In addition, the phase noise contribution arising from spectral channel crosstalk on the color camera is quantified.


Simultaneous two-wavelength transmission quantitative
phase microscopy with a color camera
Matthew T. Rinehart,
* Natan T. Shaked,
Nathan J. Jenness,
Robert L. Clark,
and Adam Wax
Department of Biomedical Engineering, Fitzpatrick Institute for Photonics, Duke University, Durham, North Carolina 27708, USA
Department of Mechanical Engineering and Materials Science, Duke University, Durham, North Carolina 27708, USA
*Corresponding author:
Received April 13, 2010; revised June 11, 2010; accepted June 24, 2010;
posted July 8, 2010 (Doc. ID 126768); published July 28, 2010
We present a quantitative phase microscopy method that uses a Bayer mosaic color camera to simultaneously
acquire off-axis interferograms in transmission mode at two distinct wavelengths. Wrapped phase information
is processed using a two-wavelength algorithm to extend the range of the optical path delay measurements that
can be detected using a single temporal acquisition. We experimentally demonstrate this technique by acquiring
the phase profiles of optically clear microstructures without 2π ambiguities. In addition, the phase noise contribu-
tion arising from spectral channel crosstalk on the color camera is quantified. © 2010 Optical Society of America
OCIS codes: 090.5694, 090.4220, 100.5088, 180.3170.
Transmission-geometry quantitative phase microscopy
(QPM) has been developed for three-dimensional mea-
surement and characterization of a wide variety of trans-
parent samples, such as transparent optical elements
(e.g., microlens arrays) [1], optical fibers [2], and living
cells [3,4]. This interferometric measurement of optical
path delays (OPDs) provides quantitative contrast arising
from both the physical height of the sample and its refrac-
tive index changes. While QPM provides diffraction-
limited lateral resolution and nanometer-scale axial
resolution of OPDs, the axial range over which the phase
can be unambiguously determined is limited to 2π, which
corresponds to one full wavelength of the illumination
To solve the 2π ambiguities in the acquired phase pro-
files and determine the correct relative OPDs across a
field of view, two-dimensional unwrapping algorithms
are typically employed. Classic unwrapping algorithms
are based on gradient minimization, which adds multi-
ples of 2π at specific points in the phase map [5]. While
these algorithms can accurately recover a smooth and
slowly varying phase map, they frequently fail to accu-
rately reconstruct objects containing a phase difference
greater than π between adjacent image pixels.
An alternative approach to overcoming the limitations
of phase unwrapping is the use of multiple illumi-
nation wavelengths. By processing the phase profiles ob-
tained from different wavelengths, it becomes possible to
synthesize a single phase profile that is unambiguous
over the range of the beat wavelength [6,7], which is
larger than the unambiguous range that results from
using a single wavelength independently. Because this
approach is based on an analytical solution and not an
iterative numerical method, two-wavelength phase un-
wrapping is particularly useful when classic unwrapping
algorithms fail to correctly unwrap across sharp phase
Two-wavelength phase unwrapping has been pre-
viously employed in reflection-geometry QPM using
both sequential [8] and simultaneous [9,10] illumination/
detection schemes to accurately reconstruct surface
profiles of highly reflective structures. Two-wavelength
phase unwrapping has also been used for transmission-
geometry phase microscopy [1,11]; however, these
methods use sequential illumination and detection,
which require multiple temporal acquisitions and careful
synchronization of illumination switching and image
acquisition. In this Letter, we present a transmission-
geometry optical microscope that uses a Bayer mosaic
color CCD camera to capture off-axis interferograms
at each of two wavelengths in a single exposure. This
method simplifies both the optical setup and the process
of data acquisition substantially, and guarantees that the
unwrapping process will not be affected by differing mo-
tion artifacts. The wrapped phase information retrieved
from these interferograms is input into a two-wavelength
unwrapping algorithm to unambiguously reconstruct the
OPD map.
