2868
J. Phys. Chem. 1993,97,2868-2870
Fluorescence Quantitation Using Digital Microscopy Diether Recktenwald,' Janette Phi-Wilson, and Ben Verwer Becton Dickinson Immunocytometry Systems, 2350 Qume Drive, San Jose, California 95131 Received: September 30, 1992;In Final Form November 23, I992
A method for the absolute quantitation of particle fluorescenceon a digital microscope has been characterized using commonly available imaging software. The method uses solid glass beads in a known concentration of dye solution. The dye volume displaced by the beads and the drop in fluorescenceintensity are measured. The intensity drop associated with the number of dye molecules displaced is given by the negative integral of the dark spots (beads) in the image. As expected, results show a linear correlation between intensity drop and bead volume for a given concentrationand a linearcorrelationbetween intensitydrop over bead volumeand concentration of dye. Precision and accuracy of the method were determined with serial dilutions of (R)-phycoerythrin and fluorescein. Once a microscope is calibrated with a dye, an absolute fluorophore quantity can be determined from other fluorescence measurements. The sensitivity of the CCD camera can also be determined from the calibration method. Problems and limitations currently encountered are discussed. The method is equally applicable to flow cytometry.
Introduction
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The widespread use of measurements of fluorescent signals from singlebiologicalcellsby flow cytometryor digitalmicroscopy creates a need for relating the measured fluorescent signal to fluorophor quantities. Cell parameters typically measured are cellular DNA for ploidy,' cellular RNA for red cell maturity,* cell protein content: enzymeactivitieswith fluorescentsubstrates: or other cell constituents, which can be identified by fluorescent labeled antibodies5or nucleic acid probes.6 Particle fluorescence is also of interest in the material sciences for investigating fluorescence inclusions in glasses and for fluorescent powders. Even though quantitative measurements by fluorescence are routine for bulk determinations, presently most of those single cell studies only report the fraction of cells exceeding a relative fluorescence intensity level, or they compare fluorescence from sample to sample in experiment-specific relative units, making comparisons from experiment to experiment hard or impossible. Some attempts have been made to overcome this limitation of particle fluorescence measurements. Some5 used antibodies doublelabeled with a radioisotope and fluorescein to determine the number of lymphocyte surface markers; others7 measured the number and fluorescence of particles in a cuvette and by flow cytometry to calibrate instruments for measuring absolute fluorescencequantitiesin a microscopic particle, a method limited in precision by the particle light scatter of the cuvette measurement, and the particle counting accuracy. All of the aboveapproachesare either specificfor an application, inaccurate, or hard to perform. This paper describes an easy method for the calibration of particle fluorescence for any soluble fluorophor. The purpose of this calibration is to provide absolute measurements, rather than relative. Our approach links the output of a CCD camera (which at each pixel is proportional to the number of incoming photons) to the number of fluorescent molecules in a solution. In order to quantify the number of molecules we we a displacement method. Part of the solution of a fluorescent dye is displaced by glass beads, resulting in an image of dark spots in a bright field (Figure 1). The volume of the beads can be measured from their area in the image. Together with thedye concentration,this gives the number of dye molecules displaced. The number of CCD units associated with that number of dye molecules is given by the negative integral of the dark spots. The advantage of this method is its directness. Only 0022-3654/93/2097-2868$04.00/0
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Figure 1. Basic approach.
