Journal of Microscopy, Vol. 165, P t 3, March 1992, pp. 347-365. Received 11 June 1991; revised and accepted 11 October 1991
The quantification of fluorescent emission from biological samples using analysis of polarization
by A. E N T W I S T Land E M. NOBLE,The Ludwig Institute f o r Cancer Research, Courtauld Building, 91 Riding House Street, London W l P 8 B T , U.K.
KEY
w o R D s. Fluorescence quantification, immunofluorescence, confocal microscope.
SUMMARY
The quantification of fluorescent emission from biological specimens can only be carried out in cellular regions where the relationship between fluorophore concentration and fluorescent emission is linear. Using a confocal scanning laser microscope, we show that quantification of fluorescent emission from biological samples labelled with fluorescein and fluorescein analogues mounted in a viscous medium can be readily achieved. Where the distribution of fluorophore is highly localized, for example in cells labelled for immunofluorescence analysis, we demonstrate that analysis of fluorescence depolarization can identify regions in which fluorophore concentration exceeds the range in which the relationship to fluorescent emission is linear. We also demonstrate that, under the conditions examined, depth-dependent effects, fading and quenching are either small enough to be ignored or can be corrected for mathematically when quantifying fluorescent emission. INTRODUCTION
The widespread use of fluorescent compounds to visualize specific molecular structures, for example within cells, offers the opportunity to use quantitative analysis to obtain information about differences between two (or more) structures which are both fluorescently labelled. The quantification of fluorescence emission with conventional microscopes is frequently beset with problems, such as excessive fading (Johnson et al., 1982; Kasten, 1989), fading at different rates in different parts of the object (Benson et al., 1985), fading whose kinetics necessitates complex mathematical corrections (McKay et al., 1981; Koppel et al., 1989) and apparent local variations in quenching (Shapiro, 1988). Many of these problems arise from, or are exaggerated by, noise and out-of-focus signals collected over the large depth of field presented in conventional microscope images. Moreover, when quantifying fluorescence with conventional microscopes, it is difficult to analyse fluorescence with large volumes of fluorophore at concentrations p 1 0 j i (van ~ Oostveldt & Bauwens, 1989). These difficulties result from inner-filter and re-absorption effects (Tanke et al., 1982) over the large depth of field examined; the inner-filter effect is a progressive reduction in intensity of the stimulating illumination by the fluorophore with increasing depth, and re-absorption is the absorption of fluorescent emission from lower layers by upper layers. As confocal microscopes reject light that does not emanate from, or close to, the plane
84 in white. (D) Original image of cells shown in (C).(E),(F)Cells displayed in (D)where pixel intensity is 11-84 with polarization ratios (E)greater than or equal to 2 mM F I T C or (F)greater than or equal to 2 mM fluorescein, displayed in white and the remainder in black. (G),(H)Cells displayed in (D) where pixel intensity is 2 85 with polarization ratios (G)greater than or equal to 2 mM fluorescein, or (H) greater than or equal to 4 mM fluorescein, displayed in white and the remainder in black. Scale bar = 25 Ltm.
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absorption-purified FITC-conjugated polyclonal anti-mouse Ig antibody, and an excess of both the primary and secondary antibodies was used. A suitable cell was selected (Fig. 3A), pairs of images collected, and a final image was created in which pixels where the polarization ratio was less than expected (i.e. less than the polarization ratio from a 2 mM F I T C standard) were mapped to black and pixels where the polarization was greater than or equal to that expected were mapped to white. Regions where the total intensities indicated that the apparent polarization ratio was significantly affected by noise were mapped to grey (Fig. 3B); the majority of these pixels were clearly not parts of the cell (Fig. 3C). Of the pixels examined in the fainter regions of the cell, almost all ( > 99-9O,) were white, indicating that the polarization ratios were above those of a 2 mM F I T C standard and in the range where the relationship between fluorophore concentration and pixel grey level was linear (Fig. 3C, D). In the more intensely stained regions, a higher proportion of black pixels (%2c)0),where the polarization ratio was less than that of a 2 mM F I T C standard, was observed (Fig. 3C, D, upper left). I n a similar image generated to compare the values observed with those generated from a 2 mM fluorescein standard (not shown) the black pixels virtually disappeared ( < 0.1O 0 ) ; however, this level of polarization was very close to the levels where it was no longer possible to assume that fluorescent emission was linear with respect to fluorophore concentration (Entwistle & Noble, 1992). Unlike the results obtained with anti-GalC antibody, the polarization of the emitted light obtained when a polyclonal primary antibody, anti-GFAP, was used indicated that the pixel grey values observed could no longer be assumed to be linearly related to the concentration of F I T C . However, using the polarization ratio values, it was possible to discriminate between regions where quantification could and could not be carried out. An excess of both anti-GFAP and FITC-conjugated anti-rabbit Ig was used to label methanol-fixed cells derived from a human glioma (specifically, a glioblastoma multiformae). Pairs of images representing the two orientations of polarization were again collected but here the polarization ratios in two regions of different pixel intensity were processed separately. We first examined less intensely stained regions, where the absolute pixel grey level was between 11 and 84 (Fig. 3D, grey pixels). Then examination of the more intensely stained regions, where the grey level was above 84 (Fig. 3D, white pixels), was carried out. With the pixels in the range 11-84, polarization ratios less than those generated from a 2 mM F I T C standard predominated (Fig. 3E, black pixels). Indeed, for large numbers of these pixels the polarization ratio was less than those generated from a 2 mM fluorescein