Fluorescence detection in (sub-)nanoliter microarrays
L.R. van den Doel, M.J. Vellekoop , P.M. Sarro , S. Picioreanu ,
R. Moerman , J. Frank , G. van Dedem , K.T. Hjelt , L.J. van Vliet, and I.T. Young
DIOC-IMDS,
Pattern Recognition Group, Faculty of Applied Sciences,
Lorentzweg 1, Delft University of Technology,
NL-2628 CJ, Delft, The Netherlands,
phone: +31-15-278-1416,
fax: +31-15-278-6740,
Electronic Instrumentation Laboratory, DIMES,
Electronic Components, Technology, and Materials, DIMES,
Bioprocess Technology Group
email: fL.R.vandenDoel,I.T.Young,[email protected]
ABSTRACT
The goal of our TU Delft interfaculty research program is to develop intelligent molecular diagnostic systems (IMDS)
that can analyze liquid samples that contain a variety of biochemical compounds such as those associated with
fermentation processes. One specic project within the IMDS program focuses on photon sensors. In order to
analyze the liquid samples we use dedicated microarrays. At this stage, these are basically miniaturized micro titre
plates. Typical dimensions of a vial are 200 200 20 m3 . These dimensions may be varied and the shape of the
vials can be modied with a result that the volume of the vials varies from 0:5 to 1:6 nl. For all experiments, we
have used vials with the shape of a truncated pyramid. These vials are fabricated in silicon by a wet etching process.
For testing purposes the vials are lled with rhodamine solutions of various concentrations. To avoid evaporation
glycerol-water (1 : 1; v=v) with a viscosity of 8:3 times the viscosity of water is used as solvent. We aim at wide
eld-of-view imaging at the expense of absolute sensitivity: the eld-of-view increases quadratically with decreasing
magnication. Small magnication, however, implies low Numerical Aperture (NA). The ability of a microscope
objective to collect photons is proportional to the square of the NA. To image the entire microarray we have used an
epi-illumination uorescence microscope equipped with a low magnication (2:5 = 0:075) objective and a scientic
CCD camera to integrate the photons emitted from the uorescing particles in the solutions in the vials. From
these experiments we found that for this setup the detection limit is on the order of micromolar concentrations of
uorescing particles. This translates to 108 molecules per vial.
Keywords: microarrays, uorescence microscopy, wide eld imaging
1. INTRODUCTION
One of the most widely exploited elds of research during the last decade is that of combinatorial chemistry. The
stated goal of this approach has been to drastically reduce the time and cost that is required for obtaining new lead
compounds. Currently, many samples are tested against a large number of targets in microarrays for high-throughput
screening. In the future, this technology will lead to a miniaturized lab-on-a-chip. At this moment, however, there
are still many challenges to be solved:
New types of chemistry need to be developed, because the possible targets need to be very sensitive and selective
for high-throughput screening.
Liquid handling needs to be modied to the picoliter range. Ultra small volumes must be fast and automatically
dispensed in (sub)-nanoliter vials in an accurate, precise, and reproducible manner.
M. Ferrari (eds.), Micro- and Nanofabricated Structures and Devices for Biomedical Environmental Applications II, Proc. SPIE,
Progress in Biomedical Optics, vol. 3606, 1999, 28{39.
28
A lab-on-a-chip is more than just a miniaturized micro titre plate: microarrays will contain vials with built-in
intelligence, i.e. integrated actuators and sensors
Many (uorescent) signals can be simultaneously generated. Therefore, state of the art detectors and detection
methods are necessary in order to perform quantitative analysis.
High-throuphput screening generates enormous amounts of data. In order to extract useful information, it is
essential to develop special data analysis systems. Furthermore, data interpretation systems must be developed
to derive new insight into the underlying biochemical principles.
The IMDS program aims to nd solutions for the challenges listed above. The goal of this research program is to
develop systems that can analyze liquid samples that contain a variety of biochemical compounds such as those
associated with fermentation processes. In contrast to most of the research done in this eld, our program does not
focus on applications related to DNA.
