AC Research
Accelerated Articles Anal. Chem. 1996, 68, 1073-1080
Chemiluminescent Low-Light Imaging of Biospecific Reactions on Macro- and Microsamples Using a Videocamera-Based Luminograph Aldo Roda* and Patrizia Pasini
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy Monica Musiani
Institute of Microbiology, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy Stefano Girotti and Mario Baraldini
Institute of Chemical Sciences, University of Bologna, Via San Donato 15, 40127 Bologna, Italy Giacomo Carrea
Institute of Hormones Chemistry, C.N.R., Via Bianco 9, 20131 Milano, Italy Anna Suozzi
Institute of Oncology “F. Addarii”, Viale Ercolani 4, 40138 Bologna, Italy
The analytical performance of a low-light imaging luminograph for quantitative luminescence analysis was evaluated in terms of sensitivity, spatial resolution, accuracy, precision, and sample geometry, at the macrolevel and in combination with optical microscopy. The system allows for the detection of 400 amol of enzymes such as alkaline phosphatase and horseradish peroxidase using 1,2-dioxetanes and luminol/p-iodophenol or acridancarboxylate esters, respectively, as chemiluminescent substrates. Enzymatic activity and spatial distribution of nylon net immobilized-alkaline phosphatase was studied; the system permits the quantification of the immobilized enzyme with a spatial resolution as low as 1 µm. Other applications, such as the alkaline phosphatase localization in 8 µm intestinal mucosa cryosections, quantitative immunocytochemistry, and dot blot DNA hybridization reactions, were studied and optimized. The system was also employed for in situ hybridization assay of cytomegalovirus DNA in infected human fibroblasts. The presence of a viral genome was revealed with digoxigeninlabeled probes and alkaline phosphatase-labeled antidigoxigenin antibody, using chemiluminescent substrate 0003-2700/96/0368-1073$12.00/0
© 1996 American Chemical Society
for this enzyme. The luminescent signal was intense and stable, and the probe was imaged and quantified within single cells with higher intensity in the nuclei, with a spatial resolution as low as 1 µm and very low background. The results show that this technique is an ultrasensitive and potent analytical tool to localize and quantify biomolecules at microscopic level, and it is suitable for many bioanalytical applications.
Chemiluminescence is the light emission produced by a chemical reaction in which chemically excited molecules decay to the ground state. This phenomenon is utilized in various analytical techniques in which small amounts of analytes or enzymes can be detected and quantified by measurement of the light emitted by bio- or chemiluminescent reactions.1,2 Chemiluminescent substrates for alkaline phosphatase (ALP), such as 1,2(1) DeLuca, M. A. In Methods in Enzymology; Academic Press: New York, 1978; Vol. 57. (2) Kricka, L. J.; Stanley, P. E.; Thorpe, G. H. G.; Whitehead, T. P. Analytical Applications of Bioluminescence and Chemiluminescence; Academic Press: London and New York, 1984.
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dioxetane phosphate,3,4 for horseradish peroxidase (HRP), such as luminol/p-iodophenol/H2O2 (ECL)5,6 and for acridancarboxylate ester derivatives,7 which allows for the detection of enzymes with very high efficiency, have recently become available. Ultrasensitive assays using these enzymes as labels in immunoassays8,9 and blotting10,11 and gene probe assays12,13 have been developed: the kinetics of the light emission is a steady-state glow type, which simplifies sample handling, and since the signal