of 8
The application of Fresnel zone plate based
projection in optofluidic microscopy
Jigang Wu
*
, Xiquan Cui, Lap Man Lee, and Changhuei Yang
Department of Electrical Engineering, California Institute of Technology, Pasadena, California, 91125
*
Email:
jigang@caltech.edu
Abstract:
Optofluidic microscopy (OFM) is a novel technique for low-
cost, high-resolution on-chip microscopy imaging. In this paper we report
the use of the Fresnel zone plate (FZP) based projection in OFM as a cost-
effective and compact means for projecting the transmission through an
OFM’s aperture array onto a sensor grid. We demonstrate this approach by
employing a FZP (diameter = 255
μ
m, focal length = 800
μ
m) that has been
patterned onto a glass slide to project the transmission from an array of
apertures (diameter = 1
μ
m, separation = 10
μ
m) onto a CMOS sensor. We
are able to resolve the contributions from 44 apertures on the sensor under
the illumination from a HeNe laser (wavelength =
633 nm). The imaging
quality of the FZP determines the effective field-of-view (related to the
number of resolvable transmissions from apertures) but not the image
resolution of such an OFM system – a key distinction from conventional
microscope systems. We demonstrate the capability of the integrated system
by flowing the protist
Euglena gracilis
across the aperture array
microfluidically and performing OFM imaging of the samples.
©
2008 Optical Society of America
OCIS codes:
(110.0110) Imaging Systems; (050.1970) Diffraction and gratings: Diffractive
optics; (110.1220) Apertures.
References and links
1.
X. Heng, D. Erickson, L. R. Baugh, Z. Yaqoob, P. W. Sternberg, D. Psaltis, and C. Yang, “Optofluidic
microscopy – a method for implementing a high resolution optical microscope on an chip,” Lab on a chip
6
,
1274-1276 (2006).
2.
X. Cui, L. M. Lee, X. Heng, W. Zhong, P. W. Sternberg, D. Psaltis, and C. Yang, “Lensless high-resolution
on-chip optofluidic microscopes for
Caenorhabditis elegans
and cell imaging,” Proc. Natl. Acad. Sci.
105
,
10670-10675 (2008).
3.
X. Heng, E. Hsiao, D. Psaltis, and C. Yang, “An optical tweezer actuated, nanoaperture-grid based
optofluidic microscope implementation method,” Opt. Express
15
, 16367-16375 (2007).
4.
G. M. Whitesides, “The origins and the future of microfluidics,” Nature
442
, 368-373 (2006).
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A. G. Michette,
Optical systems for soft X rays
(Plenum Press, 1986), chapter 8.
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W. Chao, E. Anderson, G. P. Denbeaux, B. Harteneck, J. A. Liddle, D. L. Olynick, A. L. Pearson, F.
Salmassi, C. Y. Song, and D. T. Attwood, “20-nm-resolution soft x-ray microscopy demonstrated by use of
multilayer test structures,” Opt. Lett.
28
, 2019-2021 (2003).
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E. Di Fabrizio, F. Romanato, M. Gentili, S. Cabrini, B. Kaulich, J. Susini, and R. Barrett, “High-efficiency
multilevel zone plates for KeV X-rays,” Nature
401
, 895-898 (1999).
8.
R. M. Henkelman and M. J. Bronskill, “Imaging extended objects with a Fresnel-zone-plate aperture,” J.
Opt. Soc. Am.
64
, 134-137 (1974).
9.
M. Young, “Zone plates and their aberrations,” J. Opt. Soc. Am.
62
, 972-976 (1972).
10.
F. Wyrowski, “Diffractive optical elements: iterative calculation of quantized, blazed phase structures,” J.
Opt. Soc. Am. A
7
, 961-969 (1990).
11.
A. R. Jones, “The focal properties of phase zone plates,” J. Phys, D: Appl. Phys.
2
, 1789-1791 (1969).
12.
S. W. Hu, X. Ren, M. Bachman, C. E. Sims, G. P. Li, and N. Allbritton, “Surface modification of
Poly(dimethylsiloxane) microfluidic devices by ultraviolet polymer grafting,” Anal. Chem.
74
, 4117-4123
(2002).