One of the main products obtained from human donor blood is Human Serum Albumin (HSA). Due to the risk
of transmission of viral diseases, such as Hepatitis and AIDS, there is a strong need for an alternative source of
human albumin. Unfortunately, human blood plasma is a very rich source of HSA ( 65 g=l) and the current price
of HSA is low ($ US 2 ; 3 =g). This will make it very hard to replace it through any recombinant organism. Pichia
pastoris, which is a yeast strain, produces HSA under a methanol-oxidase promotor. The expression of HSA contains
contaminants, which must be removed during purication. The objective of this project is to nd correlations between
quantities measured in the fermentation tank and the quantity and the quality of the HSA produced. The primary
goal is to increase the productivity of HSA under the following constraints. First, the obtained quantities may not
be puried at extremely high cost, and second, the product must be very pure, because it is normally administered
in relatively high doses. In order to optimize the production under the given constraints, high-throughput screening
is necessary: a sample that is taken from the fermentation tank will be tested against several targets. In order to
analyze such a liquid sample, dedicated microarrays will be developed. In this stage of the project the microarrays
are just miniaturized micro titre plates with a volume on the order of one nanoliter. The fabrication and design
parameters of the microarrays will be described in section 2.2. In the future, the microarrays will be equipped with
sensors and actuators. In each vial a sensor that measures the injected volume in the vial can be integrated. Other
examples of sensors are temperature or pH sensors. Furthermore, optical waveguides might be integrated to direct
the excitation light immediately into each vial. The microarray can be read out in parallel with a CCD camera.
The experimental setup will be described in section 2.1. Redundancy is built in by performing each test at multiple
locations on the microarray during the same screening procedure. The readout of the microarray results in high
dimensional data: images combined with data from the other sensors. This enormous amount of data correlated
in time and space must be further processed to extract valuable information. The information includes the type of
molecules, the concentration of each type, reaction speed, etc. Expert systems will be used to derive new insight
in the biochemical processes. The acquired knowledge and expertise will be used to give feedback not only to the
present situation in the fermentation tank, but to the entire underlying chemistry. This means that the fermentation
process can be optimized, as well as the whole screening methodology.
2. MATERIALS
One specic project within this interfaculty research program focuses on a sensor system for the acquisition of the
uorescent signals that will be generated in the vials of the microarrays. Our rst approach to develop such a system
employs wide eld-of-view imaging, i.e. to image the entire microarray on a CCD camera with low magnication
optics. In this section the experimental setup, the design of the microarrays, and the liquid handling system will be
described.
2.1. Detection system
The experimental setup is built around a Zeiss Axioskop epi-illumination microscope system. This microscope
is equipped with a fully automated xyz;stage (Ludl Electronics Products Ltd, Hawthorne, NY, USA). A KAF
1400 Photometrics Series 200 CCD camera is mounted on the microscope via a 1:0 camera mount (Diagnostic
Instruments Inc., Sterling Heights, MI, USA). The CCD element contains 1317 1035 pixels with a pixel size of
6:8 6:8 m2 . This CCD camera is Peltier cooled to ;42C. Due to this cooling and a slow readout rate (500kHz)
this camera is photon limited. The characteristics of this camera in terms of Signal-to-Noise Ratio are excellent.1 This
29
(a) Silicon, circular, wet etched
(b) Silicon, square, wet etched
(c) Silicon, circular, dry etched
(d) Silicon, square, dry etched
(e) Glass, circular, wet etched
(f) Glass, square, wet etched
Figure 1. This gure shows the six dierent types of vials that have been fabricated. The diameter of the circular
masking pattern is 200 m. The width of the square masking pattern is 200 m. The depth varies from 40 m for
the vial in Figure 1(a) to 17 m in Figure 1(e) and 1(f).