intensity is proportional to enzyme activity or concentration, precise and accurate quantitative analysis can be achieved. Coupled enzymatic bio- and chemiluminescent analytical methods have also been developed: in particular, ATP-involving reactions (kinases) have been coupled with the firefly luciferin-luciferase system and NAD(P)H-producing or -consuming enzymatic reactions (dehydrogenases) with bacterial luciferases, and the luminol/H2O2/horseradish peroxidase system has been coupled with oxidase enzymes.14-18 Continuing improvements in chemiluminescent substrates have been paralleled by new developments in photon-imaging instrumentation such as ultrasensitive luminographs, based on an intensified Vidicon or CCD videocamera. These instruments not only allow quantification at the level of a single photon but also permit localization of the chemiluminescent emissions by displaying them with adequate spatial resolution on a target surface (gel, membrane, tissue). These emissions can derive from biospecific reactions or nearly all analytical situations to which light-emitting processes can be linked.19-25 By connecting the luminograph to (3) Bronstein, I.; Edwards, B.; Voyta, J. C. J. Biolumin. Chemilumin. 1989, 4, 99-111. (4) Beck, S.; Ko ¨ster, H. Anal. Chem. 1990, 62, 2258-2270. (5) Thorpe, G. H. G.; Kricka, L. J. Methods Enzymol. 1986, 133, 311-354. (6) Thorpe, G. H. G.; Kricka, L. J. In Bioluminescence and Chemiluminescence; Scholmerich, J., Andreesen, R., Kapp, A., Ernst, M., Woods, W. G., Eds.; Wiley: Chichester, England, 1987; pp 199-208. (7) Akhavan-Tafti, H.; DeSilva, R.; Arghavani, Z.; Eickholt, R. A.; Handley, R. S.; Schaap, A. P. In Bioluminescence and Chemiluminescence; Campbell, A. K., Kricka, L. J., Stanley, P. E., Eds.; Wiley: Chichester, England, 1994; pp 199-202. (8) Kricka, L. J.; Scott, R. A. W.; Thorpe, G. H. G. In Complementary Immunoassays; Collins, W. P., Ed.; Wiley: Chichester, England, 1988; pp 169-179. (9) Ashihara, Y.; Saruta, H.; Ando, S.; Kikuchi, Y.; Kasahara, Y. In Bioluminescence and Chemiluminescence; Campbell, A. K., Kricka, L. J., Stanley, P. E., Eds.; Wiley: Chichester, England, 1994; pp 321-324. (10) Schneppenheim, R.; Rautenberg, P. Eur. J. Clin. Microbiol. 1987, 6, 4951. (11) Bronstein, I.; Voyta, J. C.; Lazzari, K. G.; Murphy, O.; Edwards, B.; Kricka, L. J. BioTechniques 1990, 8, 310-314. (12) Matthews, J. A.; Batki, A.; Hynds, C.; Kricka, L. J. Anal. Biochem. 1985, 151, 205-209. (13) Bronstein, I.; Voyta, J. C.; Edwards, B. Anal. Biochem. 1989, 180, 95-98. (14) Roda, A.; Girotti, S.; Ghini, S.; Carrea, G. J. Biolumin. Chemilumin. 1989, 4, 423-435. (15) Carrea, G.; Bovara, R.; Mazzola, G.; Girotti, S.; Roda, A.; Ghini, S. Anal. Chem. 1986, 58, 331-333. (16) DeLuca, M.; McElroy, W. D. Methods Enzymol. 1986, 133, 331-584. (17) Vellom, D. C.; Kricka, L. J. Methods Enzymol. 1987, 133, 229-237. (18) Worsfold, P. J.; Nabi, A. Anal. Chim. Acta 1986, 179, 307-313. (19) Bra¨uer, R.; Lu ¨ bbe, B.; Ochs, R.; Helma, H.; Hoffmann, J. In Bioluminescence and Chemiluminescence; Szalay, A. A., Kricka, L. J., Stanley, P., Eds.; Wiley: Chichester, England, 1993; pp 13-17. (20) Hooper, C. E.; Ansorge, R. E. TrAC, Trends Anal. Chem. 1990, 9, 269277. (21) Hooper, C. E.; Ansorge, R. E.; Browne, H. M.; Tomkins, P. J. Biolumin. Chemilumin. 1990, 5, 123-130. (22) Wick, R. A. BioTechniques 1989, 7, 262-268. (23) Hooper, C. E.; Ansorge, R. E. In Bioluminescence and Chemiluminescence; Stanley, P. E., Kricka, L. J., Eds.; Wiley: Chichester, England, 1991; pp 337-344. (24) Scott, R. Q.; Inaba, H. J. Biolumin. Chemilumin. 1989, 4, 507-511.
