MULTISPECTRAL ABSORBANCE PHOTOMETRY WITH A SINGLE
LIGHT DETECTOR USING FREQUENCY DIVISION MULTIPLEXING
1
G.K.Kurup1 and Amar.S.Basu1,2,*
Electrical and Computer Engineering Department, 2Biomedical Engineering Department
Wayne State University, Detroit MI, USA
ABSTRACT
Multispectral photometry is often required to distinguish samples in cytometry and other forms of high throughput
screening. The cost of multiple light detectors (PMT, avalanche photodiode, cooled CCD), their respective biasing elements (high voltage supply, resistor network, peltier cooler), and optical filters contributes to the high cost of multicolor
detection systems. This paper describes frequency division multiplexing (FDM), a simple and scalable approach for performing simultaneous multi-wavelength absorbance photometry with a single light detector. Optical emissions from
multiple LED light sources are encoded into unique frequency channels, which are later demodulated using phasesensitive electronics. This paper discusses the theory, characterizes crosstalk and frequency considerations, and demonstrates 3 color absorbance detection in solutions and in flowing droplet microreactors. This technique can potentially reduce the cost of multicolor photometry by replacing expensive optical components with lower cost electronic filters.
KEYWORDS: Multispectral absorbance photometry, Multiplexing, Microreactors, LED, Lock in detection
INTRODUCTION
Spectrophotometry is the most ubiquitous technique in analytical chemistry. Although single-wavelength photometry is useful for many types of basic analyses, it cannot differentiate between multiple analytes. Multi-wavelength photometry can offer the required specificity; however, it requires a more complex optical design, including gratings and arrayed light detectors (CCDs, photodiode arrays). In addition to the added cost and complexity, multi-wavelength
systems have lower sensitivity due to losses in the grating and the poor low-light performance of CCD based detectors
compared to photomultiplier tubes (PMTs). In microfluidic systems, the reduced sensitivity is particularly problematic
due to the small path lengths; and the integration of multiple optical paths is also challenging [1]. This paper presents
frequency division multiplexing (FDM), an electronic filtering technique which enables sensitive, multicolor photometry
using a single light detector and a single flow cell. Since the light detector and flow cell are the most expensive parts of
a detection system, FDM offers an economical and sensitive detection method for point-of-care diagnostics.
THEORY
The
FDM
technique
is
Red
Absorbance
straightforward and analogous to raRed
10KHz
LED
dio broadcasting (Figure 1). Multiple
LEDs, each emitting a different wavelength, are independently modulated
Green
Optical
using a bank of voltage controlled osGreen
Absorbance
8KHz
Fiber
LED
cillators. Each LED is ‘chopped’ at a
+
unique frequency (similar to lock-in
fluorescence detectors [2]). Therefore, each LED is assigned a frequenBlue
Photodetector
Blue
LED
cy channel, and the channels are cho6KHz
(Photodiode
Absorbance
sen so that their Fourier spectra do
or PMT)
Other
not overlap. The combined light is
Microfluidic
Synchronous
LEDs
coupled into an optical fiber, and it
Flow Cell
Demodulators
LED Array
passes through the flow cell to the
Figure 1: Multi-spectral absorbance detection using frequency division
photodetector.
Consequently, the
multiplexing. Absorbance at red (636 nm), green (574 nm), and blue (470
photodetector signal’s Fourier specnm) wavelengths are simultaneously monitored using a simple modulation/
trum contains frequency channels
demodulation scheme. Due to the low cost of electronic components, this
representing the intensities of the reapproach can be economically scaled to include many LEDs spanning a
spective LEDs. Light absorption by
wide spectral range.
the sample modulates the amplitude
of each frequency channel depending
on the sample’s absorption coefficient at the corresponding wavelength. A bank of synchronous demodulators is used to
extract the intensity from each channel. While others have reported LED-based photometers [3-8], this is the first report
to utilize FDM for performing multicolor photometry. The advantage of FDM over time division multiplexing is that
measurements can be performed continuously. However, to avoid inter-channel crosstalk, the frequency channels must
have adequate separation so their spectra (including harmonics) do not overlap. This is analyzed in the results section.