camera is connected via a NuBus interface to a computer. A Macintosh Quadra 840AV computer takes care of the
microscope control and the image acquisition. The microscope as well as the camera can be controlled from within
the environment of an image processing package; this package is SCIL Image (TNO Institute of Applied Physics
(TPD), Delft, The Netherlands, e-mail address: [email protected]). To image the entire microarray onto the CCD
array, a low magnication objective is used: a 2:5 = 0:075 Zeiss Plan-NEOFLUAR objective. With this objective
it is possible to image an area of 3:5 2:7 mm2 onto the CCD array. Using vials with an area of 200 200 m2 and
a center-to-center distance of 300 m, i.e. there is an empty region of 100 m between two vials, it is possible to
readout 100 vials per image. Wide eld-of-view imaging with low magnication optics, however, is at the expense of
absolute sensitivity. The reason is that the light-gathering power of an objective lens is proportional to the square
of the NA: the lower the NA, the less ecient photons can be collected.2
2.2. Design and fabrication of microarrays
Six dierent types of microarrays have been manufactured at DIMES (Delft Institute for Microelectronics and Submicron Technology, Delft University of Technology, NL-2628 CD Delft, the Netherlands). These vials are fabricated
either in silicon or in glass. Dierent geometries can be made by using dierent etching mask patterns. For our vials,
one mask with circular patterns is used and another mask with square patterns. The dimensions of the patterns are
200 m in diameter or in width respectively. In the following, the fabrication process of the silicon microarrays will
be described, followed by the fabrication process of the glass microarrays.
The silicon wafers are rst covered with a thin layer of silicon nitride. On top of this layer a thin lm of photo
resist is deposited. This lm is exposed through the masking pattern of the microarrays. After removal of the
exposed photo resist, the wafer is etched using plasma etching. During this process, the silicon is uncovered at the
locations of the vials. The remaining photo resist is removed by rinsing the surface with acetone. Both wet and
dry etching techniques were employed on the silicon wafers for the realization of the vials. The wet etching was
conducted in potassium hydroxide (KOH). Because of the crystal structure of silicon, this technique results in an
anisotropic etching of the silicon. The silicon wafers have a < 100 > surface plane orientation. The etching rate for
30
the < 111 > crystal planes is much slower in KOH solution than for the other crystal planes (typically by a factor of
50). Therefore, wet etching of the silicon results in vials with the shape of a truncated pyramid, which has < 111 >
planes as side walls for circular as well as for square etching masks. These two types of vials are shown in Figure 1(a)
and 1(b) respectively. In the case of a circular window, underetching of the silicon nitride will take place resulting in
free hanging silicon nitride in the corners of the vial, as can be seen in Figure 1(a). The angle between the < 111 >
planes and the < 100 > plane is about 54:7. The depth of the vials in Si for the largest sizes is typically about
20 m. The dry etching is conducted by Reactive Ion Etching (RIE), which results in an anisotropic etching. The
RIE etched vials have a cylindrical or cubic shape after etching with a circular or square masking pattern respectively.
These vials are shown in Figure 1(c) and 1(d).
Microarrays have also been manufactured in glass. Glass wafers are covered with a thin layer of polysilicon, which
is then patterned to form the etching mask. Wet etching in a hydrouoric acid (HF) solution is utilized. Glass is an
amorphous material, and thus isotropic for etching. This process results in a cilindrical or cubic shape with rounded
edges and corners. These shapes are shown in Figure 1(e) and 1(f). In Table 1, typical depths and volumes are given
for these six shapes of vials. The dierent types of vials are lled according to the methods described in the next
section.
Table 1. Typical depths and volumes for the six dierent shapes of fabricated vials with 200 m diameter or width.
Material Etching Pattern Depth (m) Volume (nl)
Silicon wet etched circular
40
1:6
square
20
0:8
Silicon dry etched circular
23
0:7
square
23
0:8
Glass
wet etched circular
17
0:5
square
17
0:7
Each microarray contains 5 5 vials. The center-to-center distance is 600 m. The dimensions of the microarray
itself are 2 1 cm2 . In Figure 2 one of the microarrays is shown.
