Anal. Chem. 1996, 68, 4512-4514
Lifetime Imaging of Luminescent Oxygen Sensors Based on All-Solid-State Technology Paul Hartmann* and Werner Ziegler
Biomedical Research and Development, AVL List GmbH, Kleiststrasse 48, A-8020 Graz, Austria
Conventional lifetime imaging requires rather complex and expensive instrumentation, like lasers and intensified CCD-cameras. We demonstrate that an array of lightemitting diodes and a directly gatable CCD-camera can be alternatively used for special applications as, for example, imaging of oxygen concentrations on a surface covered by an optical sensor. The suitability of low-cost devices is in this case favored by the microsecond decay times and the spectral properties of highly luminescent ruthenium(II) complexes. For an illuminated sensor area of 5 cm2 and an aimed spatial resolution of 1 mm, oxygen resolution was better than ∆PO2 ) 0.4 Torr in the absence of oxygen and ∆PO2 ) 2.5 Torr at PO2 ) 100 Torr. Various branches of luminescence spectroscopy have great potential for technical and medical applications.1 The recently developed techniques of phosphorescence2,3 and fluorescence “lifetime” imaging (FLIM)4,5 combine the benefits of luminescence decay time measurements with simultaneous access to spatial dimensions. Many application examples have been described,6-9 but the required instrumentation is complex and expensive: Conventional FLIM, for example, requires laser sources, external light modulators and high-voltage driven image intensifiers to provide the necessary gain and the modulation frequencies for the nanosecond time scale of the decay of fluorescent molecules. Phosphorescence lifetime imaging requires fast flashlamps, while modulation with the less expensive chopper wheels is limited to dyes with decay times of τ ) 10 µs or more. Such dyes suffer from low quantum yields, while poor photostability is not a major problem for decay time-based measurements. In the decay time range between fluorescence and phosphorescence (τ ) 100 ns to several microseconds), Ru(II) complexes have been widely investigated.10 An important application of these dyes is oxygen sensing, which takes advantage of efficient emis(1) Fiber optic chemical sensors and biosensors; Wolfbeis, O. S., Ed.; CRC Press: Boca Raton, FL, 1991; Vols. 1 and 2. (2) Wilson, D. F.; Rumsey, W. L.; Vanderkooi, J. N. Adv. Exp. Med. Biol. 1989, 248, 109-115. (3) Marriott, G.; Clegg, R. M.; Arndt-Jovin, D. J.; Jovin, T. M. Biophys. J. 1991, 60, 1374-1387. (4) Morgan, C. G.; Mitchell, A. C.; Murray, J. G. Proc. SPIE-Int. Soc. Opt. Eng. 1990, 1204, 798-807. (5) Lakowicz, J. R.; Berndt, K. W. Rev. Sci. Instrum. 1991, 62, 1727-1734. (6) Lakowicz, J. R.; Szmacinski, H.; Nowaczyk, K; Berndt, K. W.; Johnson, M. L. Anal. Biochem. 1992, 202, 316-330. (7) Clegg, R. M.; Feddersen, B.; Gratton, E.; Jovin, T. M. Proc. SPIE-Int. Soc. Opt. Eng. 1992, 1640, 448-460. (8) Clegg, R. M.; Gadella, T. W. J.; Jovin, T. M. Proc. SPIE-Int. Soc. Opt. Eng. 1994, 2137, 105-118. (9) Szmacinski, H.; Lakowicz, J. R.; Johnson, M. L. Methods Enzymol. 1994, 240, 723-748. (10) Demas, J. N.; DeGraff, B. A. Anal.Chem. 1991, 63, 829A-837A.