Figure 1 presents the optical system, an off-axis inter-
ferometric transm ission microscope based upon the
MachZehnder geometry. Light from two distinct wave-
lengths from separate laser sources, chosen to match the
peak spectral responses of the Bayer pattern red and
green channels, is coupled into a beam splitter (BS1) and
aligned to follow identical optical paths. The two arms of
the interferometer contain matched microscope objec-
tives (MOs) that are aligned in 4f configuration with lens
L1 to magnify the sample while maintaining matched
optical wavefronts. A tilt in the path of the reference
arm produces a high frequency linear interference pat-
tern in the image plane. A Bayer mosaic color camera
1, inset) records an off-axis interferogram at each
Fig. 1. (Color online) Optical system based on a modified
MachZehnder interferometric microscope with two illumina-
tion lasers. Inset, pixel filter layout of a Bayer mosaic color
2612 OPTICS LETTERS / Vol. 35, No. 15 / August 1, 2010
0146-9592/10/152612-03$15.00/0 © 2010 Optical Society of America
wavelength simultaneously. Based on the Bayer mosaic
pixel geometry, the red and green channels have larger
spatial sampling periods than an equivalent grayscale de-
tector; therefore, the system magnification necessary to
avoid aliasing is higher for the color camera and results
in a smaller field of view [12].
Quantitative OPD maps of microsc opic samples are
obtained by the following methods: First, off-axis inter-
ferograms are simultaneously acquired at both wave-
lengths and are separately detected by color channel.
Digital spatial filtering in the Fourier domain is used to
remove the contributions from the complex conjugate
and autocorrelation terms, and the remaining frequency
content is recentered in spatial frequency space to demo-
dulate the signal [13,14]. Next, wrapped phase informa-
tion at each wavelength is recovered by taking the
angle argument of the complex data on a pixel-by-pixel
basis. Finally, the OPD map at the synthetic beat wave-
length is calculated and used as a guide to add the correct
multiple of 2π to each point of the wrapped OPD map for
one of the individual wavelengths [8] chosen to be the
shorter wavelength, since it offers slightly better diffrac-
tion-limited resolution. The resulting OPD map is unam-
biguous over the range of the synthetic beat wavelength
and retains the low noise level of the original single wave-
length wrapped image.
In our experiments, a red HeNe laser (λ
632:8 nm) and a green diode-pumped solid state (DPSS)
laser (λ
¼ 532 nm) were used as illumination sources,
which together produce a synthetic beat wavelength,
Λ,of3:334 μm(Λ ¼ λ
j)[8]. Imaging was ac-
complished using 40× objectives with an NA of 0.66 in
4f configuration with a 150 mm focal length lens, provid-
ing a sample magnification of 33:3× at the image plane.
The interferometric signal was recorded using a Bayer
mosaic color camera (12 bit CoolSNAP cf, Photometrics)
with 1392 × 1040 4:65 μm square pixels. The diffraction-
limited resolution of the system is 0:492 μm and 0:585 μm
(d ¼ 0:61 λ=NA) for the green and red illuminations,
Based on the spectral response curves of the camera,
there is a 5% predicted intensity crosstalk between the
red and green channels, which cou ld slightly increase
the phase noise in each interferogram when both are cap-
tured simultaneously. When uniformly illuminated with
only the red laser, the red-to-green crosstalk was found
to be 4.3%; when illuminating with only the green laser,
the green-to-red crosstalk was found to be 5.4%. These
crosstalk figures indicate that a Bayer mosaic filter pro-
vides high wavelength selectivity. To assess the effects of
intensity crosstalk on phase measurement, interfero-
grams with no sample were acquired individually with
either red or green illumination, and then simultaneously
with both illumination sources. OPD maps were calcu-
lated using the methods described above, and the OPD
standard deviation, σ,ofa14 × 14 μm region was com-
pared. Comparing individual to simultaneous illumina-
tion, σ
changed from 15:5 to 15:9 nm, and σ
changed from 13:7 to 13:8 nm. Since neither σ
increased significantly, we conclude that the Bayer
mosaic pattern is highly effective for separating simulta-
neously acquired interferograms at two wavelengths.
To verify axial sensitivity obta ined with this method,
we measured the phase profile of a 20 μm diameter
polystyrene microsphere (Duke Scientific) immersed in
index-matching oil (n ¼ 1:515). The simulated two-
dimensional OPDs induced by an equal-diameter ideal
sphere were subtracted from the OPD measurements,
yielding an rms deviation of 17:312 nm, or λ= 30, across
the entire area covered by the microsphere. This error
incorporates not only error arising from phase noise
but also any surface imperfec tions of the microsphere,
and therefore represents an upper bound on the axial
sensitivity of this system. The diameter as calculated
from the measured OPD at the center of the bead was
found to be 20:105 μm, which is in close agreement with
the Na tional Institute of Standards and Technology
(NIST) -certified diameter of this microsphere sample
of 19:99 0:20 μ m.
