CMOS contact imager for monitoring cultured cells
Honghao Ji, Pamela A. Abshire
Mario Urdaneta, Elisabeth Smela
Department of Electrical and Computer Engineering
Institute for Systems Research
University of Maryland
College Park, Maryland 20742, USA
Department of Mechanical Engineering
University of Maryland
College Park, Maryland 20742, USA
Abstract— There is a growing interest in developing low cost,
low power, highly integrated biosensor systems to characterize
individual cells for applications such as cell analysis, drug
development, environmental monitoring, and medicine. In such
micro-systems, it’s desirable to track individual cells in real time
in order to steer cells using on-chip micro-actuators or monitor
the movement of motile cells. To address this requirement,
we are developing an embedded optical image sensor, called a
contact imager, for imaging of a biological specimen directly
coupled to the chip surface. The designed CMOS image sensor
comprises an array of active pixel sensors (APS), logic and
control signal generation, and readout circuits. The pixel layout
has a pitch of 8.4 µm (24 λ). The design was fabricated in a
commercially available 0.5 µm CMOS technology. The imager
was first characterized on the bench as a normal CMOS image
sensor, and then as a contact imager with microbeads (16 µm)
placed directly on the chip surface. After further packaging with
bio-compatible material, the chip was tested with cells cultured
directly on the chip surface. Test results confirm successful
detection of both beads and cells.
I. I NTRODUCTION
Integrated systems for probing individual cells, known as
“biolabs-on-a-chip” [1], [2], offer many advantages over their
macro-scale counterparts, including low cost, portability, high
throughput, and novel analysis capabilities. An embedded optical image sensor will augment such miniaturized biosystems
with the ability to detect individual cells in real time in
order to steer cells onto different sensor sites or to track
the movement of motile cells. “Contact imaging” [3] takes
advantage of the small pixel sizes possible in modern CMOS
technologies and the increased collection efficiency offered by
eliminating intermediary optics. While contact imagers cannot
rival the spatial resolution of an optical microscope, they can
provide the means for sophisticated cell handling outside the
realm of the traditional cell biology laboratory. To address
this requirement, this paper describes a custom CMOS image
sensor designed and fabricated in a commercially available
three-metal two-poly 0.5 µm CMOS process. We are currently
working to integrate this sensor together with other previously
reported electrical sensors [2], [4], [5] and other sensors and
control circuitry under development. This suite of circuits will
support the real-time integration of sensing, actuation, and
control necessary to enable sophisticated applications in cell
steering, cell monitoring, and biochemical detection. The rest
of this paper is organized as follows: section II introduces
the design and operation of the contact imager; section III
0-7803-8834-8/05/$20.00 ©2005 IEEE.
describes test procedures and experimental results; section IV
summarizes the work.
II. S YSTEM D ESIGN AND O PERATION
The contact imager has been implemented as an array of
CMOS active pixel sensors (APS) [6], [7]. In contrast with
regular CMOS image sensors, the resolution of a contact
imager is solely determined by its pixel size rather than the
size of pixel array. Our design focused on achieving a small
pixel size while maintaining the noise and speed performance
of an APS. First, the signal generated by a monolayer of
cells is modelled in section IIA. The pixel circuit is then
described in section IIB. Finally the architecture and operation
are described in section IIC.
A. Modelling of contact imaging
To design a CMOS image sensor for individual cell detection, we first examine how the presence of a cell may
affect the optical signal received by a sensor pixel. Unlike
in a natural scene, where the dynamic range of illumination
may be greater than 100 dB, the illumination condition of
an integrated biosensor system can be well controlled. For
example, using a commercially available LED having an
illumination power density of 50 mcd at 555 nm wavelength, a
photon flux of 2.04 × 106 photon/(um2 ·sec) will be received
by a pixel sensor placed 10 mm away from the LED. Since
most cells are nearly transparent, the visibility of a monolayer
of cells can be significantly enhanced by staining the cells
using neutral red dye, which has an extinction coefficient (Ec )
of 39000 cm−1 · M −1 . A dye concentration (C) of 0.1 M
can be established in live cells. At such a concentration, the
transmission rate (T ) of illumination through a monolayer of
cells 2 um thick (l) can be calculated as
−4
T = 10−Ec ×C×l = 10−39000×0.1×2×10
= 0.166.