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an optical microscope, it is possible to perform quantitative immunochemistry or in situ hybridization assays or to localize molecules such as enzymes, antibodies, receptors, or small organic compounds immobilized on solid supports.26-30 Luminescent detection techniques are more sensitive than colorimetric methods, and more importantly, they allow in situ quantification of the substance undergoing analysis. Only a few papers dealing with low-light quantitative luminescent imaging have so far been published, hence information about this potent analytical tool is scarce.28 The aim of this work was to evaluate the analytical performance of a Saticon-based low-light imaging apparatus (luminograph LB 980, EG&G Berthold) in terms of sensitivity, resolution, accuracy, and precision. The application of this system to the quantitative detection of ALP and HRP utilizing different chemiluminescent substrates was studied in terms of background, sample size, and geometry. The luminograph was also connected to an optical microscope to detect and quantify ALP in solution, chemically immobilized on nylon net, in organized biological structures (cryosections of intestinal mucosa), and as a label for immunological detection of digoxigenin-labeled probes for cytomegalovirus DNA in dot blot and in situ hybridization reactions. Methodological problems regarding quantitative light measurement and spatial resolution of the related images are delineated for the above-mentioned analytical model systems. EXPERIMENTAL SECTION Chemicals. Alkaline phosphatase (type VII-T, from calf intestine, 2000 IU/mg) and peroxidase (type VI-A, from horseradish, 1100 IU/mg) were purchased from Sigma (St. Louis, MO). The chemiluminescent substrates used for ALP detection were Lumi-Phos Plus (Lumigen, Inc., Southfield, MI) and CSPD (Tropix, Inc., Bedford, MA). The primary chemiluminescent compound in both preparations is adamantyl-1,2-dioxetane phosphate. For HRP detection we used the enhanced chemiluminescent luminol reagent ECL (Amersham, Amersham, UK) and Lumigen PS-3 (acridancarboxylate ester/H2O2) (Lumigen, Inc.) following the manufacturer’s instructions. Polyclonal anti-digoxigenin Fab fragments conjugated to alkaline phosphatase were purchased from Boehringer Mannheim (Mannheim, Germany). Nylon net (100 ( 3 threads/cm, thread diameter 20 µm) was supplied by Assoprint s.r.l. (Modena, Italy). All other chemicals were of high-quality analytical grade. Imaging System. Instrumentation. The luminograph used (LB 980, EG&G Berthold, Bad Wildbad, Germany) is a highperformance low-light imaging system able to detect any type of luminescent emission (400-700 nm) over a wide range of intensities (sensitivity range 50 plx-10 lx at 490 nm). The video system consists of a high dynamic range videocamera (1 in., Saticon) which is a Vidicon-type tube with Se-As-Tl light target photoconductor (Siemens, Karlsruhe, Germany) linked to an image intensifier by high-transmission lenses. An objective focuses the luminescent signal on a photocathode in the image intensifier, which converts photons to electrons, which are then (25) Leaback, D. H.; Haggart, R. J. Biolumin. Chemilumin. 1989, 4, 512-522. (26) Hawkins, E.; Cumming, R. J. Histochem. Cytochem. 1990, 38, 415-419. (27) Lorimier, P.; Lamarcq, L.; Labat-Moleur, F.; Guillermet, C.; Bethier, R.; Stoebner, P. J. Histochem. Cytochem. 1993, 41, 1591-1597. (28) Mueller-Klieser, W.; Walenta, S. Histochem. J. 1993, 25, 407-420. (29) Hiraoka, Y.; Sedat, J. W.; Agard, D. A. Science 1987, 238, 36-41. (30) Mueller-Klieser, W.; Walenta, S.; Paschen, W.; Kallinowski, F.; Vaupel, P. J. Natl. Cancer Inst. 1988, 80, 842-848.
amplified. Another lens projects the image to the Saticon tube, and the final image is then processed. The samples are placed in a light-tight box to prevent interference by external light. The system was also connected to an optical microscope (Model BH-2, Olympus Optical, Tokyo, Japan) using a C-mount adapter. The whole microscope is enclosed in a homemade dark box. The system is controlled by a PC provided with software for quantitative image analysis. Spatial Resolution. The luminograph LB 980 has a 500 × 360 pixel detection screen. The resolving power is given by the ability to discriminate between light deriving from two adjacent pixels, and it is strictly related to the pixel size. Pixel size was determined by placing a graduated millimetric scale under the macroobjective and measuring the area (µm2) corresponding to 1 pixel. Pixel demagnification was achieved by connecting relay optics to the objective, which improved the resolution obtained with the macroscale (mm size). When the system was connected to an optical microscope, pixel size was determined with a 1/100 mm stage micrometer (µm scale), under objectives of different magnifying power (×20, ×40, ×100, Olympus Optical, Tokyo, Japan). The cross-talk of the light signal between adjacent pixels was evaluated using a calibrated nylon net (100 ( 3 threads/cm, thread diameter 20 µm) on which alkaline phosphatase was immobilized and revealed with a chemiluminescent substrate (see below). Light Measurement. The light emitted from the sample is accumulated for a given period of time and integrated on the camera tube’s target at fixed time intervals. For quantitative analysis, the system is able to perform various instrumental adjustments, including