1.0 cm
2.0 cm
5x5 square
wet etched vials,
200x200x20 µm3
Figure 2. A microarray with 5 5 vials.
2.3. Fluid injection
One of the diculties involved in the miniaturization of high-throughput screening technologies is liquid handling.
Fluid volumes less than a nanoliter need to be injected into small vials. The volumes of the vials described in
Subsection 2.2 ranges from 0:5 nl to 1:6 nl. An Eppendorf Transjector 5246 (Eppendorf, Netheler-Hinz GmbH,
22331 Hamburg, GE) is used to inject such small volumes into the vials. This machine is primarily used for In
31
Exponential pressure
decay
Sudden pressure
build-up
Pressure
Injection Pressure
Injection time
Compensation Pressure
Time
Figure 3. The operation of the Transjector: to inject small liquid samples, the Transjector suddenly builds up
a pressure (typically about 150 hPa), which is kept during a certain injection time (typically about 1:5 s). After
injection, the presure is exponentially released to the compensation pressure.
Vitro Fertilization (IVF), in which sperm cells are injected through the cell membrane into an oval cell. In order
to inject sperm cells into an oval cell or in our case to deliver small liquid volumes into a nanoliter vial, a special
capillary the Femtotip II is used. The Femtotip II is about 5 cm long and the outlet of the capillary has an inner
diameter of less than 0:5 m. The capillary tapers o to the outlet. The problem with this feature is that the ow
is not constant towards the outlet of the capillary. Therefore it is not possible to control the injected volume. Other
groups have used the same or similar apparatus but with their own pulled capillaries.3{5 These pulled capillaries
have a constant diameter. This enables good control of the ow. As a result, the injected volumes with these kind
of capillaries are much smaller: 10 ; 100 pl. In order to inject a small liquid sample into a vial, the Transjector
builds up a sudden injection pressure. This pressure is held during the injection time. After injection the pressure is
exponentially released to the compensation pressure. It is necessary that the tip of the capillary touches the surface
of the vial in order to release the liquid drop from the tip and to get the liquid in the vial. The operation of the
Transjector is shown in Figure 3.
The compensation pressure must always be applied because of the capillary force. Experiments have been done to
partially ll the vials, but the variation in the injected volume for a partially lled vial is quite large. Therefore the
vials are always lled completely. From the experiments described in Section 4.1 follows that the volume variation
of a completely lled vial is about 6%. It is obvious that this kind of liquid handling does not meet our demands.
With this device it is only possible to ll a single vial at a time with just one liquid sample. We, however, want to
ll an entire microarray at once with many dierent chemicals. Figure 4 shows the experimental setup for lling the
vials with a solution.
2.4. Chemical solutions and solvents
For the experiments we have used several dierent solutions of rhodamine and resorun. In the previous section we
have shown that the volume of the vials is on the order of one nanoliter. If a droplet of water of this size is pipetted
into a vial, it evaporates in less than a few seconds. In order to reduce the evaporation rate we have used solutions
of glycerol : water (1:1, v/v) with a viscosity of 8:30 , where 0 is the viscosity of water. With this high viscosity
the evaporation process of the liquid is extended to more than 45 minutes. To lower the surface tension a detergent
is added to the solutions.
The solutions of Rhodamine B (Merck, Ge) were prepared by serial dilution in a glycerol : water (1:1, v/v)
mixture containing 0.005% (w/v) Sarkosyl NL-97 detergent (Cyba-Geigy). The solutions of Resorun were prepared
also by serial dilution in a glycerol : 50 mM sodium phosphate buer, pH 7.4 (1:1, v/v) mixture containing 0.005%
(w/v) Sarkosyl NL-97 detergent (Cyba-Geigy). The Sodium phosphate buer and resorun (sodium salt) were
purchased as parts of the Amplex Red Glucose Assay kit (A-12210, Molecular Probes, Eugene, OR, USA). Millipore
demineralized water and glycerol (analytical grade reagent, BDH, England) were used for all experiments.