concentration and light are measured. Our method is inspired by a method to find cell volume from a fluorescence light loss! Experimental Methodology We used a fluorescence microscope (Zeiss, Oberkochen, Germany) with a 1OX objective and a 0.25 numerical aperture, illuminated with a 50-W mercury arc lamp through a 485-nm band-pass excitation filter and a 510-nm dichroic beam splitter, imaged through the same beam splitter and a 520-nm long-pass emission filter, equipped with a liquid-cooledCCD camera system (Series 200 with a Thompson 384 X 576 chip, cooled to -35 OC, Photometrics Ltd., Tucson, AZ). The dyes measured were (R)-phycoerythrin(strain P5, MW = 240000 QuantaPhy Inc., Santa Cruz, CA) and fluorescein (Sigma, USA). Emission maximum wavelengths were at 580 and 520 nm, respectively. Concentrations of stock solutions of dyes were determined spectrophotometricallywith a Varian DMS 90 using an extinction coefficient of 80 000 at 485 nm for fluorescein and 1.96 X 106 at 565 nm for (R)-phycoerythrin. Glass beads (5% weight/volume, 5-20 pm diameter, Polysciences, Warrington, PA) were suspended in 4% bovine serum albumin (BSA, Sigma) in Dulbecco's phosphate buffered saline (DPBS, Sigma) for 24 h at 4 OC. To minimize the number of multiples, the suspension was sonicated (Heat Systems Ultrasonics, Model W- 10, power and tune level 3) and vortexed prior to pipeting. An 800 nM solution of R-PE was suspended in 4% BSA in DPBS. Four serially diluted solutions in 25% glycerol in DPBS of 320, 160,80, and 40 nM were prepared. The fluorescein samples were prepared in four serial dilutions of 21.6-2.7 pM. To test sample reproducibility, the beads were
0 1993 American Chemical Society
Fluorescence Quantitation Using Digital Microscopy
The Journal of Physical Chemistry, Vol. 97, No. 12, 1993 2869 0, r
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Figure 3. Intensity drop versus volume for four solutions of fluoreacein (numbcrsinparenthcscsaftersloptestimotararecoeffcientsofvariation). Inset shows intensity drop over volume versus concentration. Relation is 0.0038 CCD units/molecule.
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Figure 2 (a, top) h i r e d boundary for integral: the background image. (b, middle) Smoothed original. (c, bottom) Smoothed original after maximum and minimum filtering.
also suspended in 10independently prepared tubes of 5.4 pM. Of the same set, two images were taken out of focus. Four microliters of thesamples was put on standard microscope slides and spread under 4.84-cm2 coverslips which were subsequently sealed with rubber cement (Carter'srubber cement, Boise Cascade, IL) to minimize evaporation. The beads were brought into focus using transmitted light (0.1 s exposure time). The exposure time to the fluorescence light was 10 s. For all measurements, the dark current of the CCD was subtracted from the images (measured with 10-s exposure time and the shutter closed). Aarlytid Methodology
The image processing contained two stages. In the first stage the volume of the dark spots was estimated. In the second stage the drop in fluorescenceintensity. All image processing was done on a Macintosh Quadra (Apple, Cupertino, CA) using the image processing package TCL-Image (TPD, Delft, The Netherlands). To estimate a bead volume we measured the bead area from an image. To measure the bead area we thresholded the image and counted pixels. For reproducibility,thresholding was done automatically with a histogram method9after shading had been removed by smoothing the image with a large round uniform filter (diameter 51 pixels) and subtracting it from the image. Overlapping beads, out of focus beads, and other artifacts were removed manually. To estimate the drop in fluorescence light, we needed, apart from the area over which to integrate, which we already defined, the level from which the drop occurred. Figure 2a shows the level we wanted toestimate. Our procedure involved three steps: smoothingof the original to removenoise, maximum and minimum filteringto fill the dark spots,and a final smoothing step to remove artifacts of the maximum and minimum filtering. Parts b and c of Figure 2 highlight the successive steps. We call the image