background, γ, geometric, and flat-field corrections. In particular, the camera is set to a nonlinear transfer function (γ characteristic) which amplifies the dark parts of the image more than the bright ones, thus allowing higher dynamic in the image data; this function needs to be numerically corrected by γ correction to perform quantitative analysis. Measurements can be limited to specific areas of the field under study; the results can be achieved as integrated brightness of area or as either maximum or average brightness per pixel; they can be expressed in photons per second, watts, relative light units, or percent. The image can be processed with different functions, including two-dimensional filtering, or with a nonlinear gray scale to modify the contrast. A pseudocolor function converts the different gray shades to colors. The quantification can be expressed as the measurement of light from a given area or as either a linear or 3D plot of light intensity. Evaluation of Analytical Performance. Sample Geometry and Size. Fixed volumes (50 µL) of steady-state chemiluminescent solutions (ECL system) were analyzed inside white and black polystyrene microtiter wells (Dynatech Laboratories, Inc., Chantilly, VA), as a drop forming a convex surface on a flat unwettable support and as a thin film obtained by sandwiching the solution between two conventional glass microscope cover slips. The light emission from these samples was measured simultaneously in order to assess the influence of sample geometry on photon quantification. The effect of sample size was evaluated by determining emission values from increasing volumes (25, 50, 100, 200 µL) of the same steady-state chemiluminescent mixture dispensed in microtiter wells and calculating the light emission values (expressed in photons/s per well) for given volume units. The experiment was performed in three experimental sets with
chemiluminescent solutions emitting 102, 104, or 106 photons/s per well. Precision and Accuracy. The precision was evaluated by measuring steady-state chemiluminescent samples (ECL system) at three different light levels (102, 104, 106 photons/per well) in 12 subsequent acquisitions. Mean values and standard deviations were then calculated. In order to evaluate the system’s accuracy, light emission from same samples (n ) 50) was also measured with a photomultiplier tube-based microplate reader (Amerlite Analyser, Kodak Clinical Diagnostic Ltd., Amersham, UK). The data were correlated using a linear least-squares regression reporting the correlation coefficient, the slope of linear curve, and the y-intercept with the corresponding standard deviations. The graph shows also the confidence hyperbola for the linear curve and the data. Calibration Graph. The detection limit, i.e., the minimum quantity of light detectable with a signal to noise ratio of 3:1, and the sensitivity, i.e., the slope of the curve given by graphing light emission against concentration, were determined using the ALP/ 1,2-dioxetanes, HRP/ECL, and HRP/Lumigen PS-3 systems on serial dilutions of enzymes. For comparative evaluation, the same solutions were also analyzed with the instrument coupled with an optical microscope and the light emission values were expressed in photons per second per pixel. With the macrosetup, the light-emitting areas correspond to the wells and cover many pixels. The background was calculated on microtiter wells without the trigger chemiluminescent enzyme, thus a signal/noise of g3 is considered as the detection limit. With the microscope setup, the image covers most of the field of view including few pixels and the light signal derives from unknown portions in which the background may have different values. A fluctuation of five standard deviations of its mean value in a single pixel, calculated in several fixed negative areas, must be the threshold for a light signal to be considered significant.31 Analytical Applications. Analysis of Macrosamples. Luminescent Assay of Enzymatic Activity in Solution. Different concentrations ranging from 0.25 to 10 ng/mL ALP were made in a buffer solution containing 0.1 mol/L diethanolamine, 1 mmol/L MgCl2, and 0.02% sodium azide, pH 10. Similar solutions were made for HRP using 0.1 mol/L Tris-HCl buffer, pH 8.6. All the measurements were performed by adding 80 µL of these solutions to 100 µL of chemiluminescent substrate at 25 ( 1 °C using thermostated microtiter plates. Light emission was measured for time periods of different lengths. Luminescent Dot Blot Hybridization Reaction. A hybridization reaction was performed as previously described.32 Briefly, different amounts (ranging from 1 to 1000 fg) of cytomegalovirus (CMV) DNA from the Towne strain of human CMV were filtered with a Bio-Dot apparatus (Bio-Rad Laboratories, Richmond, CA) onto nylon membranes (Amersham) equilibrated in distilled water. After hybridization with digoxigenin-labeled CMV DNA probes, the target hybrid was revealed immunoenzymatically employing ALP-labeled anti-digoxigenin Fab fragments. The membranes were immersed in Lumi-Phos Plus or CSPD solutions and incubated for 45 min, after which the light output was measured. (31) Hooper, C. E., Ansorge, R. E., Rushbrooke, J. G. J. Biolumin. Chemilumin. 1994, 9, 113-122. (32) Musiani, M.; Zerbini, M.; Gibellini, D.; Gentilomi, G.; Girotti, S.; Ferri, E. Anal. Biochem. 1991, 194, 394-398. (33) Hornby, W. E.; Goldstein, L. Methods Enzymol. 1976, 44, 118-134.
