Fluorescence lifetime biosensing with DNA
microarrays and a CMOS-SPAD imager
Gerard Giraud,1,* Holger Schulze,2 Day-Uei Li,3 Till T. Bachmann,2 Jason Crain,1,4
David Tyndall,3 Justin Richardson,5,8 Richard Walker,3 David Stoppa,6
Edoardo Charbon,7 Robert Henderson,3 and Jochen Arlt1
COSMIC & School of Physics and Astronomy, SUPA, The University of Edinburgh, The King’s Buildings,
EH9 3JZ Edinburgh, UK
Division of Pathway Medicine, College of Medicine and Veterinary Medicine, The University of Edinburgh,
Chancellor’s Building, Little France Crescent, EH16 4SB Edinburgh, UK
3
The Institute for Integrated Micro and Nano Systems, School of Engineering and Electronics, The University
of Edinburgh, The King’s Buildings, EH9 3JL Edinburgh, UK
4
National Physical Laboratory, Hampton Road, Teddington, Middlesex TW11 0LW, UK
5
Imaging Division, ST Microelectronics, Edinburgh EH12 7BF, UK
6
Smart Optical Sensors and Interfaces, Fondazione Bruno Kessler, Trento, Italy
7
EEMCS Faculty, Delft University of Technology, Mekelweg 4, 2628CD Delft, Netherlands
8
Currently at Selex Galileo, A Finmeccanica Company, 2 Crewe Road North, Edinburgh, EH5 2XS, UK
*gerard.giraud@ed.ac.uk
1
2
Abstract: Fluorescence lifetime of dye molecules is a sensitive reporter on
local microenvironment which is generally independent of fluorophores
concentration and can be used as a means of discrimination between
molecules with spectrally overlapping emission. It is therefore a potentially
powerful multiplexed detection modality in biosensing but requires
extremely low light level operation typical of biological analyte
concentrations, long data acquisition periods and on-chip processing
capability to realize these advantages. We report here fluorescence lifetime
data obtained using a CMOS-SPAD imager in conjunction with DNA
microarrays and TIRF excitation geometry. This enables acquisition of
single photon arrival time histograms for a 320 pixel FLIM map within less
than 26 seconds exposure time. From this, we resolve distinct lifetime
signatures corresponding to dye-labelled HCV and quantum-dot-labelled
HCMV nucleic acid targets at concentrations as low as 10 nM.
©2010 Optical Society of America
OCIS codes: (170.2520) Fluorescence microscopy; (170.3650) Lifetime-based sensing;
(120.3890) Medical optics instrumentation; (280.1415) Biological sensing and sensors;
(250.1354) Avalanche photodiodes (APDs).
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Received 10 Aug 2010; revised 12 Sep 2010; accepted 30 Oct 2010; published 4 Nov 2010
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1. Introduction
DNA microarrays are important tools for biomolecular detection. Widely used for gene
expression profiling, disease screening, mutation and forensic analysis, they also hold much
promise for the future development of personalized drugs and point of care testing devices
[1,2].
In general terms, microarray technology exploits molecular recognition between a probe
molecule attached to a substrate and a complementary target. The strength of the technique
lies in its unique multiplexing capabilities, whereby a very large number of target molecules
can be interrogated by a multitude of reporter probe spots printed on a single chip. The read
out of microarrays can be achieved using a variety of detection techniques [3], however,
fluorescence is the most commonly used transduction method [4]. Recently, fluorescence
lifetime imaging (FLIM) has been reported as a powerful multidimensional technique that
offers a number of benefits for microarray reading in term of sensitivity, specificity and
multiplexing capabilities [5,6]. Nonetheless, the relative complexity of the method, combined
with bulky and expensive equipment has yet prevented its integration into microarray based
biosensing devices.
FLIM can be performed using various methods either in time or in frequency domain
[7,8]. The respective advantages and disadvantages associated with different approaches have
been reviewed in recent publications [9,10]. The choice of the given technique is driven by
specific applications. In the case of DNA microarray reading, time-correlated single-photon
counting (TCSPC) based methods present some critical advantages over other techniques that
include single-photon sensitivity combined with unlimited dynamic range and high temporal
resolution. The major draw back of TCSPC lies on its inherently low duty cycle and
associated long data acquisition time, incompatible with real-time monitoring and highthroughput screening.