resulting from this procedure the background image. (For the first smoothing step we used a median filter of 3'3 followed by a round uniform filter with a diameter of the largest spot in the image. The maximum and minimum filters used a filter size twice that diameter. The final smoothing was done with a Gaussian filter, u = 3 pixels.) Once the base level had been determined, we corrected for uneven lighting by multiplying all pixels in the image by a factor. The factor for each pixel equaled the average of the background image divided by the background level at that pixel. The final steps summed, for each bead, the corrected gray values under the previously defined mask. The background level had to be estimated from the data itself, because the largedepth of focus led to different background levels for each image, dependent on the distance between slide and coverslip. Another step in the calibrationwas the pixel sizedetermination. For that purpose we used a hemacytometer (Neubauer hemacytometer, AO,Bright-Line, Buffalo, NY) with a well-defined distance of 50 pm between two lines. The image was taken and thresholded and the lines were estimated using a least-squares estimator. This resulted in a pixel size of 0.594 I m with a coefficient of variation of less than 0.2%. Within this accuracy the pixels were square. This sampling density leads to an intrinsic errorlo in the areameasurementsof 196forourbeadsizes (fl.S%volumeerror). RHultS
Figure 3 shows the correlation of calculated bead volume to number of units of fluorescence displaced for four solutions of fluorescein. Surface staining was clearly visible during experimentation, rendering the intensity drop measurement incorrect. By suspending the glass beads, prior to addition of dye solution, in 4% BSA for 24 h at 4 OC, the staining was blocked. The volume of the beads is determined by measuring the area of the dark spots (in pixels) and converting it to a volume using a 0.594pm pixel size and assuming a spherical shape of the beads. Each point on a line represents a bead. The beads show a good linearity of intensity drop to bead volume. The legend of Figure 3 gives the slope value and its standard deviation for each concentration. The inset shows the correlationof slopevalue with concentration. Using all data points acquired gives a sensitivity of 0.0038 CCD units/molecule. The coefficientof variationfor this measurement was 1.2%. Figure 4 shows the results for phycoerythrin. Using all data points acquired gives a sensitivity of 0.30 CCD units/molecule. The coefficient of variation was 1.6%. To test reproducibility, we took images of 10 independently prepared 5.4 pM solutions of fluorescein (one image of each solution, number of beads per images 3G70). The sensitivity estimates for nine images are shown in Figure 5 (one sample was
2870 The Journal of Physical Chemistry, Vol. 97, No. 12, 1993
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F'ignre 4. Intensity drop versus volume for four solutions of (R)phycoerythrin (numbers in parentheses) after slope estimates are coefficients of variation. Inset shows intensity drop over volume versus concentration. Relation is 0.30 CCD units/molecule.
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Figure5 First-order estimates (solid lines) of intensity drop and volume for nine images of independently prepared solutionsof 5.4 pM fluorescein. Also shown are data points and estimates (dotted lines) for two images taken out of focus.
removed because the microscope slide it was put on was contaminated). The mean for the estimates in the slope values is 12.3 CCD unitslpm3 with a coefficient of variation of 0.86 (6.9%). One way to improve on this would be to use the a priori information that thebeadsareroundand fit amodel (with position and size as parameters) to the data for each visible bead. Also shown in Figure 5 are the data points and line estimates of two images heavily out of focus. The main effect of the focus error is that the line estimatesare shiftedvertically. Furthermore, a nonlinear effect can be seen as the slope levels off for larger beads. However, the slope estimates are still only 1.1 and 1.6 standard deviations from the mean of the other nine. This shows that small focus errors are negligible. To measure the limit of detection, we made two exposures of one image, containing only beads in PBS. The images were subtracted to eliminate pixel variability." The variance of the difference image was 2.8, giving a standard deviation for the blank image of 1.2 A safe limit of detection is 3 times the standard deviation, giving 4 CCD units (should be integer valued). For fluorescein and phycoerythrin this gives limitsof detection of 1.1 X IO3 and 13 molecules/pixel, respectively.
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l.nacumim We havedescribed a method for thecalibrationof a quantitative fluorescence micrcwcope for the absolute quantitation of particle fluorescence, Le., the determination of the number of fluorescent molecules associated with a particle. Only the fluorophor concentration in the preparation under the microscopeand the sizeof a pictureelement in the microscopic image have to be known for the calibration. All other information is obtained directly from the microscopic image.