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Received 17 Jul 2008; revised 6 Sep 2008; accepted 11 Sep 2008; published 18 Sep 2008
(C) 2008 OSA
29 September 2008 / Vol. 16, No. 20 / OPTICS EXPRESS 15595
13.
X. Heng, X. Cui, D. W. Knapp, J. Wu, Z. Yaqoob, E. J. McDowell, D. Psaltis, and C. Yang,
“Characterization of light collection through a subwavelength aperture from a point source,” Opt. Express
14
, 10410 (2006).
1. Introduction
The optofluidic microscope (OFM) method [1-3] developed recently enables the construction
of high-resolution and low-cost chip-level microscopes. Combined with the appropriate high
flow-velocity microfluidic techniques [4], OFM systems can potentially address a large
number of biomedical applications, such as image-based cytometry, blood parasite diagnosis
and water quality inspection. The OFM imaging approach differs significantly from the
conventional microscopy approach; the design choice is motivated by the fact that the
conventional microscope design, with its requisite high precision optical elements, is difficult
to miniaturize cost-effectively.
In its typical format, an OFM system consists of three parts: an array of apertures
patterned on a metal-coated linear array sensor, a microfluidic channel emplaced on the
aperture array such that the aperture array spans the channel floor in a skewed pattern, and a
uniform light field projected through the microfluidic channel to the aperture array. As a
sample flows through the channel and across each aperture, it interrupts light transmission
through the aperture. Each transmission time trace in effect represents a line scan across the
object. By ensuring that the microfluidic channel is arranged such that the separation of
adjacent apertures perpendicular to the channel flow axis is smaller than the aperture
diameter, we can ensure that each point on the sample is scanned [2]. By compiling all of the
line scans appropriately, we can create a microscopy image of the sample in which the
optimal resolution is fundamentally limited by the aperture size. This lensless design allows
us to build highly compact and high-resolution microscope systems with commercially
available CMOS and CCD sensor chips in a simple and cost-effective way.
While the typical OFM design calls for the aperture array to be patterned directly onto a
metal-coated linear array sensor, there are certain scenarios for which a different OFM design,
where the aperture array and the sensor are separated, would actually be better. Here are two
of the more important scenarios: 1) Recycling/retrieval of optical sensor is desired.
The
typical OFM structure is not particularly easy to dismantle. If the optical sensor employed is
inexpensive, this would not be an important consideration as the complete replacement of an
entire expended OFM system would be more cost-effective. On the other hand, if the cost of
optical sensor employed is high, an OFM design that allows easy disassembly and recovery of
the optical sensor would be highly desirable. 2) Cooling of the optical sensor is desired.
Operating an optical sensor at a low temperature can help reduce noise and improve signal-to-
noise ratio. While cooling the OFM optical sensor to sub-freezing temperatures is easily
achievable, the close proximity of the aperture array and the microfluidic channel to the
optical sensor implies that that such cooling will likely freeze the microfluidic channel content
as well. As such, an OFM design in which the aperture array and the microfluidic channel are
well separated from the optical sensor is desirable in such a scenario.
In the paper, we report an OFM design that incorporates a Fresnel zone plate (FZP) to
relay the light transmissions through the aperture array to an optical sensor. This design,
termed FZP-OFM, achieves the desired separation of the aperture array and the optical sensor
without compromising on the resolution of the OFM system. The FZP can be used to focus
the light and form images [5-7]. However, direct imaging by FZP suffers from multiple
diffractions and serious aberrations for extended objects [8, 9]. These disadvantages prevent
the FZP from being widely used in imaging applications, especially in the visible regime
where better conventional refractive optics is available. Nevertheless, as the FZP in our FZP-
OFM design is only used to project the transmissions from the apertures onto an optical
sensor, the OFM resolution (dependent on the aperture size) is unaffected by the FZP’s
aberration characteristics as long as the transmission projections from the apertures are
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(C) 2008 OSA
29 September 2008 / Vol. 16, No. 20 / OPTICS EXPRESS 15596
differentiable. Finally, we note that it is possible to design a general diffractive optical
element (DOE) other than the FZP to increase the imaging performance in non-paraxial region
[10]. However, the resulting DOE is generally more complicated and requires more efforts to
design and fabricate.
As the FZP can be cost-effectively fabricated with current microfabrication techniques
such as optical or electron-beam lithography, the manufacturing cost associated with this
OFM design is anticipated to be low.