(1)
Thus, 83.4% of the incoming light will be blocked. When
the optical area of a pixel is comparable to or less than the
cell size, an individual cell close to the pixel surface blocks a
photon flux of 1.70 × 106 photon/(um2 ·sec). Assuming 40%
quantum efficiency, a photodiode under a stained cell with a
parasitic capacitance of 0.5 f F/um2 will generate a signal of
43 V /sec, which differs from the brighter background signal
by 218 V /sec.
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Vdd
Reset
Vdd
M1
Row_select
M2
M3
Vbias
M4
Fig. 1.
A schematic of photodiode type CMOS active pixel sensor (APS).
B. Pixel Design
A schematic for the CMOS photodiode type APS is shown
in Figure 1. Several techniques have been used in order to
achieve a small pixel size and are illustrated in the pixel
layout shown in Figure 2. First, all three MOS transistors
are N-type transistors. A Nplus Psub photodiode is used to
avoid minimum Nwell spacing requirements. To reduce the
number of contacts, there is only one Vdd contact per pixel.
We designed the layout of pixel array in a staggered style
so that one Vdd contact can be shared by the source follower
input transistor of one pixel and the reset transistor of another.
Thus, a small pixel size with maximum optically active area
is achieved by routing the reset signal through a row using
only Poly1. We used the MOSIS scalable CMOS (SCMOS)
design rules for a double poly, three metal layer, Nwell process
(λ = 0.35 µm). A pitch size of 8.4 µm (24 λ) is achieved.
Metal3 used for routing Vss blocks light from all but the
photodiode active area. The fill factor, calculated as the ratio
of uncovered photodiode active area to the total pixel area, is
17%.
Fig. 2.
The layout of two staggered pixels. SF: source-follower input
transistor; PD: photodiode.
Fig. 3.
Photomicrograph of the fabricated contact imager chip.
C. Contact Imager Architecture And Operation
Figure 3 shows the photomicrograph of the fabricated
imager chip. The system consists of a 96 × 96 APS array,
row and column scanners, column-wise readout circuits, and
buffers and switches for input control and clock signals. The
row and column scanner is implemented using a closed-loop
shift register where each stage is a positive-edge triggered
dynamic D-flipflop. The output of the first stage of the row
scanner serves as the clock signal for the column scanner.
The complete chip including the pad frame fits on a standard
1.5 mm × 1.5 mm die.
A schematic diagram of one pixel together with circuits
for row logic and control, and correlated double sampling
(CDS) readout chain is shown in Figure 4 along with a timing
diagram. Three clocks are required to operate the imager:
ph 1, ph 2, and ph clamp. They share the same frequency and
must satisfy the phase relationships indicated by the dashed
lines in Figure 4. The clock signal for the row scanner is
ph 1. The output of one stage of the row scanner serves as
the Row select signal for all pixels in the corresponding row.
The Reset signal initializes the integrated pixel value and is
generated by performing a logic AND operation on the signals
ph 2 and Row select.
To suppress 1/f noise and fixed pattern noise (FPN) due
to threshold variations of source-follower input transistors,
column-wise CDS [8] is performed. After the pixel is selected
by Row select and before Reset goes high, clock ph clamp
is high. Thus, Vout (t1 ) = nbias is read out from the column
amplifier. Clock ph clamp then becomes low right before the
positive edge of the Reset signal. This turns the input of the
readout amplifier into a floating node capacitively coupled
to the output of the selected pixel. After Reset goes high,
Vout (t2 ) = Vsignal + nbias is sampled again, where Vsignal is
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Vdd
Q
Vdd
Row_select
Reset
ph_2
ph_1
D
Vph
Column Bus
Ccol_in
Vbias
+
Vout
ph_clamp
−
nbias
ph_clamp
ph_2
Fig. 5. Comparison of images of microbeads on chip surface taken by (a) a
camera, and (b) the contact imager. An overlapped view is also shown in (c).
ph_1
Row_select
A. Test On Bench
Reset
Vsignal
Vph
Vout
time
t1 t2
Fig. 4. A schematic diagram of row logic and readout chain for one pixel,
and timing of corresponding signals.
the difference of pixel outputs before and after the photodiode
is reset as shown in Figure 4. To perform CDS properly, the
three clock signals must satisfy the following phase shifts.
Clock ph 1 is an inverted and slightly delayed copy of clock
ph 2 so that pixels won’t be reset right after they are selected.
Clock ph clamp is an inverted and slightly advanced version
of ph 2. It’s especially important that the rising edge of ph 2
must fall behind the falling edge of ph clamp. Otherwise,
Vsignal won’t be coupled to the column output. In order to
illustrate these phase shifts clearly, the clock signals shown in
Figure 4 are not shown to scale.