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Analysis of Microsamples. Luminescent Assay of Enzymatic Activity in Solution. The ALP and HRP activities were measured in standard solutions by placing 10 µL of the chemiluminescent mixtures on a microscope slide and distributing them as thin films. The results were expressed as photons per second per pixel. ALP Immobilized on Nylon Net. ALP was chemically immobilized on nylon net using a slightly modified method described by Hornby and Goldstein.33 The nylon nets (4 × 4 cm) were first activated with triethyloxonium tetrafluoroborate and, after treatment with 0.86 mol/L 1,6-diaminohexane and 0.5 mol/L glutaraldehyde, were allowed to react at room temperature for 3 h with the enzyme solution (10.000 IU/mL of 0.1 mol/L potassium phosphate buffer, pH 7). Next, the nets were thoroughly washed with 0.1 mol/L potassium phosphate buffer, pH 7, containing 1 mmol/L DTT and 0.1% BSA, to remove any protein not covalently linked. For stability studies, nets were stored in buffer or water at either 4 or 25 °C. The ALP activity and its spatial distribution were evaluated by placing a small portion (5 × 5 mm) of the net on a glass microscope slide and adding 20 µL of the chemiluminescent substrate. The light output was then recorded using the optical microscope-videocamera setup. ALP Activity in Tissues. Cryosections of rabbit intestine, 8 µm thick, were obtained and observed at ×20, ×40, and ×100 magnification. The chemiluminescent detection was performed by adding few drops of substrate solution for ALP to the glass slides; after 15-30 min of incubation the solution was removed and the light image recorded. Immunocytochemistry. Immunocytochemistry was performed for the search of specific human IgM to Epstein Bar virus-induced virus capsid antigens (VCA). P3-HR1 cells, which express VCA in from 10 to 15% of the cells, were dropped onto glass slides and air-dried. Cells were fixed in acetone and treated with serial dilutions of sera from patients with infectious mononucleosis. Then peroxidase-conjugated anti-IgM antibody was added and detected with ECL chemiluminescent substrate. In Situ Hybridization Assay. In situ hybridization was performed as previously described.34 Briefly, CMV-infected human fibroblasts were fixed in 4% paraformaldehyde, treated with pronase, and dehydrated by ethanol washes. Dehydrated cells were overlaid with 10 µL of the hybridization mixture (50% deionized formamide, 10% dextran sulfate, 250 µg/mL calf thymus DNA, and 2 µg/mL digoxigenin-labeled probe DNA in 2× SSC buffer). Cell samples and the hybridization mixture containing the digoxigenin-labeled probe were denaturated together by heating in an 85 °C water bath for 5 min and were then put to hybridize at 37 °C for 3 h. After hybridization, cells were washed three times under stringent conditions. Hybridized probes were detected using polyclonal anti-digoxigenin antibody conjugated to ALP and chemiluminescent substrates for ALP. RESULTS AND DISCUSSION Analytical Performance. The pixel sizes obtained using the macroconfiguration with relay optics and with the videocamera connected to the optical microscope are reported in Table 1. Using the manufacturer’s configuration, the pixel size is 240 µm, which is reduced to 160 and 125 µm with relay optics. When the video system is connected to the microscope, the pixel size is reduced to 2, 1.1, and 0.4 µm using ×20, ×40, and ×100 (34) Gentilomi, G.; Musiani, M.; Zerbini, M.; Gallinella, G.; Gibellini, D.; La Placa, M. J. Immunol. Methods 1989, 125, 177-183.
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Table 1. Optical Characteristics of the Luminograph Used in the Standard Setup, with Relay Optics and When Connected to an Optical Microscope magnification factor 1.6 2.6 3.4 185 (×20)a 360 (×40)a 800 (×100)a a
pixel size (µm) Macro Setup 240 160 125 Microscope 2 1.1 0.41
max field of view (mm2) 120 × 80 75 × 50 57 × 38 1 × 0.67 0.51 × 0.34 0.21 × 0.13
Objective magnification.
magnification objectives respectively. Under these conditions, the highest resolving power of the chemiluminescent image (0.4 µm) is comparable to that obtained with the optical microscope, on the real image, which is 0.25 µm. With this resolution, it is potentially possible to localize a chemiluminescent signal in a tissue section or within a single cell and it is particularly suited for analysis at subcellular level. When a videocamera system is used to measure light, the sample geometry is critical because the light scattering and internal reflectance can modify the amount of photons reaching the objective. When 50 µL of a steady-state chemiluminescent solution was analyzed as a drop placed on a black surface, it gave much less light than the same solution contained in a black microtiter well with a flat surface. This was due to total internal reflection and interference of light, as observed by the formation of interference fringes. When the same 50 µL volume was analyzed as a thin film between two microscope cover slips, the amount of the measured light was even less. Moreover, when a white polystyrene microtiter well, instead of a black one, was used, much more light was measured as a result of reflection and diffusion of the light toward the objective direction. The edge effect observed in both drops or thin films is considerable and due to total internal reflection phenomena. The edge effect was also observed when a drop of the chemiluminescent solution was placed on a reflecting aluminum foil. These results show that the sample geometry is critical when a quantitative analysis and comparative studies are performed, and the reference standards must be analyzed under the same experimental conditions. The effect of the sample volume on light emission is negligible, and similar results were obtained when the sample volume was increased from 25 to 200 µL maintaining the same area (well), i.e., the same number of pixels. The light emission from the different chemiluminescent solutions, once expressed per unit of volume, is similar, suggesting that internal quenching- or lightscattering phenomena are not relevant, at least within the selected range of volumes and with a colorless solution. With a standardized sample geometry, the instrument presents good precision as showed by the data obtained when replicates (n ) 12) of three luminescent solutions were analyzed. The coefficients of variation were always below 4% in samples with medium [(1.19 ( 0.04) × 104 photons/s per well] and high [(2.31 ( 0.07) × 106 photons/s per well] intensities and slightly higher (5-6%) in low-light [(1.92 ( 0.1) × 102 photons/s per well] emitting samples. This agrees with the rule that the precision increases with the light intensity at a given integration time.35 The (35) Bernroider, G. J. Biolumin. Chemilumin. 1994, 9, 127-133.