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sion quenching by oxygen. The absorption spectra are suitable for the superbright blue light-emitting diodes (LEDs),11 while the emission spectra meet typical spectral response curves of CCDs. Especially, when Ru(II) complexes are immobilized in a polymer matrix, their quantum yields are high.12 The optimum frequencies for phase angle-resolved measurements are in the range between f ) 30 kHz and f ) ∼500 kHz, which is too high for low-cost modulation devices such as chopper wheels but accessible to the direct modulation capabilities of CCD-cameras.13 In the following we describe a setup based on all-solid-state technology for lifetime imaging of Ru(II) complexes and its application to oxygen sensing. EXPERIMENTAL SECTION Oxygen Sensor. Tris(1,10′-phenanthroline)ruthenium(II) chloride (Aldrich, Steinheim, Germany) was adsorbed to silica gel particles (Whatman, Clifton, NJ), which were immersed in fillerfree silicone (ABCR, Darmstadt, Germany).14 The sensing layer was attached to a transparent polyester sheet (DuPont, Bad Homburg, Germany) and covered by an optical isolation layer of black silicone (Wacker, Burghausen, Germany). Instrumentation. Eight serially connected LEDs (Nichia, Nu¨rnberg, Germany) were driven by a square-wave function of a synthesized function generator (SRS DS345, Sunnyvale, CA). The light output was filtered and homogeneously directed to the sensor membrane. Homogeneity of the illumination is not required for lifetime-based sensing but is recommended to achieve a uniform signal-to-noise ratio (S/N). The luminescence response was filtered and detected by a directly gatable prototype CCD-camera (Variomod, PCO, Kelheim, Germany): While acquisition and data readout cycles of the camera alternate at the video frequency (25 Hz), a high-frequency square-wave modulation (frequencies up to f ) 500 kHz) can be superimposed electronically for gating. During the 20 ms acquisition period, the modulation provides a periodical response of the CCD, which is suppressed during the 20 ms data readout period. The CCD was gated by a square wave of the same shape and frequency as the modulation of the excitation source (homodyne principle). Data were transferred via a frame grabber (Bitflow Raptor, Stemmer, Munich, Germany) to a PC (Figure 1). To improve the S/N of the images, several frames were averaged. (11) O’Keeffe, G.; MacCraith, B. D.; McEvoy, A. K.; McDonagh, C. M.; McGilp, J. F. Sens. Actuators B 1995, 29, 226-230. (12) Hartmann, P.; Leiner, M. J. P.; Lippitsch M. E. Anal. Chem. 1995, 67, 88-93. (13) Reich, R. K.; Mountain, R. W.; McGonagle, W. H.; Huang, C. M.; Twichell, J. C.; Kosicki, B. B.; Savoye, E. D. Proc. IEEE 1991, 91, 171. (14) Wolfbeis, O. S.; Leiner, M. J. P.; Posch, H. E. Mikrochim. Acta 1986, 3, 359-366. S0003-2700(96)00540-9 CCC: $12.00
© 1996 American Chemical Society
Figure 1. Schematic representation of the instrumental setup.
Figure 3. Lifetime image (ratio k) of an oxygen sensor attached to a surface supplying gases of various oxygen content (0, 28, 100, and 14% O2).
Figure 2. Luminescence response to a square wave excitation.
We constructed a device that is able to provide spatially separated regions of defined oxygen concentrations in a sensor membrane of an area of several square centimeters. A planar aluminum block with a number of separated gas outlets was flooded with gases of different compositions. The sensor was attached to the surface of the block via a network of vacuum channels, which further prevented gas cross-talk between adjacent sensor regions. Measurement Technique. The applied principle of decay time determination15 is given in Figure 2: The LEDs are periodically switched on and off. After switching on, the sensor luminescence starts to rise immediately (the molecular rise time of the luminescence is in the picosecond range), but the concurrent luminescence decay results in a delay of the sensor response (see rise period in Figure 2). Thus, an idealized square-wave excitation gives rise to a near-exponential increase of the sensor luminescence while the LEDs are on. Switching off results in a near-exponential decay. The shape of this shark fin function can be described with the help of a Fourier series of sine functions phase-shifted with respect to the excitation (in practice, rise and fall times of the LEDs of ∼100 ns lead to deviations from this idealized behavior; however, blue LEDs with significantly reduced rise and fall times are already available on the market). For a single acquisition period (20 ms) the adjustable phase of the square wave modulating the CCD response was set to acquire incoming photons while the LEDs were switched either on or off. For the first image, the open period of the CCD-camera modulation was set in phase with the light source, integrating the luminescence in the phase interval Φ ) 0 - π (rise period; see Figure 2). For the second image, the phase of the gate was shifted to integrate the luminescence between Φ ) π and 2π (decay period). The obtained images also contain contributions (15) Khalil, G.; Gouterman, M. P.; Green, E. U.S. Patent 5,043,286, 1991.