The following sections are structured as follows. In section 2, we show the system setup
and experiment methods. In section 3, we demonstrate the capab
ility of our system by
imaging the protist
Euglena gracilis
, and the resulting images are compared with images
acquired by FZP directly and conventional microscope images. Finally, we summarize our
work in section 4.
2. Experiment methods
The FZP was designed to have a focal length of
f
= 800
μ
m. The radius of the
m
th
zone was
calculated by [5]
f
m
r
m
λ
=
(1)
where
λ
is the wavelength of illumination light. Our FZP has 32 zones with an outer diameter
of 255
μ
m. The imaging scheme of the FZP is shown in Fig. 1(a). The aperture array located
at 1 mm (equivalent distance in glass is 1.5 mm because of the glass refractive index of 1.5)
from the FZP was imaged onto the imaging plane located at 4 mm from the FZP. The
magnification (M) of the system is M = 4. The coordinate system is also shown in the figure.
Fig. 1. (a) Imaging scheme of the FZP; (b) Illustration of the FZP-OFM system setup. (c)
PDMS microchannel aligned and attached to the aluminum layer with tiny aperture array. (d)
Microscope image of the fabricated FZP; (e) Image of the aperture array by the FZP.
The system setup is shown in Fig. 1(b). The system consists of two separated parts: the
FZP-OFM chip with poly(dimethylsiloxane) (PDMS) microfluidic channel and an imaging
sensor. To make the FZP-OFM chip, a glass wafer was coated with 300-nm-thick aluminum
layer by thermal evaporator (Veeco 7760) and an array of tiny apertures (diameter = 1
μ
m,
separation = 10
μ
m) was drilled by focused ion beam (FEI Nova
200). The glass wafer was
then attached to another glass slide where a FZP was fabricated on the other side using
photolithography (Karl Suss MJB3) with 500-nm-thick SU-8 photoresist. Finally a 200-nm-
thick poly(methyl methacrylate) (PMMA) layer was spin-coated onto the aluminum layer to
protect the aperture array. The PDMS microchannel (width = 25
μ
m, height = 16
μ
m) was
fabricated by transferring the microfluidic structure from the prefabricated SU-8 mold to the
PDMS channel
Flow
Apertures
Aluminum layer
Glass
Fresnel zone plate
Imaging sensor
Illumination
(b)
(d)
(c)
α
-hole
β
-hole
Backup
α
-hole
20
μ
m
50
μ
m
Air
(e)
50
μ
m
...
...
...
...
(a)
x
z
1mm
4mm
255
μ
m
θ
PDMS channel
Flow
Apertures
Aluminum layer
Glass
Fresnel zone plate
Imaging sensor
Illumination
(b)
(d)
(c)
α
-hole
β
-hole
Backup
α
-hole
20
μ
m
50
μ
m
Air
(e)
50
μ
m
...
...
...
...
(a)
x
z
1mm
4mm
255
μ
m
θ
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(C) 2008 OSA
29 September 2008 / Vol. 16, No. 20 / OPTICS EXPRESS 15597
PDMS elastomer. Then the microchannel was aligned to the aperture array and attached to the
aluminum layer of the FZP-OFM chip under microscope. The optical sensor (Lumenera Lu-
080M, with 9.9-
μ
m pixel size) was located at the imaging plane of the FZP and the aperture
array was imaged by the FZP onto the sensor. The system was illuminated by an elongated
laser beam (~5 W/cm
2
) transformed by cylindrical lenses from a HeNe laser (wavelength =
633 nm, Melles Griot 25-LHP-928-249). The different components of the system are shown in
Fig. 1(c)(d)(e).