Figure 1. Correlation between chemiluminescent signals detected using the luminograph LB 980 and a PMT luminometer. Results are expressed as percentage of maximum light intensity. The equation shows the standard deviation of the slope and the y-intercept.
light emission values obtained with this instrument were in good agreement with those obtained with a conventional photomultiplier tube-based luminometer, as documented by the high regression coefficient (r ) 0.999) and the low standard deviations of both the slope and y-intercept, suggesting good accuracy of the instrument (Figure 1). The light was measured as photons per second per well with 2 s integration time for a 1 min accumulation period, and with this acquisition mode the accumulation time is critical, being the instrumental background noise inversely related to the square root of accumulation time. Consistently with the steady-state light emission of the chemiluminescent systems used, 1 min accumulation time, with 2 s integration time, is sufficient to reach acceptable data with a light emission greater than 102 photons/s per well. Chemiluminescent Assay of Enzyme Activity. The ALP activity in solution has been evaluated using both CSPD and LumiPhos Plus chemiluminescent substrates. The two substrates exhibit different kinetics profiles as shown in Figure 2. With both of them the kinetics is very slow, reaching a steady-state plateau only after 20-30 min; with the CSPD substrate the plateau is reached earlier (20 min). The light output at the plateau is linearly related to enzyme concentration or activity with a dynamic range from 50 to 800 pg of ALP for both substrates and with a sensitivity higher for the CSPD substrate (3.7 photons s-1 pixel-1 pg-1) in respect to LumiPhos Plus (0.38 photons s-1 pixel-1 pg-1). The detection limit (signal/noise 3:1) using CSPD is lower in respect to Lumi-Phos Plus and as low as 200 amol of enzyme can be detected (ALP specific activity 2000 IU/mg), in respect to 500 amol using LumiPhos Plus as a substrate. Figure 3 shows the kinetics profiles of HRP activity evaluated with ECL and Lumigen PS-3 substrates. Both substrates exhibit fast kinetics, and the steady-state light output is reached within few minutes; the signal is stable for about 20 min with Lumigen PS-3 and for a shorter time with ECL. The light output is linearly related to enzyme concentration or activity for both substrates with a dynamic range from 50 to 800 pg of HRP and with a higher sensitivity for the ECL substrate (1.18 photons s-1 pixel-1 pg-1)
Figure 2. Kinetics profiles of ALP activity using CSPD (upper panel) and Lumi-Phos Plus (lower panel) chemiluminescent substrates. The light emission was expressed as photons per second per pixel for both systems. The background values were subtracted: ([) 10, (9) 7.69, (2) 5, (×) 3.7, (*) 2.5, (b) 2, (|) 1.25, (-) 0.625, and (s) 0.25 ng/mL.
in respect to Lumigen PS-3 (0.47 photons s-1 pixel-1 pg-1). Using the ECL substrate, the detection limit with a signal/noise of 3:1 is 400 amol of enzyme (HRP specific activity 1100 IU/mg), and despite a higher absolute light emission using the Lumigen PS-3, the detection limit is slightly higher (about 900 amol of enzyme) as a result of a higher aspecific background signal. Dot Blot. Analysis of different dilutions of CMV DNA dotted on nylon membrane and hybridized with the gene probe shows that the light emission intensity is proportional to DNA concentration. Quantitative values were calculated by surrounding the spots with circles covering a known and fixed number of pixels; background was calculated on unspotted adjacent zones. The detection limit (signal/noise 3:1) is 10 fg of homologous DNA using both chemiluminescent substrates, despite a higher absolute light intensity with Lumi-Phos Plus than with CSPD. Replicate analysis of the same specimens indicates that the method is reproducible. When compared with previously developed colorimetric assay using the same probe,32 the chemiluminescent method exhibits a lower detection limit (50 times). Moreover, the main advantage of the chemiluminescent method is the possibility of performing quantitative analysis, which cannot be easily and accurately performed using colorimetric methods. Quantitative Optical Microscope Luminescence. When the videocamera is connected to the optical microscope (×40 objective) and the luminescent detection of ALP and HRP activity in solution is performed, the sensitivity is similar to that of the macrosetup and the precision, as evaluated by five consecutive Analytical Chemistry, Vol. 68, No. 7, April 1, 1996
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Figure 3. Kinetics profiles of HRP activity using ECL (upper panel) and Lumigen PS-3 (lower panel) chemiluminescent substrates. The light emission was expressed as photons per second per pixel for both systems. The background values were subtracted ([) 10, (9) 7.69, (2) 5, (×) 3.7, (*) 2.5, (b) 2, (|) 1.25, (-) 0.625, and (-) 0.25 ng/mL.