III. E XPERIMENTAL R ESULTS
First, the chip was aligned with a camera objective and its
operation as an imager was verified. Edge effects within the
pixel array cause dark pixels along the edges. Future versions
will incorporate dummy pixels surrounding the pixel array to
address this problem. We then tested the chip as a contact
imager using microbeads placed directly on the chip surface.
After being further packaged with bio-compatible material, the
chip was tested with cells plated on chip surface. The test
procedure and results are described in sections IIIA and IIIB.
The contact imager was first tested on the bench. Three
clocks of frequency 50 kHz, with phase shifts as described in
section IIC, were generated from a microcontroller. Another
clock of frequency 100 kHz was also generated to provide
timing signals for a PC-hosted data acquisition card (DAQ)
(MCC PCI-DAS6052). Synchronization is achieved by triggering both the on-chip scanner and the data acquisition using
a pulse signal generated by the DAQ card.
We tested the contact imager with dry polymer microspheres
of diameter 16 µm placed directly on the chip surface. Figure
5 shows an image acquired with the contact imager and a
corresponding photograph taken using a camera through a
microscope. An overlapped view is also shown in Figure 5
to demonstrate that the contact imager is capable of tracking
cell-size particles in a precise manner.
B. Test With Cells
The imager chip is packaged in a standard 40 pin ceramic
dual in-line package (DIP). In order to test the contact imager
with cultured cells directly coupled to the chip surface, the
chip must be further packaged both to protect the bond
pads and wires from being corroded and shorted by cell
culture medium and to protect cells from toxic materials in
the chip packaging. We used a photo-patternable polymer
(LoctiteR 3108) to encapsulate all bonding pads and wires
and leave an opening about 1mm × 1mm large in the center
of the die. Our experiments showed that LoctiteR 3108 is
suitable only for short-term packaging since it swells in water,
which tends to detach the bonding wires from the chip.
On top of the LoctiteR packaged chip, a piece of plastic
tube was glued to form a well. The well is sufficiently large to
contain enough cell culture medium to prevent the cells on the
chip surface from drying out. The finished test fixture ready for
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Fig. 6. Photographs of (a) test fixture ready for cell plating, and (b) a close-up
view of packaged contact imager.
Fig. 7. Pictures of live cells coupled to chip surface are taken using (a) a
camera and (b) the contact imager. The overlapped view is shown in (c).
TABLE I
S UMMARY OF SENSOR PERFORMANCE
Process
resolution and sensitivity for monitoring cells on chip surface.
We are working to integrate the contact imager into a “Biolabon-a-chip” system in the near future.
AMI05 (SCMOS design rule, λ = 0.35um)
Power supply
5V
Maximum signal
1.2 V
Conversion gain
22 uV /e
ACKNOWLEDGMENT
Meas. pixel noise
σ = 2.5mV over 2 ms
The authors thank Nicole Nelson for her valuable help in cell
culture and cell plating, Ramesh Subbaraman for assistance in testing
the chip, and Dr. Marc Cohen for technical discussions and assistance.
The authors thank MOSIS for chip fabrication; this chip will be used
to teach an undergraduate course in mixed signal VLSI design. This
research was supported by the National Science Foundation (NSF)
through Awards 0225489 and 0238061, and by the Laboratory for
Physical Sciences (LPS).
Dynamic range
53.6 dB
Dark signal
0.46 V /sec
plating cells is shown in Figure 6 along with a close-up view of
the LoctiteR packaged chip. We used bovine aortic smooth
muscle cells (BAOSMC) purchased from Cell Applications,
Inc. for our experiments. These cells were stained using neutral
red dye to increase their visibility. Figure 7 shows an image
acquired by the contact imager as well as a photograph taken
with a camera through a microscope. The overlapped view
is also shown in the same figure. In this case the images
don’t completely match. The cells did not completely adhere
to the chip surface, so that most of this mismatch is due to
rearrangement that occurred when the chip was transferred
from the test board to the microscope stage. The movement
of cell clusters due to chip movement was confirmed by visual
observation through the microscope as well. Reconfiguring the
test fixture so that images can be acquired while the test board
is on the microscope stage will eliminate this artifact.
IV. C ONCLUSION
We have designed, fabricated, and tested a chip which
functions on the bench as a standard imager and as a contact
imager with microbeads, and in vitro as a contact imager with
cells directly coupled to the chip surface. Major characteristics
of the fabricated chip are summarized in Table I. We have
demonstrated that a CMOS imager can achieve sufficient
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