32
3. THEORETICAL ASPECTS OF CONCENTRATION MEASUREMENTS
In this section we will briey describe some aspects related to our experimental setup for measuring concentrations
of uorescing particles in solution. It is possible to divide these aspects into two parts: on one hand the eciency
of exciting the uorophores, and on the other hand the eciency of collecting and detecting the emission light from
the uorophores.
3.1. Excitation eciency
The strength of the emission light depends on two properties of the uorophore: the quantum eciency or quantum
yield and the absorption cross section of the uorophore. The latter property is comparable to the molar extinction
coecient or the absorptivity in a bulk solution. The absorption cross section is the eective area of a uorophore
which is illuminated by an incident beam of light and which is capable of absorbing photons. The quantum eciency
of a uorochrome is dened as the ratio between the number of emitted photons to the number of absorbed photons.2
The quantum yield of a uorophore must be high, at least up to 30%.6
Furthermore, the excitation eciency is aected by the illumination of the excitation light beam. In Kohler
illumination the light beam emerging from the light source is focused in the back focal plane of the objective. This
results in a parallel bundle of light rays in the image plane of the objective. As a result the eld-of-view is evenly
illuminated. The area of the eld of view is proportional to 1=M2, where M is the magnication of the objective. As a
result, the light intensity per unit area in the eld-of-view is proportional to M2 . With wide eld-of-view imaging, the
total energy of the incoming light is spread out over a large region, where the light is not wanted. Given a microarray
with vials of sizes 200 200 m2 and a 600 m center-to-center distance, the area of interest is just 10%. In the
remaining 90% of the eld-of-view reections from the surface of the microarray occur. These reections cause a high
background signal. A second source of inuence is stray light which originates in the optical system by reections
(are) at air-lens interfaces and scattering at diuse surfaces within the optical system (glare). Finally, the third
source of inuence is (Raman) scattering in the solvent and autouorescence in the objective. These phenomena are
mentioned here, because they originate in the excitation path of the microscope system, although they inuence the
detection of the uorescence signal.
3.2. Detection eciency
The detection eciency is mostly inuenced by the Numerical Aperture of the objective. The light gathering power
of an objective in Kohler illumination is proportional to NA2 =M2 . Under the assumption that there is a linear
relation between excitation and emission, we can say that the intensity of the emission light is proportional to M2 ,
as shown in the previous section. Combining these results, it follows that the light gathering power of an objective
(with epi-illumination adjusted to Kohler illumination) is proportional to NA2 . For our experiments we have used
a 2:5 = 0:075 Zeiss Plan-NEOFLUAR objective. The cone of light that can be collected with this objective is
Figure 4. The microscope setup for lling the microarrays. The capillary is placed under the 2:5 objective (working
distance 9:3 mm) at an angle. The 5 5 200 200 m2 microarray is clearly visible by the scattered excitation light.
At the right a "Dutch Dime" (15 mm in diameter) is placed to indicate the size.
33
Coefficient-of-variation (%)
Coefficient-of-variation (%)
8
7
6
5
4
3
2
1
8
7
6
5
4
3
2
1
1
2
3
4
5
6
7 8 9 10 11 12 13
Number of filling procedure
(a) Inter-vial error: 5:9%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Label of vial
(b) Intra-vial error: 5:9%
Figure 5. The left graph shows the inter-vial error for successive lling procedures. This error is expressed as the
coecient-of-variation. On average this error is 5:9%. The right graph shows the intra-well error for dierent vials
on the microarray. This error is equal to the inter-vial error.