Figure 1(c) shows the image of a PDMS microchannel attached to the aperture array. The
microchannel was oriented at a small angle
θ
=
3.3
o
with the aperture array. In the experiment,
the channel spanned 44 apertures. In addition to the aperture array, an
α
-hole and a backup
α
-
hole were drilled on the glass wafer with the same technique. The isolated
α
-hole had the
same size as other apertures and was positioned at the center of the microchannel as shown in
the figure. During the experiment, the
α
-hole and the corresponding
β
-hole (distance between
them is 170
μ
m) in the aperture array downstream will scan the same part of the sample. By
comparing the transmission from the
α
-hole and the
β
-hole, we can monitor the flow stability
and measure the flow speed. The flow stability can be characterized by the correlation
between time scan signals of the
α
,
β
-hole. Note that successful OFM imaging relies on the
stability of sample flowing, so we must ensure that the sample does not rotate or change shape
during the imaging process. The correlation of the transmissions through
α
,
β
-hole provides a
good criteria for rejecting images of unstable samples. The flow speed
V
can be calculated by
τ
d
V
=
(2)
where
d
and
τ
are the distance and the travel time of the sample between the
α
,
β
-hole,
respectively. We further note that the rotation of spherical samples in the microfluidic flow
can be significantly reduced through the use of electrokinetic actuation and this approach has
been employed in other versions of the optofluidic microscope [2].
Figure 1(d) is a microscope image of the fabricated FZP. The FZP was a phase reversal
FZP where the optical thickness of adjacent zones differed by half of the laser wavelength.
Given the refractive index of SU-8, n = 1.596, the thickness of spin-coated SU-8 was set to be
near
nm
n
t
531
)
1
(
2
=
=
λ
(3)
The phase reversal FZP has improved diffraction efficiency for the principle focus and is
especially suitable for monochromatic illumination [5]. Note that in our case, the error in
diffraction efficiency will be less than 5% if the thickness error in spin coating is less than 50
nm [11], which is achievable with spin-coating.
To reduce stray light from transmitting through the peripheral of the FZP, we proceeded
to coat the peripheral with a layer of aluminum. The process is as described here. First, the
entire wafer (on the FZP side) was coated with a thin layer of aluminum (thickness = 300 nm).
Next, a layer of SPR220 photoresist was spin-coated on this aluminum layer by the spinner
WS-400A-6NPP/LITE (Laurell Technologies). The photoresist directly on top of the FZP was
then removed by photolithography. Finally the aluminum covering the FZP was removed by
aluminum etchant (Transene Company) and the remaining SPR220 was washed away with
acetone.
The image of the aperture array projected by the FZP onto the optical sensor is shown in
Fig. 1(e). We can see that the transmission of all apertures, except those blocked by the edges
of the microchannel, were well projected and distinguishable from each other. Note that the
detected optical power from different apertures varies and, as such, we need to normalize the
power of the apertures during data processing.
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Before the experiment, the surface of the channel was coated with Poly(ethylene glycol)
(PEG) to promote smooth microfluidic flow [12]. During the experiment, the channel was
oriented vertically to actuate microfluidic flow by gravity. Note that other techniques, such as
electroosmosis flow [2] or optical tweezer [3], can also be used to drive the samples scanning
in the OFM system and maybe more suitable for other specific applications. While the sample
was flowing through the microfluidic channel, the transmitted light of the aperture array was
detected by the imaging sensor and each aperture effectively acquired a line scan of the
sample.
The principle of image reconstruction was similar as our previous OFM systems [1-3]
and can be briefly described as follows. Each line of the reconstructed image is actually a
time-shifted line scan from the corresponding aperture. The time shift between adjacent line
scans
Δ
t
can be expressed as, using equation (2),
θ
τ
θ
cos
cos
d
L
V
L
t
=
=
Δ
(4)
where
L
is the separation between adjacent apertures. In the final OFM image, the pixel size
in x-direction (
δ
x
) and in y-direction (
δ
y
) can be calculated as
θ
δ
δ
sin
,
L
y
F
V
x
=
=
(5)
where
F
is the frame rate of the imaging sensor. Suppose the length of the sample is
l
, then the
image acquisition time can be expressed as
V
l
t
j
V
l
jL
T
+
Δ
=
+
=
θ
cos
(6)
where
j
is the total number of apertures that scan the sample.