Additional experimental validation of simultaneous
two-wavelength transmission QPM with a Bayer mosaic
CCD was performed by measuring phase profiles of
optically clear microstructures. These microstructures
were created by maskle ss holographic micropatterning
of UV-cured optical adhesive (NOA63, Norland Products)
onto glass microscope coverslips [15]. We first imaged
the microstructures with a scanning electron microscope
(SEM) in both en face Fig. 2(a) and 45° tilt Fig. 2(b)
geometries in order to visualize the structures. Note that
while these SEM images allow visualization of the struc-
tures, quantitative axial measurements can only be
inferred. Next, these microstructures (n ¼ 1:56) were
immersed in index-matching oil (n ¼ 1:515), producing
a relative refractive index difference of Δn ¼ 0:045 and
imaged using the QPM methods described above. A back-
ground phase image was acquired from an adjacent area
containing only the coverslip and index-matching oil and
used to correct for wavefront curvature in the imaging
system before applying phase unwrapping techniques.
To demonstrate the efficacy of our technique, we com-
pared the results of a well-known quality-map guided
phase unwrapping algorithm [6] to two-wavelength un-
wrapping. The quality-map guided algorithm iteratively
adds multiples of 2π to the wrapped phase until the global
phase gradient has been minimized. Figure 3(a) shows
the OPD map produced using this method, and Fig. 3(c)
shows a cross-section plot of the calculated physical
object height. One of the four objects appears to be
accurately reconstructed at a larger OPD than the back-
ground, while the other three objects are almost indistin-
guishable from the background. When examining the
Fig. 2. SEM images of optical adhesive microstructures
(2170× magnification): (a) en face view with 15 μm lateral scale
bar; (b) 45° tilt view with 15 μm vertical scale bar.
August 1, 2010 / Vol. 35, No. 15 / OPTICS LETTERS 2613
SEM images from Fig. 2, it is clear that the quality-map
guided unwrapping of the latter structures is incorrect.
The difference in unwrapping algorithm success among
the objects is due to variability in the edge slope and true
height of the fabricated microstructures; erroneous ob-
ject unwrapping likely arises from microstructures with
a significant number of sharp edges, where the OPD
changes by more than π between neighboring pixels.
Figure 3(b) shows the results when using our simulta-
neous two-wavelength unwrapping approach in which
the beat wavelength map has been used as a guide for
adding multiples of 2π to the original green illumination
wrapped phase map. A specific cross-section plot of the
physical object height (after dividing by Δn) from two-
wavelength unwrapping is shown in Fig. 3(d). As can
be seen from Fig. 3(d), the object heights are between
10 and 15 μm, which is in agreement with estimates from
the SEM images in Fig. 2. The remaining errors at the
edges of the microstructures arise from high local noise,
which causes an extra multiple of 2π to be added to
the single wavelength phase map in the final refinement
process [9,11].
In summary, we have presented a method of using the
channels of a Bayer mosaic color camera for simulta-
neously capturing interferograms at multiple distinct
illumination wavelengths. Experimental results confirm
that color channel crosstalk is minimal and does not
noticeably affect two-wavelength unwrapping of recov-
ered phase information. Because this technique extends
the measurement range of transmission-geometry QPM
using only a single acquisition, it should be especially
useful for analysis of transparent samples in which dy-
namic processes are of interest, such as microscopic bio-
logical phenomena in microfluidic devices or microchip
assays. Although sequential speed of acquisition with a
monochromatic camera and gating of the illumination
source can compete with the speed of acquisition of a
single exposure from a color camera seen here, our tech-
nique is more useful for imaging highly dynamic samples,
as will be explored in our future work.
The authors acknowledge the support of the
McChesney Fellowship in Biomedical Engineering from
Duke University, the Bikura Postdoctoral Fellowship
from Israel, and National Science Foundation (NSF)
grants CMMI-0609265 and CBET-0651622.