less than one percent of the total solid angle of 4. Under the assumption that the emitted photons radiate in all
directions, the light collection eciency of such a low NA objective is less than one percent. The photons collected
by the objective fall onto the CCD array and are converted to photoelectrons. This conversion factor is the quantum
eciency of the camera and depends on the wavelength of the light. In the UV range of the spectrum the quantum
eciency is virtually zero, whereas the quantum eciency towards the red part of the spectrum goes up to about
40 ; 50 % for this CCD camera. Another important characteristic of the CCD camera is the electronic gain factor :
the electronic gain for the KAF 1400 Photometrics CCD camera is 0:126 #ADU=e; , i.e. an increase of the intensity
with a single grayvalue means that about 8 photoelectrons have been collected.1 The lowest measurable signal with
our system is limited by the readout noise of the camera: the statistical distribution of the detection limit should lie
well above the distribution of the readout noise in order to discriminate between noise and signal. The readout noise
is additive and has a Gaussian distribution. It is therefore expressed in terms of its standard deviation in number of
electrons. Due to the slow readout rate of this CCD camera the readout noise is as low as 11.7 electrons.1
4. EXPERIMENTAL SECTION
In this section, we will describe a number of experiments that have been performed with the microarrays. All
experiments described below are performed with the vials with the shape of a truncated pyramid (See Figure 1(b)).
These vials have dimensions of 200 200 20 m3 . This corresponds to a volume of 0:8 nl. Section 4.1 describes
the experiment to measure the variation of the manual lling procedure of the vials. Section 4.2 describes the
measurement of the detection limit of our system. In Section 4.3 we will show the inuence of certain modications
to the system. Finally, in Section 4.4 we will show some aspects of evaporation processes that occurred after lling
the vial with a solvent with (too) low viscosity.
4.1. Calibration of the injected volume
As described in Section 2.3 the various solutions are manually injected into the vials with a special capillary. In this
experiment we want to calibrate the injected volume in terms of a lling error. The lling procedure is quite tedious.
The lighting conditions are bad and the microarrays must be moved under the capillary to ll vial after vial. For this
reason this experiment is only performed for one particular shape of vial. The vials used in this experiment have the
shape of a truncated pyramid with dimensions 200 200 20 m3 (See Figure 1(b)). We expect that the geometry
of the vial has some inuence in the error, however, we assume that this contribution to the error is small with respect
to the contribution of the method of lling the vials. In order to measure the lling error, 4 4 vials (corresponding
to an area of 2 2 mm2 ) were lled with the same Resorun solution (1:0 M resorun in glycerol/water). From each
vial the average intensity was measured. The measured intensity is directly proportional to the number of molecules
34
Average vial intensity (#ADUs)
1000
500
100
50
blank
10
10-8
10-6
10-4
Rhodamine concentration (M)
Figure 6. A typical response of the detection system to dierent concentrations of rhodamine. For all six vials the
detection limit is on the order of 10;7 ; 10;6 M.
in the vial, which is directly related to the amount of liquid in the vial. From these values a lling error can be
computed: this is the lling error that occurs after lling a number of vials on the same microarray. Part of this error
is caused by the lling method and part of this error is caused by dierences in volume of the vials. The variation
in volume of the vials is expected to be quite small. This error is the inter -vial error. This experiment is repeated
with the exact same microarray more than ten times, which results in a better estimate of the inter-vial error. From
these results, a second error can be dened: the intra -vial error. Only the lling procedure itself contributes to this
error. In Figure 5 the results of this experiment are shown.
Both error measures are expressed in terms of the coecient-of-variation, which is dened as = 100%. As
can be seen in Figure 5, the inter-vial error is equal to the intra-vial error 5:9%, which corresponds to about 50 pl.
These results imply that the variation of the volume of the vials is negligible with respect to the error of the lling
procedure. Although the method to ll the vials is quite tedious, the error is acceptably small.
4.2. Measurement of detection limit
The detection limit for a similar kind of setup with high Numerical Aperture objectives and laser induced uorescence
is on the order of 10;9 M.7 We have measured the detection limit of our system by lling the vials with solutions
with varying concentrations of rhodamine and measuring the signals from the vials. This experiment is performed
for the six dierent vials as shown in Figure 1. A typical graph of this experiment is shown in Figure 6. This graph
corresponds to the experiment with the vials of the type as shown in Figure 1(b).