Recktenwald et al. To overcome the inaccuracies associated with the calculation of the image-transfer function, a volume was defined by inert sphericalparticles with a diameter of severalmicrometers (ideally, the indices of refraction of the beads and solutions should be the same). Depth of focus error contributions to the measurements were minimized by using a low magnification, low numerical aperture objective. These errors are expected to result in a nonlinearity of the fluorescence signalversus volume relationship which was not observed. Focus errors will give a nonlinearity as well, as was shown in Figure 5. But the errors are negligible. The method can be used with all fluorescent systems that form stable solutions, stable transparent suspensions, or transparent glasses, in which inert transparent particles can be embedded. Those particles must not show any significant enrichment of the fluorescent system under investigation at their surface, and they must be 'hard" to avoid penetration of fluorescent compound into the particle volume. From our measurements, sensitivities were found of 0.0038 and 0.30 CCD units/molecule for fluorescein and phycoerythrin, respectively. The difference in the two numbers is explained by the differencein number of chromophores (PEhas approximately 40 more than FITC), the difference in quantum yields ( f l .O for PEand f0.55 for FITC) and the wavelengthdependent sensitivity of the CCD camera (f1.2 times more sensitive at the maximum emission wavelength of PE (580 pm) than at that of FITC (520 pm)). The difference in photobleaching effects between the two dyes is insignificant because the integration time was less than half the photobleaching decay times (data not shown). In future studies,we will apply this method for the quantitation of the fluorescence, endogenous and acquired by reaction with fluorescent reagents, of biological cells, for the calibration of fluorescent particle standards, and for the determination of sensitivitiesand limitsof detection of systemsfor the measurement of fluorescent particles. More intensive image analysis methods are also being considered, such as using the a priori knowledge of the shape of the dark spots in segmenting the image. This method can be used in flow cytometry if the cytometer is capable of measuring sizeand intensitydrops. Once calibrated with beads, that will naturally also allow accurate volume measurements of cells.
References and Notes (1) Michaelson, M.J.; Price, H. J.; Ellison, J. R.; Johnston, J. S . Am. J . Botany 1991, 78(2), 183. (2) Serke, S.;Huhn, D. Br. J . Haemarol. 1992,81,432. Lee, L.; Chen. C.-H.; Chiu, L. Cyrometry 1986, 7, 508. (3) Freeman,D. A.;Crissman, H. A.Stain Tcchnol. 1975,50,279. StOhr, M.; Vogt-Schaden, M.; Knobloch, M.;Vogel, R. L.; Futterman, G . Srain Technol. 1978, 53, 205. (4) Malin-Berdel, J.; Valet, G . Cyromerry 1980, I , 222. Dolbcare, F. A.; Smith, R. E. Clin. Chem. 1977, 23. 1485. (5) Ledbetter, J. A.; Frankel, A. E.; Herzenberg, L.A.; Herzenberg, L. A. Monoclonal Antibodies and T-cell DlfjrrcntiarionAntigens: Quanriratiue Expression on Normal Lymphoid Cells and Cell Lines. In Monoclonal Antibodies and T-cell Hybridomas, Perspectives and Technical Notes; Haemmerling, G., Haemmerling, U.. Kearney, J., W.; Elsevier North Holland: New York, 1981. Roe, R.; Ronbins, R. A,; Laxton, R. R.; Baldwin, R. W. Mol. Immunol. 1985,22,11. Dux, R.;Kindler-ROhrborn, A.; Lennartz, K.; Rajewsky, M. F. Cyromerry 1991, 12, 422.
(6) Nederlof, P.M.;Robinson,D.; Abukncaha,R.; Wiegand, J.;Hopman, A. H. N.; Tanke, H. J.; Raap, A. K. Cyromerry 1989, 10, 20. Bianchi, D. W.; Harris, P.;Flint, A,; Latt, S.A.; Cyromerry 1987,8, 197. ( 7 ) Schwartz, A. Monograph: Fluorescent Microbead Standards; Flow Cytometry Standards Corp.: Research Triangle Park, NC, 1988. Vogt, R. F.;Crcas, G. D.; Phillips, D. L.; Henderson, L. 0.;Hannon, W. H. Cyromerry 1991, 15, 525. Brown, M.; Hoffman, R.; Kirchanski, S.; Ann. N.Y.Acad. Sci. 1986, 468, 93. (8) Bccker, P. L.; Fay, F. S.Biophys. J. 1986.49, 465A. (9) Zack, G. W.;Rogers, W. E.; Latt, S . A. J. Histochcm. Cyrochem. 1977, 25, 741. (IO) Young, 1. T. Anal. Quant. Cytology Histology 1992, IO, 269. (1 1) van Vliet. L. J. Proceedings of Workshop on Fluorescence Insrcmentation in Support of In Situ Hybridization; Delft University of Technology: Delft, The Netherlands, 1992; p 6.