978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS
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14th International Conference on
Miniaturized Systems for Chemistry and Life Sciences
3 - 7 October 2010, Groningen, The Netherlands
EXPERIMENTAL
The prototype FDM system (Figure 2) utilizes low cost LEDs, requires no optical filters, and has a total cost <$20. A bank of three voltage controlled oscillators (Texas Instruments SN74LS629N) are configured to operate at 10 KHz, 8 KHz, and 6 KHz, respectively. The
oscillator outputs are connected to a bank of buffer amplifiers (National Semiconductor) which drive a tri-color LED (Optek). The red,
green, and blue LEDs are driven with 10, 8, and 6 KHz signals, respectively. Three potentiometers are used to adjust the relative intensity of
the colors. Optical emissions are measured using a fiber optic spectrometer (Ocean Optics). The multicolor light is fed into the fiber optic flow cell described in [9], consisting of two 1.5 mm diameter optical fibers (Industrial Fiber Optics) affixed to opposite ends of a cross
junction (Value Plastics). The path length for this cell, defined by the
separation between the fibers, is approximately 1.5 mm. Light from
the receiving fiber is coupled to a photodetector (Industrial Fiber OpFigure 2: Photograph of the FDM detectics). A transimpedance amplifier (National Semiconductor) converts
tion system including the flow cell.
the photodetector current to a voltage signal, which is acquired using a
data acquisition card (National Instruments). The time domain measurements are converted to frequency domain using
Matlab. Three phase sensitive demodulators (AD630, Analog Devices) are connected to the photodiode signal, and each
is also connected to the respective modulation signal from the VCOs. The demodulators are followed by a single pole
passive low pass RC filters. The signals from the red, green and blue channels is recorded using three channel DAQ.
|x(f)|
|x(f)|
Optical Emission (Arb)
RESULTS AND DISCUSSION
Encoding LED emissions into frequency channels. The optical
Blue
spectra of the red, green, and blue LEDs shows non-overlapping emis470 nm
sions at 470, 574, and 636 nm, respectively. Typical optical bandGreen
Red
widths are 100 nm. By oscillating each LED at a different frequency,
574 nm 636 nm
the optical spectra are converted to frequency spectra (Figure 3). The
difference between the theoretical and measured spectra is due to interchannel crosstalk.
To further characterize the inter-channel crosstalk, a single channel
450
550
650
750
A 350
(ie, a single LED) is activated and then the presence of that signal in
Wavelength
(nm)
the other channels is measured. In Figure 4, for example, the red LED
x 10
3
is modulated at a frequency of 10 KHz with other two LEDs are turned
Theoretical Spectra
off. The reference frequency of the blue channel demodulator is va0.43s @ 100 KHz
2
ried from 2 to 12 KHz, and the blue channel output is recorded. As
expected, there is a high interference at 10 KHz due to frequency over1
lap with the red LED. In general, there exists an inverse exponential
relationship between time constant of the RC filters and channel
0
bandwidth (see inset in Figure 4). Here, the RC time constant is varied
5
6
7
8
9
10
B 4
Frequency (KHz)
from 10 ms to 100 ms and the corresponding inter-channel crosstalk is
x 10
3
measured. The results illustrate the fundamental tradeoff in FDM: as
Measured Spectra
the time constant increases, cross-talk decreases, but it also increases
0.43s @ 100 KHz
2
the response time of the circuit. A fast response time necessitates a
large frequency separation between channels to prevent crosstalk. Due
1
to the high speed of LEDs (> 1 MHz), the system could theoretically
support hundreds of frequency channels with adequate channel separation. Additional channels can be implemented by adding an additional
C 04
5
6
7
8
9
10
oscillator, LED, and demodulator for each wavelength.