This experiment is repeated with resorun in the same type of microarrays. In this experiment the concentrations
of resorun were restricted from 1:0 to 20:0 M. In Figure 7 an image of the lled microarray with resorun is shown
and the response of the system to the dierent concentrations.
4.3. Inuence of modications to the system
In this section we will describe some preliminary results of experiments that have been performed to measure the
inuence of certain modications to the microscope system. With our microscope system it is easy to adjust the
illumination to Kohler illumination or to critical illumination. Futhermore, the eld stop can be adjusted to change
the eld of view. Finally, objectives with higher Numerical Apertures could have been used to measure its inuence.
For the latter modication, we do not have any results as yet. The results of the rst two modications will be shown
below.
Kohler vs. Critical illumination. In Kohler illumination the image of the arc lamp is focused in the back focal
plane of the objective. This results in a parallel bundle of light in the specimen plane of the objective. This
type of illumination leads to a uniformly illuminated eld-of-view. When the illumination system, however, is
adjusted to critical illumination, the image of the arc lamp is imaged in the specimen plane. In this case the
eld-of-view is very brightly illuminated in the center, but the intensity drops o rapidly towards the border
of the eld-of-view. It is obvious that Kohler illumination is required to readout the vials of the microarray in
parallel. To measure the inuence of the illumination adjustment we lled vials with dierent concentrations
35
Average vial intensity [#ADUs]
1000
800
600
400
200
5
(a) Image of microarray
10
15
20
Concentration resorufin [µMolar]
(b) System response to resorun
Figure 7. The image on the left side shows a microarray lled with solutions of varying concentrations of resorun:
each column contains one specic concentration. The data of this image is shown in the right graph. This graph
shows the averaged vial intensity for dierent concentrations of resorun 2:5 ; 20 M.
Critical illumination (#ADUs)
of rhodamine and measured the uorescence signals as a function of the illumination type. The results of this
experient are shown in Figure 8. It can be seen in this graph that with critical illumination 5 times more light
comes out of the vial than with Kohler illumination. This does not necessarily mean that the detection limit
of the system will move towards lower concentrations.
2000
1000
500
200
100
50
20
20
50
100
200
500
Kohler illumination (#ADUs)
Figure 8. Critical illumination vs. Kohler illumination: With critical illumination 5 more light comes out of the
vials than with Kohler illumination. The dierent grayvalues of the dots correspond to dierent concentrations.
The eect of closing the eld stop. In Section 3 we mentioned that with the current microarrays 90% of the
total area is illuminated from which no valuable signals are acquired. This extraneous illumination causes stray
light in the optical system. One way to reduce the amount of stray light is closing the eld stop in the excitation
light path. In this experiment we measured the uorescent signal from the vials and from the background,
when the size of the eld-of-view was adjusted by opening or closing the eld stop. In Figure 9 the results of
this experiment are shown. It follows from these results that stray light can be avoided to a certain extent by
closing the eld stop.
36
Average intensity Vial (#ADUs)
Background
35
30
Vial
25
20
15
10
5
closed
open
intermediate filled
Opening of the Field Stop
Figure 9. Closing the eld aperture reduces the amount of stray light in the optical system. Note that the signal
from the vial is lower than the signal from the background, which is the empty area of the microarray. Closed is
the smallest opening, open is the widest opening, lled means that the edge of the eld stop lls the aperture of the
eyepiece. The intermediate opening is between the closed and lled opening.
4.4. Evaporation of solvents
A droplet of water of 1 nl injected into one of the vials evaporates within a few seconds because of its low viscosity.