To characterize the imaging quality of the FZP, we used a 1-
μ
m pinhole as our object and
recorded its image while translating it along x-direction, as shown in Fig. 2(a). We then
measured the image spot sizes at full-width-half-maximum (FWHM) in both x and y-direction
by fitting the spot with a Gaussian profile and plotted them against the x position of the
object, as shown in Fig. 2(c). As expected, the spot size and the error of the fitting along x-
direction increased as the pinhole moved away from the center and the imaging spot became
larger and more irregular. In contrast, the spot diameter along y-direction remained
approximately the same as there was no y-displacement of the pinhole. We also note that the
spot size for on-axis pinhole was approximately 10
μ
m, corresponding to a resolution of 10/M
= 2.5
μ
m in object space. This is in fair agreement with the theoretical resolution of ~2.4
μ
m,
which can be calculated based on the diameter of the FZP. In Fig. 2(c), we also show the
pinhole images at three different x positions. According to this experiment, we can see that
there is a resolution limit for extended object if the FZP is used for direct imaging. To
maintain the best resolution, the image can not be extended more than 100
μ
m away from the
axis, corresponding to an object size of less than 100/M * 2 = 50
μ
m. With the image scheme
of OFM, however, the aberration issue is not important as long as the image light spots
associated with the transmissions from the apertures can be resolved and separated from each
other. As shown in Fig. 2(b), in FZP direct imaging, we will need to resolve object points with
a separation of the on-axis resolution of FZP to achieve the optimal resolution, and the
corresponding imaging spot separation is 2.5*M = 10
μ
m. While in the FZP-OFM scheme,
the FZP projection will only need to resolve adjacent apertures which usually have a larger
separation (10
μ
m in our case) than the on-axis resolution of the FZP. Thus the FZP-OFM can
accept an aberrated imaging spot size of the aperture image separation, and the corresponding
imaging spot separation is 10*M = 40
μ
m. This resolution is needed to perform high-
resolution OFM imaging, as indicated in Fig. 2(c). In this case, the imaging resolution is
determined by the size of the aperture and the distance between the sample and the aperture
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29 September 2008 / Vol. 16, No. 20 / OPTICS EXPRESS 15599
instead of the imaging quality of FZP. As such, it is still possible to achieve high resolution
imaging for larger field of view. The extension of our effective aperture array is 440
μ
m, i.e.,
220
μ
m away from the axis, and is well within the capability of our FZP-OFM system. As
expected, the acquired OFM images shown in the next section is much better than the images
acquired by direct FZP imaging.
Fig. 2. (a) Experimental scheme to characterize the imaging quality of the FZP; (b)
Demonstration of resolution needed to perform FZP direct imaging and FZP-OFM imaging; (c)
Measured FWHM spot size of the image in x and y-direction versus the x position of a 1-
μ
m
pinhole. Pinhole images in three different positions are shown. The resolution limit of the FZP
and the resolution needed for the FZP-OFM imaging are indicated.
3. Imaging results
As a demonstration, we acquired images of the protist
Euglena gracilis
(Carolina biology
supply company) with the FZP-OFM system. We
immobilized the protists by treating them in
a 90
o
C heat bath for 15 second. The solution containing the protists was then injected into the
input port of the FZP-OFM system. By tilting the FZP-OFM vertically, we were able to
induce the solution to flow through the microfluidic channel. During the imaging process, the
flow speed of the protists was measured by the
α
,
β
-hole pair to be
V
~ 400
μ
m/sec. The
imaging sensor was operated at a frame rate of
F
= 1350 frames/sec. The pixel spacing (or
size) of the OFM images was
δ
x
= 0.3
μ
m and
δ
y
= 0.6
μ
m. The acquisition time for the
protists images is ~1 sec. The resolution of the OFM system can be characterized by the point
spread function (PSF) associated with the imaging aperture. We can achieve an optimal
0
50
100
150
200
250
300
0
10
20
30
40
50
60
70
x(
μ
m)
FWHM spot size (
μ
m)
y-direction size
x-direction size
Resolution limit of
the FZP
x
z
pinhole
Imaging sensor
Fresnel zone plate
Extension of the OFM
aperture array
(b)
(c)
(a)
FZP Direct imaging
Resolution needed for
our OFM scheme
FZP-OFM imaging
10
μ
m
40
μ
m
0
50
100
150
200
250
300
0
10
20
30
40
50
60
70
x(
μ
m)
FWHM spot size (
μ
m)
y-direction size
x-direction size
Resolution limit of
the FZP
x
z
pinhole
Imaging sensor
Fresnel zone plate
Extension of the OFM
aperture array
(b)
(c)
(a)
FZP Direct imaging
Resolution needed for
our OFM scheme
FZP-OFM imaging
10
μ
m
40
μ
m
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resolution determined by the aperture size when the sample is very close to the aperture (in
the optical near field regime). Since the width of the PSF increases as the height of the point
object increases, the resolution of our OFM system will degrade with increasing distance
between the sample and the aperture. The PSF can be measured by laterally scanning a near
field scanning optical microscope (NSOM) tip across the aperture at different height
H
and
measure the transmitted signals through it [13]. We measured the width of the PSF for the 1-
μ
m aperture used in our experiment to be 0.9
μ
m when
H
= 0.1
μ
m and 3
μ
m when
H
= 2.5
μ
m. Thus the ultimate resolution of our OFM system is 0.9
μ
m.