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Fig. 3. (Color online) Microstructure OPD maps and profiles:
(a) 532 nm OPD map after quality-map guided unwrapping,
15 μm lateral scale bar; (b) 532 nm OPD map after two-
wavelength unwrapping, 15 μm lateral scale bar; (c) incorrect
object height profile, from the dotted line in (a); (d) object
height profile from two-wavelength unwrapping, from the
dotted line in (b). The quantitative height measurements ob-
tained from QPM agree with the SEM images in Fig. 2.
2614 OPTICS LETTERS / Vol. 35, No. 15 / August 1, 2010
    • "One direct method is to employ multiple lasers with different wavelengths in QPM [16,71,[81][82][83][84]. White light illumination equipped with various spectral filters [19,85] and second-harmonic generation (SHG) [75,86,87] also have been used. A color image sensor has been used for multispectral imaging channels so that spectroscopic quantitative phase images can be simultaneously measured [71,84,88]. As a representative example, spectroscopic phase microscopy (SPM) [19] has been used for noninvasive label-free quantification of Hb protein in living human red blood cells (RBCs). "
    [Show abstract] [Hide abstract] ABSTRACT: The identification and quantification of specific molecules are crucial for studying the pathophysiology of cells, tissues, and organs as well as diagnosis and treatment of diseases. Recent advances in holographic microspectroscopy, based on quantitative phase imaging or optical coherence tomography techniques, show promise for label-free noninvasive optical detection and quantification of specific molecules in living cells and tissues (e.g., hemoglobin protein). To provide important insight into the potential employment of holographic spectroscopy techniques in biological research and for related practical applications, we review the principles of holographic microspectroscopy techniques and highlight recent studies.
    Article · Sep 2014
    • "Digital holographic microscopy (DHM) is a promising 3D imaging technology that allows the investigation of the shape of engineered surfaces or tomography of biological samples with microscale lateral resolution and nanoscale axial precision [1] . Over the past decades , thanks to newly available CCD cameras, high performance of computers, and advances in offline numerical processing techniques, DHM has been fully developed and successfully applied in diverse applications; for instance, in quantitative biology microscopy234567, surface relief characterization of MEMS or other microstructures89101112, topography of micro-optics13141516, and so on17181920. Usually, coherent light sources, i.e., lasers, are adopted in DHM as illumination light, and the quality of holograms is inevitably degraded by the inherent coherent noise. "
    [Show abstract] [Hide abstract] ABSTRACT: A reflection mode digital holographic microscope with light emitting diode (LED) illumination and off-axis interferometry is proposed. The setup is comprised of a Linnik interferometer and a grating-based 4f imaging unit. Both object and reference waves travel coaxially and are split into multiple diffraction orders in the Fourier plane by the grating. The zeroth and first orders are filtered by a polarizing array to select orthogonally polarized object waves and reference waves. Subsequently, the object and reference waves are combined again in the output plane of the 4f system, and then the hologram with uniform contrast over the entire field of view can be acquired with the aid of a polarizer. The one-shot nature in the off-axis configuration enables an interferometric recording time on a millisecond scale. The validity of the proposed setup is illustrated by imaging nanostructured substrates, and the experimental results demonstrate that the phase noise is reduced drastically by an order of 68% when compared to a He-Ne laser-based result.
    Full-text · Article · Dec 2013
    • "It can be seen that the quantitative experimental result of the proposed method is in agreement with the standard four-step PSI result. The slight difference between profiles can be attributed to the phase noise due to the aforementioned intensity crosstalk between the red and green channels [26]. In addition, a non-plane wave reference [23], laser coherent noise [15], incomplete subtraction of DC terms [25] and miscalibration of the piezoelectric transducer encourage this difference in the comparison. "
    [Show abstract] [Hide abstract] ABSTRACT: In this work, we propose a dual-wavelength in-line digital holographic microscopy (DHM) configuration in order to eliminate the conjugate image and reach the maximum resolution of CCD. By using this configuration, two holograms are acquired in one shot. Our method is based not only on the recordings of two holograms at slightly different planes, but also on the diffraction patterns formed with two wavelengths. With this experimental setup, we are able to analyze fast dynamic processes at the microscopic scale in real time. Theoretical evaluation, computer simulations and experimental results validate our proposal. The experimental results are obtained using a phase-amplitude object and compared with those calculated from the well-established phase-shifting interferometry technique. As far as we know, in-axis configuration with a single exposure has not been used in DHM, as we present in this paper.
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