In order to prevent evaporation or at least to extend this process suciently, we have used solvents with a higher
viscosity. As described in Section 2.4 glycerol/water (1:1,v/v) with a viscosity of 8:3 times the viscosity of water is
used as solvent. Mixtures of ethyleneglycol and water in dierent ratios have a viscosity of about 50 . Once a vial is
lled with this solvent, it evaporates in less than 20 minutes. We have monitored this process by acquiring an image
every 30 seconds. Five images of this process are shown in Figure 10.
(a) t = 0 min:
(b) t = 5 min:
(c) t = 10 min:
(d) t = 15 min:
(e) t = 20 min:
Figure 10. A series of images showing the evaporation process of ethyleneglycol-water (90%=10%; v=v) in a vial at
dierent times.
At t = 0, just after injection of the liquid, the signal from the vial is uniform, besides some geometrical eects
along the sidewalls and in the corners of the vial. This implies that the meniscus of the liquid is virtually at and
the vial is completely lled. After lling the vial, the liquid is "pinned" to the edge of the vial. During evaporation,
the pinning of the liquid ensures that the evaporation from the edge is replenished by liquid from the center part of
the liquid in the vial.8 This can be seen qualitatively in Figure 10: the uorescent signal gets weaker in the center,
until the bottom of the vial is reached. The signal disappears from that area sometime between 10 and 15 min.
While the evaporation continues, the liquid is stuck in the four corners of the vial. At these spots the concentration
of uorescing particles is increasing. Finally, when the solvent is completely evaporated, the particles loose their
uorescent behavior and the signal disappears. In Figure 11 the average uorescence signal from the vial and the
signals from the center, from one of the sidewalls and from one of the corners of the vial is shown as a function of
time.
It can be seen in this graph that the average uorescence signal from the vial remains almost constant during
37
intensity in #ADUs
800
600
Intensity in corner
400
Average vial intensity
200
Intensity at
sidewall
Intensity in centre
5
10
15
20
time in minutes
Figure 11. The average uorescence signal from the vial, the signals from the center of the vial, the sidewalls of
the vial and the corner of the vial as a function of time. It can be seen that, besides some geometrical eects, the
average signal of the vial does not decrease during the evaporation process.
evaporation. The slight increase of the signal in the beginning is caused by geometrical eects. These eects are
less than 10% of the signal. Furthermore, it can be seen that the signal in the corners of the vial increases steadily,
because of the accumulation of the uorescent particles at these spots.
Note that with our microscope system the three dimensional liquid-air interface, the meniscus, is projected onto
a plane. Therefore it is not possible to retrieve the shape of the meniscus during the evaporation. It is possible to
monitor this with a confocal scanning laser microscope.
5. CONCLUSIONS AND DISCUSSION
In this phase of our research program, we use miniaturized micro titre plates fabricated in silicon. In the future, these
microarrays will have integrated intelligence by means of several sensors and actuators. The volume of each vial is on
the order of one nanoliter. In order to pipette such a small volume, a special apparatus is used. With this machine
it is possible to manually ll the microarrays. To avoid evaporation, solutions with a high viscosity must be used.
We have used glycerol/water as solvent with a viscosity of 8:30 . To lower the surface tension a detergent is added
to the solutions. The lling error is 5:9%, which corresponds to about 50 pl. In this phase we have aimed at wide
eld-of-view imaging at the expense of absolute sensitivity. We have therefore used an epi-illumination microscope
equipped with low magnication optics and a scientic CCD camera. With this setup the detection limit is on the
order of micromolar concentrations. The system can be improved by reducing the amount of stray light. There are
several options to solve this. One option is to separate the excitation path from the emission path in the optical
system. A second option is to insert a grating right after the light source. This grating will only pass the light that
reaches the area of the vials. A third option is to integrate the light source into the vials. This can be achieved
by means of optical waveguides or with light-emitting polymers. At this moment these polymers are only used in
displays of portable computers.
ACKNOWLEDGMENTS
This work is supported by the Delft Interfaculty Research Center Intelligent Molecular Diagnostic Systems (DIOCIMDS).
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