Frequency (KHz)
Multispectral photometry of FD&C dyes. Figures 5A-C show the
Figure 3: (A) Optical emission spectra of
ability of the system to simultaneously perform 3 wavelength absor3 LEDs. Each LED optical band is transbance measurements without crosstalk. Each dye is diluted to several
lated into a corresponding frequency
concentrations, and the absorbance signals on each channel are recordchannel. (B) theoretical spectra, calcued. The experiment is repeated for each dye. The results show, as exlated using MATLAB. The measured
pected, that FD&C yellow dye absorbs 470 nm (blue) light, but not
Fourier spectra at the photodetector is
574 nm (green) and 636 nm (red) light. Similarly, FD&C red strongly
shown in (c).
absorbs blue and green light, but not red light. FD&C blue strongly
absorbs all colors. These results agree with the known absorbance
spectra of the dyes. This experiment validates the system in performing low bandwidth measurements.
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0.6
0.2
0
A
470 nm
574 nm
636 nm
0
20
40
60
80
100
Yellow Dye Concentration (%v)
100 ms
50 ms
20 ms
10 ms
0.8
0.6
0.4
0
9.6
0.2
0.1
0
50
100
Time Constant (ms)
9.8
10
10.2
Frequency (KHz)
10.4
Figure 4: Analysis of interchannel crosstalk.
Main figure: blue channel output voltages versus
the demodulator reference frequency, when the red
channel is active at 10 Khz. Inset: Channel bandwidths for several different time constants, set by
the low pass filter.
2
1
1.5
0.6
0.4
470 nm
574 nm
636 nm
0.2
B
0.3
0.2
0.8
0
Bandwidth (Khz)
Time Constant
Absorbance (AU)
0.8
0.4
1
1.2
Absorbance (AU)
Absorbance (AU)
1
Normalized Voltage (V/V)
Flow through analysis of droplet microreactors. The use
of the system for time-dependent droplet analysis is shown in
Figure 6. When performing experiments which require high
speed measurements, the time constant and frequency separation between channels must be set appropriately. For example, this experiment is configured for a 100 Hz measurement.
Accordingly, the RC time constant of the low pass filters are
set to 100 Hz (10 ms), and based on Figure 4, the minimum
frequency separation between channels is determined to be
~200 Hz. A 2 KHz channel separation is therefore more
than sufficient. When blue-dyed droplets are flowed past the
detector, the temporal variation in absorbance can be observed in all three channels. The red channel shows the largest modulation not only due to the high absorbance of red,
but also due to i) the high intensity of the red LED, and ii)
the high spectral sensitivity of the detector at red wavelengths. These systematic offsets can handled by calibration
constants, and by adjusting the relative intensity of the LEDs.
0
1
470 nm
574 nm
636 nm
0.5
20
40
60
80 100
Red Dye Concentration (%v)
0
C
0
20
40
60
80
100
Blue Dye Concentration (%v)
Green
.4V
Red
1.6V
CONCLUSION
Conceptually, the FDM system performs spectral filtering using
electronics instead of optical filters. This system can potentially provide several benefits: 1) Scalability: additional channels can be added
economically. LEDs with a wide spectral range (200-2000nm) are
commercially available. 2) Low Cost: achieved by using a single light
detector, low-cost optoelectronics, and fewer optical components. In
addition to the cost, it is simple, compact and consumes less power. 3)
Sensitivity: Lock-in detection inherently reduces measurement noise up
to 100dB, enabling nanomolar sensitivity [2]. We are presently using
the system for multiplexed analysis of droplet microreactors in high
throughput screening. Future efforts will expand this technique to multiplexed fluorescence detection using a single PMT.
Blue
.9V
Figure 5: Multiplexed absorbance measurements of food dyes at 3 wavelengths. (A) FD&C Yellow absorbs blue
light, but passes red and green. (B) FD&C Red absorbs blue and green light, but passes red. (C) FD&C Blue absorbs all three wavelengths. The results are in agreement with the known absorbance spectra of the 3 food dyes.
0
0.2
0.4
0.6
0.8
1
Time (sec)
Figure 6: Inline analysis of droplet miREFERENCES
croreactors using the FDM photometer.
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CONTACT
*Dr. Amar Basu, tel: 313-577-3990; abasu@eng.wayne.edu
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