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Received 10 Aug 2010; revised 12 Sep 2010; accepted 30 Oct 2010; published 4 Nov 2010
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Improvements to wide-field photon counting cameras based on multi-channel plates have
led to increased count rates up to 500 kHz with good spatial resolution [11] but the global
detection scheme of one photon event at the time in the whole field of view limits the
scalability of this approach. The emergence of a new type of imager based on single-photon
avalanche diode (SPAD) arrays built on CMOS technology [12,13] is enabling high
parallelization of TCSPC acquisition in a compact and cost effective manner. Such a device
has been utilized as an active array substrate to perform the time-resolved Forster resonance
energy transfer detection of a DNA assay, demonstrating a reduction in the transduction signal
dependence to the DNA probe surface density [14]. More recently our CMOS-SPAD device
was used to demonstrate real-time FLIM based on an onboard lifetime estimation method [15]
which works well for bright samples with mono-exponential decays, such as the reported
mixing of highly concentrated dye solutions. However, samples encountered in the context of
biosciences are typically much dimmer, which reduces the advantage of lifetime estimation
both in terms of speed and data compression. Biological samples also often show more
complex decays which require photon arrival time histograms for more detailed analysis. In
the present article we show fast FLIM detection at low fluorophore concentrations relevant to
DNA binding assays. By adding an onboard memory controller to our CMOS-SPAD device
[12,15] we can capture the arrival time of every detected photon, allowing rapid FLIM based
DNA microarray readout and the recording of arrival time histograms confirming multiexponential decays. This study lays the foundations for high-throughput multiplex FLIM
detection and fluorescence lifetime kinetic studies.
2. Material and methods
2.1 Microarray preparation
We prepared a DNA microarray spotted with two different DNA probes and hybridized with
their distinct complementary artificial targets. The detailed procedure of production and
incubation has been reported in a previous publication [5]. In short, the microarray consisted
of 16 spots, incubated with a) hepatitis C virus (HCV) probe, b) human cytomegalovirus
(HCMV) probe and c) a 1:1 molar ratio of HCV and HCMV probes.
The array was hybridized with a solution of complementary target containing 10 nM of
Alexa Fluor 430 (Alexa430) labeled HCV target and 10 nM biotinylated HCMV target, which
was further incubated with quantum dot (QDs) streptavidin-conjugate solution, Qdot525.
Qdot525 and Alexa430 were chosen for their overlapping absorption and emission spectra
combined with a distinctly different excited state lifetime, approximately 4.2 and 22 ns
respectively. Those properties permitted FLIM measurements using both a single excitation
wavelength (415 nm) and a single read out channel (515 to 565 nm band-pass).
2.2 DNA oligonucleotide sequences
The sequences of the HCV probes (P1) and targets (T1) obtained from Metabion (Martinsried,
Germany)
are
as
follows:
P1
5TGCACGGTCTACGAGACCTCCCGGGGCACTCGCAAGCACCCTATCAGGCAGTACC
ACAAGGCCTTTCGC-3[5]
=
SH,
T1
5GCGAAGGCTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTC
GTAGACCGTGCA [5] = Alexa 430. The sequences of the HCMV probes (P2) and targets
(T2) obtained from Eurogentec (Seraing, Belgium) are as follows: P2 5CAAATACCGTGGGACGACACGCACCGGCAGTGCGCAGGCAGCGTCGGACACAAC
ACGCTTACGGCCCTCAACACT–3[5]
=
SH(C6),
T2
5AGTGTTGAGGGCCGTAAGCGTGTTGTGTCCGACGCTGCCTGCGCACTGCCGGTGC
GTGTCGTCCCACGGTATTTG – 3[5] = Biotin-TEG.
2.3 TIRF-SPAD array imaging Setup
The microarrays were imaged by combining a total internal reflection fluorescence (TIRF)
setup with the SPAD imager (Fig. 1). The excitation source consisted of a Ti:Sapphire laser,
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Received 10 Aug 2010; revised 12 Sep 2010; accepted 30 Oct 2010; published 4 Nov 2010
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pulse picker and frequency doubler (Coherent, Glasgow, Scotland) and produced femtosecond
pulses at 415 nm wavelength with a repetition rate of 4.75 MHz. Note however that such a
complex excitation system is not really required and could be replaced by a much more
compact picosecond diode laser. The TIRF setup used a quartz prism (Cairn Research,
Faversham, UK) attached to the condenser of a Nikon TE300 inverted microscope and placed
into contact with the undersurface of the DNA microarray using immersion oil. 2 mW of laser
light was directed below critical angle, generating a local evanescent excitation of circa 1 mm2
area on the DNA microarray. The resulting fluorescence was then collected with a 10 ×
microscope objective (Plan Apo, N.A. 0.45, Nikon, Japan), filtered with an emission bandpass filter (535/40 nm) and imaged onto the SPAD array imager. A de-magnifying image
relay (f1 = 50 mm, f2 = 8.5 mm) was used to match the excitation area to the field of view of
the sensor.
Fig. 1. Experimental setup of the TIRF-SPAD imager setup. (see text for details).
