ARTICLE |
Correspondence to: M. Ono, Dept. of Anatomy, Yokohama City U. School of Medicine, 3-9, Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. E-mail: mono@med.yokohama-cu.ac.jp
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Summary |
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Fading is one of the major obstacles to reliable observation in fluorescence microscopy. Using a confocal laser scanning microscope (CLSM) coupled to a computer, we quantitatively measured fading of fluorescence to formulate an equation, evaluated the anti-fading ability of several anti-fading media, and restored the faded images to the original level according to this equation. NIH 3T3 cells were stained with fluorescein isothiocyanate (FITC)phalloidin, mounted with several commercial and homemade anti-fade media, and observed with CLSM under repeated illumination. With any mounting medium, attenuation of fluorescence intensity at a certain pixel occurred stepwise and the decrease was proportional to the intensity of the previous scan. From these results, we formulated an equation that has three coefficients: anti-fading factor (A), indicating the ability to retard fading; fluorescent intensity at the first scan (EM1); and background fluorescence (B). The fluorescent intensity at a certain point following nth scan is given as EMn = EM1 A (n-1). This equation was available for restoring faded images to their original states, even after the image had faded to only 60% of its original intensity. (J Histochem Cytochem, 49:305311, 2001)
Key Words: anti-fading media, anti-fading factor, confocal laser scanning microscopy
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Introduction |
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IN FLUORESCENCE OBSERVATION by conventional fluorescence microscopy and confocal laser scanning microscopy (CLSM), the retardation of fluorescence fading, the high initial intensity of images, and low background noise are important factors for obtaining clear and accurate images. Fluorescein isothiocyanate (FITC) is the most widely used fluorochrome in fluorescence microscopy. However, the fluorescence of FITC is rapidly lost when it is exposed to excitation light, and FITC-stained preparations mounted in conventional buffered glycerol show prompt bleaching under illumination, especially under optimal conditions for observation: under illumination at the wavelength of maximal absorbance of the fluorochrome and with objective lenses of a high numerical aperture (Johnson et al. 1981;
Several types of mounting media are available (
In this study, we examined the fluorescence fading phenomenon quantitatively, obtained an equation to express the phenomenon, compared the ability of various anti-fading media, and tried to restore already faded images to their original state with a computer.
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Materials and Methods |
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Cell Preparation
NIH 3T3 cells were used for this study. The cells were cultured on coverslips (22 mm2) in RPMI-1640 medium containing 10% newborn calf serum in a humidified atmosphere of 5% CO2 and 95% air at 37C. Nonadherent cells were removed by rinsing the coverslips with PBS. The cells on coverslips were fixed and permeabilized with 4% paraformaldehyde and 0.05% Triton X-100 in PBS (pH 7.4) for 10 min at room temperature (RT), washed with PBS, and then stained with 0.1 mg/ml FITCphalloidin (Sigma Chemical; St Louis, MO) and stored overnight at 4C. The coverslips were washed three times with PBS and mounted with different media, as described below.
Mounting Media
We used 50% glycerol in 20 mM phosphate buffer (pH 8.5) (GB) as a glycerol-based standard medium. For experiments with homemade anti-fading reagents, 0.1% p-phenylenediamine (PPD) (Wako Pure Chemical Industries; Tokyo, Japan) (Johnson et al. 1981,
As commercial anti-fading media, we used SlowFade Light Antifade Kit (Molecular Probes; Eugene, OR), PermaFluor (Lipshaw/Immunon; Pittsburgh, PA), FluoroGuard Antifade Reagent (Bio-Rad Laboratories; Hercules, CA), and ProLong Antifade Kit (Molecular Probes). The commercial media were used within 1 month because those were newly opened. All specimens were prepared in the same way before observation. The plastic tapes that opened the rectangular hole were used as spacers for cultured cells on coverslips. The spacer was placed on a glass slide. Then the hole was filled with each mounting medium. The coverslips were put upside down on the spacer, then, were fixed with glue. The mounted specimens were incubated for 1 hr in the dark before use.
Confocal Laser Scanning Microscopy
We used a confocal laser scanning microscope (LSM GB-200; Olympus, Tokyo, Japan) coupled with an IEEE-488 interface to an IBM PS/V 486-66M computer (IBM). The excitation source was an argon ion laser with a 20 mW output power at 488-nm line and 1%, 3%, and 10% transmittance normal-density (ND) filters. The fluorescence emission was split by a dichroic mirror DM488 and was measured by a detector placed behind a BP530-nm bandpass filter.