analyses, is acceptable with coefficients of variation always below 5%. The total amount of enzyme under analysis and visualized by the camera can be calculated by knowing the field of view area at the objective magnification used and the height of the liquid film analyzed. Usually 10 µL of chemiluminescent mixture sandwiched between two glass microscope slides corresponds to a volume of 3 ( 0.1 nL of sample in the field of view. Under these conditions, the detection limit is 3.3 fg for ALP and 10 fg for HRP. These values are comparable with those obtained on the same 10 µL solution with the conventional macroassembly, once normalized to the same volume unit by considering the number of pixels under analysis and their size. Using the microscope assembly, the dynamic range is limited by that of the imaging device, while in the macrosetup, the use of a 1-32 iris enlarges the possibility of performing simultaneous measurements of light with different intensities (4-5 decades), despite the limited and fixed dynamic range of the instrument. For comparative studies, the light emission values were expressed as photons per second per pixel for both arrangements. Nylon Net-Immobilized ALP. The chemical derivatization of nylon made by introducing an arm composed of six C atoms plus the bifunctional reagent glutaraldehyde is an easy and efficient means of immobilizing proteins. Figure 4 shows the live image obtained with the optical microscope, the chemiluminescent signal, and the overlay of the two images after processing the 1078 Analytical Chemistry, Vol. 68, No. 7, April 1, 1996
Figure 4. Nylon net chemically immobilized ALP: live image ×40 (upper panel), chemiluminescent signal (middle panel), and overlay after processing chemiluminescent image (lower panel). Pseudocolor ruler on the right shows the relative light intensity.
chemiluminescent image with a pseudocolor function. More than 25 different specimens of net were studied. The obtained data show that the enzyme is homogenously distributed for at least an area of 25 mm2, with consistently more activity on the knots. This is probably due to the fact that the nylon activation procedure introduces the functional groups more efficiently onto the knots. The kinetics of the immobilized enzyme is similar to that of the ALP in solution, i.e., glowing-like; the structure of the nylon net, with 57 ( 0.5 µm distance between threads, allows fast diffusion of the substrate to the active enzyme sites. The nylon net is a suitable model to evaluate the spatial resolution and the quantitative aspects of the low-light imaging, since the background can be quantified simultaneously with the photon emission derived from the immobilized enzyme. The high resolution of the chemiluminescent image between one thread and the adjacent open area, obtained with a pixel size of 1.1 µm, permits the evaluation of the topographic distribution of the immobilized
Table 2. Quantitative Analysis of Nylon Net-Immobilized ALPa net area
photons/s per pixel (mean ( SD)
ALP (ng/mm2, mean ( SD)
background knots threads
4.6 ( 0.2 7.4 ( 0.6 6.4 ( 0.5
17 ( 3 11 ( 2
a Performed by measuring light emission on defined areas within the nets and calculating the enzyme amount by the calibration graph.
Figure 5. Spatial distribution of endogenous ALP in rabbit intestinal mucosa cryosections (8 µm) evaluated using chemiluminescent substrate; ×40. Pseudocolor ruler on the right shows the relative light intensity.
enzyme since the brightness of two adjacent pixels can be resolved at the micrometer level. The amount of immobilized enzyme was calculated on circular areas located on knots and threads (1320 pixels ) 1.57 × 10-3 mm2, n ) 20) and the values are reported in Table 2. Similar areas located within the net gave the mean background values. The mean photon emission derived from the immobilized enzyme was significantly higher than the background value considering a five standard deviation threshold. The amount of immobilized enzyme per millimeter squared of nylon net can be calculated by a calibration curve in which the light emission is related to enzyme activity; the amount is significantly higher on the knots (17 ( 3 ng/mm2) than on the threads (11 ( 2 ng/ mm2). The reported data show the extraordinary ability of chemiluminescent imaging to assess the spatial distribution of immobilized enzyme on a solid surface and to perform quantitative analysis of an enzymatic activity. This method can be applied to other enzymes such as all the oxidases (xanthine oxidase, cholesterol oxidase, etc.) using ECL chemiluminescent substrate and potentially it can be used for all dehydrogenase enzymes, using direct or indirect bioluminescent or chemiluminescent reactions. Once immobilized, antibodies, receptors, or other analytes could also be quantified by means of coupled luminescent reactions.36 ALP in Tissues. Figure 5 shows the results obtained when a cryosection (8 µm) of rabbit intestine was analyzed for the ALP activity using Lumi-Phos Plus substrate; with a ×40 objective and a corresponding pixel size of 1.1 µm we achieved a good resolution of the chemiluminescent image. Overlay of the luminescent signal (36) Campbell, A. K. Chemiluminescence: Principles and Applications in Biology and Medicine; VCH: Weinheim, Germany, 1988.