The camera is based on a 32 × 32 pixel array, fabricated in 130 nm CMOS technology,
where each pixel contains its own SPAD and 10-bit time-to-digital converter (TDC). The two
halves of this prototype chip have different implementations of the TDC, most notably leading
to different time resolutions of 54 ps and 175 ps respectively (see ref [12,16]. for technical
details and characterization of the chip). Detection of a photon starts the in-pixel TDC with
the stop signal provided by a fast photo diode monitoring the excitation beam. The sensor is
capable of supplying photon arrival time data at a variable frame rate up to 500 kHz
(potentially over 500 million photon events per second), producing data rates of up to 5Gbit/s.
In order to record full arrival time data a memory controller has been implemented on the
device which can stream up to 100,000 frames of data directly to its local memory at any
frame rate. Once the device memory has been filled up it can be transferred post-experiment
to a PC.
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2.4 Image acquisition and analysis
Data presented here only used 20 × 16 pixels on the „fast‟ TDC half, which has a time range
of 55 ns with 1024 bins of 54 ps resolution. Here, the low intensity fluorescence of the DNA
microarray samples required an increase in exposure time to 256 μs, leading to a reduced
frame rate of just under 4 kHz. The memory controller was used to acquire blocks of 100000
frames with TDC codes (~26 s total exposure time), which were then transferred to the PC
and combined into arrival-time histograms (~12 s in total). Custom software written in
Labview (National Instruments, Texas) was used to remove the background in noisy pixels
and prepare the TCSPC data for import into commercial FLIM analysis software (SPCImage
3.1, Becker & Hickl GmbH, Germany).
3. Results and discussion
Initially, TCSPC data for the three different categories of spots ((a) HCV probes, (b) HCMV
probes and (c) mixture of 50% HCV and 50% HCMV probes) was collected using subarrays
containing four identical spots. Fig. 2 shows representative decay curves for each category
obtained from an individual pixel. Least square fitting using the SPCImage software clearly
revealed multi-exponential decays for each category of spots, with bi-exponential models
leading to good fits (χ2 ~1.4) with distinct average lifetimes.
Fig. 2. Decays histograms (circles) with corresponding fit (solid lines) obtained for three
individual pixels associated with the three categories of microspots, (a) HCV, (b) HCMV-HCV
mixture and (c) HCMV.
Fig. 3 shows the average lifetimes of four subframes (16x20 pixels each) as FLIM maps
(left) and corresponding pixel lifetime distribution histograms (right). The fit of pure HCV
and HCMV decay curves resulted in average lifetimes centered on 4.2 ns and 22 ns matching
the reported emission decay values for Alexa430 and Qdot525 [5]. Both their distributions are
fairly narrow with FWHM of around 10% of the mean lifetime (610 ps and 1.97 ns for HCV
and HCMV, respectively). The distribution for HCV-HCMV mixture is markedly broader
with a FWHM of 3.5 ns centered on a mean value of 11.3 ns. Dynamics of the QDs excited
state follows complex mechanisms resulting in multi-exponential emission decays with up to
5 components [17,18] and Alexa in the complex environment of the microspot has several
decay components [5]. Decays from the HCV-HCMV mixture are a superposition of these
single label decays and the average lifetimes depend sensitively on the local relative
concentration, giving raise to the observed much wider distribution. Repeating the fitting
process with two globally fixed lifetimes (τ1 = 4.2 ns & τ2 = 22.0 ns) leads to good fits of
comparable quality for all three types of probes, as demonstrated by the FLIM map with
different spots displayed in Fig. 3(d). In this case, the relative contributions of the two lifetime
components match the probe ratio of the spots (100:0, 50:50 or 0:100 resp.). A more detailed
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Received 10 Aug 2010; revised 12 Sep 2010; accepted 30 Oct 2010; published 4 Nov 2010
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study is currently underway, as this might open the way for quantification of the probe ratios
in individual spot, especially when combined with more rigorous global analysis of the
lifetime images [19].
Fig. 3. (Left) FLIM map of DNA microarray spotted with a series of four sub-arrays
corresponding to four subframes of 16x20 pixels each: (a) HCV probes, (b) HCMV probes, (c)
four spots containing a mixture of 50% HCV and 50% HCMV probes, (d) HCV probes (top
left), HCMV probes (bottom right) and 50% probe mixture (diagonal). The array was
hybridized with a solution containing 10 nM of Alexa430 labeled HCV complementary target
and 10nM of Qdot525 labeled HCMV complementary target. (Right) Fluorescence lifetime
histograms and Gaussian fit, extracted from the four sub-arrays. Note that (d) used a biexponential model with τ1 = 4.2 ns & τ2 = 22.0 ns fixed.