Fluorescence emission was recorded through a x60 SPlan-Apo oil-immersion objective with a high numerical aperture (1.4). Each specimen was scanned 50 times and the whole scanning field was recorded for each scan. The area and the rate of laser scanning were 1024 x 768 pixels/40 sec (0.257 µm/pixel and 51 µsec/pixel). The conditions for high-voltage, gain, and offset were -800 kV, 2.0, and 2, respectively, and the same conditions were used throughout the study. The images were stored on the coupled personal computer. All measurements were performed at RT.
Data Analysis and Image Processing
We analyzed fluorescence intensity by fading in pixel units, using a Power Macintosh 7300/166 (Apple Computer; Cupertino, CA). For the analysis, we developed software (AF Analyzer) to obtain fluorescence intensity of the same coordinate pixels from every excitation. For each pixel, the mean value of initial fluorescence intensity was compared with the intensities after fading by using the AF Analyzer. These data were analyzed using the pixel data from which saturated intensity and background intensity were subtracted. The regression lines were calculated and drawn with MacCurveFit (Kevin Raner Software, Victoria, Australia; http://www. home.aone.net.au/krs/).
We performed image processing for correction of fading with FluoroFixer software (http://bioimage.med.yokohama-cu.ac.jp/confocal/fluorofixer), which we developed to enable fluorescence reconstitution calculated by the use of a fading equation.
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Results |
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The Nature of the Decrease in Fluorescence Intensity
To ascertain the ability of anti-fading reagents to reduce the decrease in normalized fluorescence intensity, we compared the time course of bleaching of FITCphalloidin-stained NIH 3T3 cells in mounting media with or without anti-fading reagents under a CLSM (Fig 1). With all the mounting media tested, the fluorescence decreased as a function of the number of scans, although the decreasing speeds and the initial fluorescence intensity were significantly different among the media. It was not possible, however, to compare quantitatively the ability of different mounting media to prevent a decrease in fluorescence with this method, because the slope of the graph changed as the number of scans increased even for a single mounting medium.
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In the method used in Fig 1, there is also the possibility that the speed of fading is different among spots with different initial fluorescence intensities. We decided to take advantage of the fact that in CLSM the intensity of each spot can be recorded digitally, and the decrease in fluorescence intensity in each pixel unit can be traced. To compare the change in fluorescence intensity between spots with different initial intensities, we measured the change in fluorescence intensities of all the spots and grouped them according to the initial intensities. Fig 2 shows the data for eight different initial intensities of a buffered glycerol (GB)-mounted specimen. The stronger the initial fluorescence intensity, the more rapidly the intensity decreases in response to laser illumination. We again recognized that the amount of fading decreases as the number of scans increases.
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An Equation to Indicate Fluorescence Fading
In the CLSM, because the specimens are illuminated intermittently, the intensity of fluorescence decreases in a stepwise manner between each scan, and the quantity of fading can be measured as the difference in the intensity between scans. We measured the difference in intensities between adjacent spots and plotted them as a function of the fluorescence intensities in the previous scan (Fig 3). This value is shown to be almost in proportion to the fluorescence intensity in the previous scan for each mounting medium, and the lines fit very well when we employed a linear least-squares fit. We determined that the relation of the fluorescence intensity and the quantity of fading by illumination is given by the following equation:
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(1) |
where EM is the decrease in fluorescence intensity, k is the inclination of regression lines in Fig 3 and thus a coefficient that indicates the extent of fading, and EM is the fluorescence intensity in the previous scan.
Equation 1 can be changed to EMn+1 = EMn (1 - k), then into:
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(2) |
where EMn is the fluorescence intensity of the nth scan. Equation 2 is a geometrical progression in which A is the common ratio. A is a constant between 0 and 1 that is specific for each mounting media.
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(3) |
When a background intensity (B) which does not fade is introduced, the equation is changed to:
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(4) |
For more general uses, we can use time (t) instead of the number of scans (n). In this case, Equation 1 can be converted to
Equation 3' can be changed to the following by setting A' = 1/k. Then the following equation can be deduced from Equation 4 by using (t) instead of (n).
A' is the time spent until fluorescence intensity becomes 1/e, which is specific for each mounting medium. The larger A and A' are, the stronger the ability of a given mounting medium to prevent fading. Therefore, we defined A as the anti-fading factor in the study. Thus, we were able to obtain an equation to evaluate the ability of a medium to retard fading. With this equation, the fluorescence intensity after a given number of scans can be calculated from the original intensities (intensities at the first scan) as described below. We call these equations, either Equation 4 or Equation 4', the fading equation in this study.