from the dark current of the CCD. Therefore it was necessary to evaluate also the gray values Id(r) of the pixels at position r of the dark background image and to subtract it from each of the luminescence images. In the next step, for each particular pixel the image ratio k(r) was formed (according to eq 1). Since the
k(r,O2) )
I2(r,O2) - Id(r) I1(r,O2) - Id(r)
(1)
shape of the shark fin luminescence and, thus, its area depend on the apparent decay time of the sensor, which in turn is a function of oxygen concentration, the ratio of the gray values I1(r,O2) and I2(r,O2) of the pixels of the luminescence images is finally related to the oxygen content of the sensing layer and further independent of the absolute luminescence intensity of the sensor. RESULTS AND DISCUSSION Figure 3 shows an image of an oxygen sensor covering areas with different oxygen concentrations. The bright grid of the vacuum channels is also apparent. The gray values represent the ratio k computed for each pixel according to eq 1 by simple image analysis (subtraction and division of images). No complex computations are required. The shorter the decay time (e.g., for increasing oxygen concentration) the lower is I2 compared to I1 (while oxygen quenching of the quantum efficiency results in lower absolute signals for both periods). Thus, the lower the ratio k the higher is the oxygen concentration in the sensing layer. Luminescence intensity and decay times are related to the quencher concentration by Stern-Volmer-type equations. From measurements in the frequency domain an apparent lifetime can be determined, which depends on the frequency of modulation. Thus, the quenching behavior also becomes frequency-dependent. This is also true for the ratio k of the applied principle of measurement, but in-factory calibration can be performed for a given frequency. A calibration graph obtained with the described Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
4513
Figure 4. Ratio k (eq 1) vs applied PO2: detection by directly modulated CCD-camera (squares); detection by gated intensified CCD-camera (stars).
setup is shown in Figure 4. The frequency used (f ) 140 kHz) was optimized for decay times of τ ≈ 1 µs. The oxygen resolution can be calculated from the calibration graph. It was better than ∆PO2 ) 0.4 Torr in the absence of oxygen and ∆PO2 ) 2.5 Torr at PO2 ) 100 Torr for an illuminated sensor area of 5 cm2 and an aimed spatial resolution of 1 mm at the sensor surface. The calibration curve obtained with the help of an intensified CCD camera (IRO, PCO),16 which is commonly used for FLIM, is shown in Figure 4 for comparison. In this case, the modulation of the camera response was achieved by applying a square-wave gating to the photocathode of the image intensifier. It is apparent that both detection systems give similar results. However, the S/N of the IRO measurement was superior due to the high gains achievable with the built-in microchannel plate. It should be (16) Hartmann, P.; Ziegler, W.; Holst, G.; Lu ¨ bbers, D. W. Sens. Actuators B, in press. (17) Wilson, D. F.; Cerniglia, G. J. Cancer Res. 1992, 52, 3988-3993. (18) Demas, J. N.; DeGraff, B. A. Proc. SPIE-Int. Soc. Opt. Eng. 1992, 1796, 71-75.
4514 Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
possible to increase the performance of the directly modulated camera by expanding the exposure time. The results demonstrate the capabilities of an analytical technique of affordable cost due to recent developments in LEDs, oxygen sensors, and CCD-camera technology. Luminescence lifetime imaging of oxygen is of great interest, for example, for medical applications. Since invasive techniques of oxygen monitoring based on time-resolved phosphorescence measurements17 are not applicable in human medicine, noninvasive oxygen sensors of the type used in the present study are expected to have a great potential for imaging in the fields of microcirculation and cutaneous respiration, which are critical mechanisms in a great variety of diseases. Besides the oxygen-sensing applications demonstrated in this work, luminescent lifetime imaging based on allsolid-state technology may prove practical for several other application fields. Luminescent temperature sensors based on Ru(II) complexes18 have been discussed recently. With the help of the described lifetime imaging setup, temperature imaging should be straightforward. Since both probe chemistry and LED development proceed quickly, favorable dyes and LEDs will probably soon lead to other promising imaging applications in the analytical sciences. In the red and near-IR spectral range, laser diodes can also be considered as excitation source to yield a better S/N, which is always a problem for lifetime imaging. ACKNOWLEDGMENT The authors thank PCO Co., Kelheim, for supplying the prototype CCD-camera, and Gerhard Holst, Bremen, for valuable discussions. Received for review June 3, 1996. Accepted September 19, 1996.X AC9605408 X
Abstract published in Advance ACS Abstracts, November 1, 1996.