Figure 3 is an image compilation of similar protists acquired with the FZP-OFM and
other imaging schemes. Figure 3(a) was acquired by simply using a FZP to directly image a
protist onto our optical sensor. We can see that the image is blurry and is negatively impacted
by the presence of a diffraction background. In comparison, the FZP-OFM images shown in
Fig. 3(b) are clearer and well-resolved. Since our FZP-OFM system used coherent laser as
illuminati
on, we believe that a comparison of the acquired images to those from a
conventional microscope operating with coherent illumination and incoherent illumination
would be helpful. Figure 3(c) and (d) show images of the protist that are acquired with a
microscope (Olympus BX41) through a 20x objective under coherent and incoherent
illuminati
on, respectively.
Fig. 3. Images of
Euglena gracilis
acquired by (a) FZP direct imaging; (b) FZP-OFM system;
(c) Conventional microscope with 20x objective under coherent illumination; (d) Conventional
microscope with 20x objective under incoherent illumination.
It is interesting to note that the FZP-OFM images appear to be more similar to the
incoherent illumination conventional microscope image than the coherent illumination
conventional microscope image, despite the fact that FZP-OFM also employed a coherent
illumination source. The speckle backgr
ound in Fig. 3(c) arose from the interference of stray
light components scattered and reflected from various interfaces in the microscope. This
pattern varied spatially but had no time varying component. The absence of this speckle
background in Fig. 3(d) is attributable to the wash-out of such interference effect in incoherent
illuminati
on. The absence of the speckle background in our FZP-OFM images can be
explained by noting that each horizontal line in the images represents a time trace of an
aperture in the array. In the absence of an object directly above the aperture, we can expect the
transmission through the aperture to remain unchanged in time. The transmission through
each aperture was normalized with respect to each other at the beginning of the experiment.
This was intended to correct for small variations in the aperture size and sensor sensitivity.
However, variations in light intensity incident on the apertures would be, likewise, normalized
by this process. As such, the impact of a speckle background in the FZP-OFM was effectively
eliminated. We further note that the effect of mutual light interference from scatterings within
the object should remain in our FZP-OFM images.
(b)
10
μ
m
10
μ
m
(a)
(d)
10
μ
m
10
μ
m
10
μ
m
(c)
(b)
10
μ
m
10
μ
m
10
μ
m
10
μ
m
(a)
(d)
10
μ
m
10
μ
m
10
μ
m
(c)
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(C) 2008 OSA
29 September 2008 / Vol. 16, No. 20 / OPTICS EXPRESS 15601
4. Summary
We have shown that the FZP can be incorporated into the OFM design to create a modified
FZP-OFM scheme. We showed that the aberrations associated with FZP did not impact or
limit the resolution of the FZP-OFM, which is limited f
undamentally by the size of apertures
employed. We further presented the use of our FZP-OFM prototype to acquire microscopy
images of protists. The absence of a speckle background in our FZP-OFM images was an
unexpected observation that can be explained by closely examining the OFM’s imaging
strategy.
As this scheme allows the separation of the OFM aperture array from the optical sensor
array, it is advantageous for a number of scenarios. Specifically, this design allows for easier
recycling of high-cost optical sensor array and it allows for the cooling of the optical sensor
with less risk of freezing shut the microfluidic channel.
Acknowledgments
The authors would like to acknowledge Dr. Xin Heng for helpful discussions. This project is
supported by the NSF career award BES-0547657.
#99006 - $15.00 USD
Received 17 Jul 2008; revised 6 Sep 2008; accepted 11 Sep 2008; published 18 Sep 2008
(C) 2008 OSA
29 September 2008 / Vol. 16, No. 20 / OPTICS EXPRESS 15602