Figure 6. In situ hybridization in CMV-infected human fibroblasts using digoxigenin-labeled DNA probe and ALP-labeled anti-digoxigenin antibody detected with chemiluminescent substrate. Pseudocolor display of the chemiluminescent signal (upper panel) and overlay of the chemiluminescent signal and the live optical microscope image (lower panel); ×40. Pseudocolor ruler on the right shows the relative light intensity.
with the live image permits us to localize ALP in the epithelial cells with good sensitivity and low aspecific signal. Diffusion of the chemiluminescent products was minimized by removing excess solution from over the cryosection, since the chemiluminescent substrate is still in excess and embedded in the tissue. Quantitative analysis of emitted light can be performed on a given area, corresponding for example to an intestinal villus and considering as significant values those higher than the reagent background plus five standard deviations. The thickness of the cryosection must be the same for all specimens under analysis because there is a linear relationship between light emission and section thickness. In 8 µm cryosections 40-60 fmol of ALP/villus can be detected. This method is superior to conventional histochemical colorimetric detection of ALP, since experiments performed on the same specimen first analyzed with chemluminescence and then stained with a colorimetric dye show that it is more sensitive and also permits a more accurate quantification of the enzyme.37 (37) Roda, A.; Musiani, M.; Pasini, P.; Suozzi, A.; Baraldini, M.; Girotti, S.; Venturoli, S.; Polimeni, C. In Bioluminescence and Chemiluminescence; Campbell, A. K., Kricka, L. J., Stanley, P. E., Eds.; Wiley: Chichester, England, 1994; pp 625-628.
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nescent assay proved more sensitive than the colorimetric immunoperoxidase assay with a sharp signal from the VCAexpressing cells. In Situ Hybridization. CMV nucleic acid was analyzed in fixed cultured human fibroblasts infected with the Towne strain of human CMV. A typical chemiluminescent signal pattern is shown in Figure 6: there is an intense signal specifically localized in the individual positive cells. As can be seen in the figure, the aspecific signal outside the cells and in smaller CMV-negative cells is absent or very low, demonstrating that diffusion of the emitting chemiluminescent species is negligible and that the digoxigeninlabeled DNA probe and the subsequent immunological detection using ALP-labeled anti-digoxigenin antibody are highly specific. Figure 7 shows a single positive cell observed at the highest magnification possible with our system, i.e., with a ×100 objective and with a pixel size of 0.4 µm. The light emission from the labeled DNA probe is confined within the cell with maximal values in an area corresponding to the nucleus.37 These results show the possibility of performing quantitative imaging at a subcellular level and offer analytical challenges for other applications.
Figure 7. In situ hybridization in CMV-infected human fibroblasts using digoxigenin-labeled DNA probe and ALP-labeled anti-digoxigenin antibody detected with chemiluminescent substrate. Single cell: 3D diagram of the chemiluminescent signal (upper panel) and overlay of the chemiluminescent signal and the live optical microscope image (lower panel); ×100, pixel size 0.4 µm. Pseudocolor ruler on the right shows the relative light intensity.
Immunocytochemistry. A sensitive detection of anti-VCAspecific IgM in infectious mononucleosis patients was achieved with chemiluminescent immunocytochemistry. The chemilumi-
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CONCLUSIONS Low-light luminescent imaging is a tool that provides unprecedented capability for ultrasensitive quantitative analysis of a given luminescent system either directly or via immunological and genic biospecific reactions. Commonly used enzymes such as ALP or HRP can be detected and imaged at submolar levels in macrosamples; the conjunction of the luminograph with an optical microscope allows localization of analytes at subcellular level, with spatial resolution as high as 0.4 µm and high detectability. This system provides a viable alternative to the use of radioactive substances and it is a unique analytical tool for performing ultrasensitive in situ quantitative analysis. Received for review October 24, 1995. Accepted January 10, 1996.X AC951062O X
Abstract published in Advance ACS Abstracts, February 1, 1996.