Each individual FLIM map (320 pixels) presented in Fig. 3(a)–3(d) was obtained within
40 s. Assessment of different technologies is difficult as they often involve very different
principle of operation, detector size, resolution and illumination. Compared with our recently
published results, measured on similar samples and obtained with a state of the art TCSPC
quadrant anode detector [5], the SPAD imager acquisition time was circa 200 times faster.
With the quadrant anode detector it took up to 30 min to collect 500000 counts per spot of the
microarray (imaging 9 to 12 of them simultaneously), whereas the SPAD imager could
acquire 1.5M counts per spot in an exposure time of only 26 s.
Evidently, the SPAD array imager has a much lower spatial resolution than the device
used in ref [5]. or other state-of-the-art FLIM systems but it is sufficient for use with
microspot arrays. For the data presented in Fig. 3 there were about 20 pixels for each DNA
microspot, sufficient to provide FLIM maps resolving the shape of individual spot as well as
providing information on the variance of the lifetime estimate. For screening application this
spatial detail is often not required and the throughput could be dramatically increased by
mapping each microarray spot to fewer (ultimately individual) pixels.
A more severe limitation of the current prototype device is its low photon detection
efficiency. The active area of the SPAD (8 µm diameter) is very small compared to the pixel
pitch of 50 × 50 μm mostly taken up by its timing and counting circuits, leading to a fill factor
of only 2% [12]. The overall efficiency is therefore below 1% and the relatively high
excitation light levels needed to achieve high count rates lead to noticeable photo bleaching of
the Alexa labels.
To address this problem, mounting of micro-lenses onto the SPAD array is currently being
investigated. This is expected to enhance light collection by at least 10-fold [20], making the
overall efficiency directly comparable to PMT based devices. This will dramatically increase
the sensitivity, making it feasible to work with lower sample concentrations and/or lower laser
intensities. Alternatively this could further decrease the data acquisition time as the frame rate
could be increased to 50 kHz (compared to ~4 kHz used for data presented here) without
suffering from distortions through pile-up.
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Received 10 Aug 2010; revised 12 Sep 2010; accepted 30 Oct 2010; published 4 Nov 2010
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Additionally, acquisition speed could also be improved by streaming data directly to the
PC using USB with data compression techniques (latest firmware/software updates can
achieve continuous transfer of data to PC at about 20000 fps). However, the huge amount of
raw data generated by such detector arrays requires large bandwidth and storage capacities,
and for better scalability with detector array size on-board data processing will be essential.
By building arrival time histograms on the device the data transfer to the PC can be reduced
dramatically while still retaining all information typically used for lifetime analysis [21].
Besides, for ultimate speed, software based lifetime determination can be replaced by an onboard method. The potential of such an approach was recently demonstrated by successfully
monitoring the real-time mixing of dye solution in microfluidics using high-speed integration
for extraction method (IEM) algorithms, implemented on field programmable gate arrays
(FPGAs) [15]. Algorithms more suitable for dim samples, such as the centre-of-mass method
[22], have since then been suggested. As the device can easily switch between full TCSPC
data acquisition and lifetime estimation mode, verification/calibration can easily be achieved
without changes to the experimental geometry. Finally, the production of a much larger SPAD
plus TDC array will allow 256 microarray spots to be measured in a single detection event,
opening true opportunities for high throughput FLIM detection.
4. Conclusion
In summary, we have demonstrated rapid TCSPC data acquisition for fluorescence lifetime
imaging and multiplexing of a DNA microarray using a CMOS-SPAD array camera.
Although results presented here were obtained using a complex excitation laser system, the
required power levels (~2 mW) are easily accessible for compact picosecond pulsed diode
lasers. The data presented has been obtained from fixed samples but the current system should
be fast enough to monitor hybridization dynamics of DNA microspot arrays which constitute
the focus of our future work. Ongoing upgrading of the SPAD imager will enable faster and
more sensitive detection on a larger sensor area. Such improvements will permit highthroughput FLIM of DNA microarrays and open the possibility to integrate FLIM
transduction on biosensing and point of care testing devices.
Acknowledgments
This work was supported by the European Community within the Sixth Framework
Programme of the Information Science Technologies, Future and Emerging Technologies
Open MEGAFRAME project (contract 029217-2, http://www.megaframe.eu). The memory
controller module was designed and implemented by Ed Duncan and Andrew Kinsey from
Pixic Ltd. (http://www.pixic.co.uk/). Justin Richardson‟s present address is Selex Galileo, A
Finmeccanica Company, 2 Crewe Road North, Edinburgh,EH5 2XS, UK.
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(C) 2010 OSA
Received 10 Aug 2010; revised 12 Sep 2010; accepted 30 Oct 2010; published 4 Nov 2010
1 December 2010 / Vol. 1, No. 5 / BIOMEDICAL OPTICS EXPRESS 1308