Comparison of Mounting Media by Fading Equation
We compared the ability of anti-fade media using the fading equation. We calculated means of intensities of all pixels in each scanned image and calculated A and EM1 values for each anti-fading medium by a nonlinear least-squares analysis (Fig 4; Table 1). The B value was adjusted to 0 by selecting an optimal offset value. Among all the anti-fading media tested, ProLong showed the highest A values under both high (99.423 at 10% transmittance ND) and low (99.635 at 1% transmittance ND) excitations, indicating the strongest anti-fading effect, but showed the lowest EM1 value, which indicates the darkest initial image. FluoroGuard showed the second highest A value (99.607) and a high EM1 value, indicating that FluoroGuard gives both a strong anti-fading effect and bright initial images. PPD and DABCO also showed good anti-fading effects comparable to some commercially available anti-fading media, but the effects were not as strong as those of ProLong or FluoroGuard. PPD appears to give higher EM1 values than DABCO. As for the diluents, glycerol and PVA did not show a big difference in A values, but PVA containing diluent seemed to be superior in terms of initial intensities (EM1).
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Correction of Fading Image by the Fading Equation
Because the alteration in fluorescence due to fading is provided by the fading equation, the initial illuminated intensities of a fluorescent image can be calculated from the intensities after a given number of scans with the following equation, which is calculated from
Equation 4:
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(5) |
We attempted to restore an original image with this equation, using a personal computer, from an image that had faded as a result of prolonged illumination. Fig 5 shows FITCphalloidin-stained NIH 3T3 cells mounted in buffered glycerol which had been faded by prolonged illumination (Fig 5B5D) and the images restored to their original luminescence with Equation 4 (Fig 5E5G). Restoration without significant deterioration of the images that had faded up to 60% of their initial fluorescence intensity was possible with this equation (Fig 5G compare with Fig 5A). The equation not only is available to indicate the abilities of anti-fading media but it can also be available for the correction of faded images.
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Discussion |
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Fading Equation
We compared the ability of various commercial and homemade anti-fading media by CLSM. In previous reports, normalized intensities of images obtained by every scan were compared (
Anti-Fading Factor
The decrease in fluorescence intensity after each scan was shown to be proportional to the intensity before the scan at the pixel unit, regardless of the fluorescence intensities (Equation 1). Consequently, the decrease of intensity by excitation was shown as an exponential decay function (Equation 2Equation 3Equation 4). This fact can be explained as follows. In CLSM, fluorochromes under incident light are excited and emit fluorescence. A certain proportion (F in Equation 1) of the excited fluorochromes are oxidized and will no longer emit fluorescence (
To prevent retardation of fluorescence intensity, three methods can be used. The first is to remove oxygen from the mounting medium. The second is to increase the viscosity of the mounting medium and retard the diffusion of oxygen, such as with the use of glycerol or PVA as a mounting medium. The third is the use of a reagent that quenches the excitation and consequently reduces the amount of oxidized fluorochromes. Azide, iodide ions, and DABCO are known to have this quenching ability (
In photochemistry, the SternVolmer equation is used to express the process of quenching (
Comparison of Anti-Fading Media and Their Practical Use in CLSM
Various mounting media were compared with their anti-fading factor (A) and initial intensity of fluorescence (EM1). Among commercial and homemade anti-fading media examined, ProLong showed the highest A value. Its A value remained high even under strong excitation. ProLong, however, has a low EM1 value. On the other hand, FluoroGuard showed the second highest A value and a relatively high EM1 value. However, the A value of FluoroGuard decreases when excitation is strong. In general, in high EM1 (bright) mounting media, more fluorochromes are excited at the same illumination intensity than in low EM1 mounting media. Accordingly, the decrease in fluorescence intensity cannot be prevented well in high EM1 mounting media. Because with CLSM the brightness and contrast of images can be controlled arbitrarily by adjusting the sensitivity of photomultipliers, gain, and offset, we can use anti-fading media with high retardation ability even though they may have low EM1 values. In this context,
Application of the Fading Equation
The application of our fading equation enables us to restore faded images. We can calculate the initial intensities of each pixel unit with our fading equation backward from the faded images. As shown in Fig 5, recovering the original state from an image faded to 60% of its original fluorescence was possible. This application is particularly important for specimens in which an anti-fading reagent cannot be used. For example, in fluorescence observation of living cells, it is difficult to use an anti-fading agent, and therefore it is difficult to distinguish physiological changes in fluorescence intensity from changes caused by fading. Another application is to reconstruct quantitative 3-dimensional images. In 3D observation of CLSM, the specimens fade during scans on various optical planes by repeated excitations. Each plane is faded during the illumination to other planes, which should be corrected (
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Acknowledgments |
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Supported in part by grant-in-aid 11770013 from the Ministry of Education, Science and Culture, Japan.
We thank Dr Akito Ishida (The Institute of Scientific and Industrial Research, Osaka University) for helpful discussions and advice on photochemistry.
Received for publication September 14, 2000; accepted September 20, 2000.
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