standard (Fig. 3F, black pixels), as seen in the diffuse processes close to the exterior of the middle regions to the bottom of the cell in the left of the image and the lower right of the cell in the right of the image. I t can also be seen in these images that the polarization ratios for regions of both diffuse and dense fibres in the interior of the cell were generally equal to or greater than those from a 2 mM fluorescein standard (Fig. 3F, white pixels) and frequently equal to or greater than those from a 2 mM F I T C standard (Fig. 3G, white pixels), suggesting that the local concentration of antibody binding was lower in the interior regions than in the peripheral regions. With pixels where the grey level was > 84 (Fig. 3C, white), many pixels in both of the cells shown in Fig. 3(C) had polarization ratios that were less than a 2 mM fluorescein standard (Fig. 3G, black pixels) and some ratios were less than a 4 mM fluorescein standard (Fig. 3H, black pixels); these results indicated that the local concentration in the very bright regions greatly exceeded the range where a linear response with respect to fluorescent emission can be demonstrated. Fading With many immunohistochemically stained preparations, quantification of large data
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sets was possible without correcting for fading since the loss in intensity was small ( < 10°o)even when a large number of images ( 350), each the average of many scans
(loo), was collected. For example, the complex and delicate network of intermediate filaments in a human glioblastoma multiformae cell was clearly visible in sixty sequential images. Vertical steps of 0.2 pm were made between each image, and each image was generated by averaging the data from scans giving a total laser exposure of ~ 0 . nWs 4 pixel-'. Despite the low laser intensities employed, the collection of highcontrast images where the filamentous nature of the staining was clearly visible was easily achieved (Fig. 4A-G). Images collected at the same plane through the cell ( & 0.2 pm), but before and after a total laser exposure of z 21 nWs pixel- (fifty images), were very similar (Fig. 4A, H) and quantitative measurements indicated that the fading was slight (i.e. < loo,,). Even when data sets large enough to induce obvious fading were required, the fading of FITC: was approximately linear over the first part of the decay process making correction simple. Intensity of emission plotted against the total exposure to the laser gave curves that were fitted by a straight line over the first 20°, of the decay process, in twenty different experiments under various conditions; and in all of these experiments, the correlation coefficient R2 was > 0.98. When correcting for fading, corrections for different rates of fading across the image field were not required as the rate was uniform throughout the object even when preparations with a complex structure were examined. Immunofluorescently stained GFAP filaments in human glioblastoma multiformae cells were again imaged (Fig. 5A) and cells were then exposed to the laser until approximately 80, 60 and 40", of the original F I T C intensity remained (Fig. 5D, G, J, respectively). T h e imaged files were renormalized to a constant mean intensity to permit direct visual comparison (Fig. 5B, E, H, K). T h e pixel values in the re-normalized image files were then subtracted from the pixel values in the equivalent image file at time zero (Fig. 5B). T h e resulting files were mapped to a new image where pixels in the re-normalized images (Fig. 5B, E, H, K) that varied from the expected value by more than 1 standard deviation of the background were mapped to white when above the mean and black when below the mean. Pixels within 2 0.33 of the standard deviation of the background were mapped to grey, and values between 0.33 and 1 standard deviation were scaled appropriately. Pixels where the fluorescence in the initial image (Fig. 5B) was less than three times above the background were also mapped to grey. Although significant variation was found in some pixels, there did not appear to be any underlying pattern to their distribution and their position was not constant from image to image. Where a pattern was evident (e.g. Fig. 51, bottom left) the alternation of black and white regions was characteristic of movement of the object following, for example, slight movement ( < 1 pm) of the stage. T h e complete data set used to generate these images took z 80 min to collect and over this time period slight loss of registration was not surprising.
'
Quenchi~g Non-specific quenching of fluorescence was not a general problem when quantifying fluorescent emission with a confocal microscope. T o examine quenching, two different ratio methods had to be employed. T h e first compared the level of fluorescence within a sample with an external standard taken outside the cell (where no sample-induced quenching was present). For example, with the six different cell preparations used here (HeLa cells, rat embryo fibroblasts immortalized with simian virus 40 large-T antigen, human rhabdomyosarcoma cells, primary chick myogenic cultures, bovine endothelial cells and primary cultures of cells derived from optic nerves of 7-day-old rats) a region of medium immediately adjacent to the cell under examination was utilized as an external standard. Cultures were fixed and carboxyfluorescein or FITC-conjugated
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Fig. 4. A human glioblastoma multiformae cell immunofluorescently labelled for intermediate filaments with low laser intensities. Sixty images were collected at 0.2-pm vertical intervals, each the average of 100 laser scans. (A-G) Images collected at 1.6-pm intervals starting from just above the cover slip (A). (H) An image collected at the end of the series at the same level i 0.2 Ltm as image (A). Scale bar = 25 pm.
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Table 1. The apparent level of fluorophore found inside six different cell types following exposure to fluorophores of different molecular weights. Cell type
Fluorophore Carboxyfluorescein Carboxyfluorescein (post-rinse) FITC-4 kDa dextran FITC-40 kDa dextran FITC-400 kDa dextran
HeLa cells
Rat embryo fibroblasts
Rhabdomyosarcoma cells
Chick myogenic cultures
Bovine endothelial cells
Rat optic nerve cultures
85.7 2 1.6
86.6 + 1.6
81.1 t 1.8
90.9 i 6.0
87.9 f 3.5
89.0 2 3.1
0.2i0.1 75.5 f 8.7 68.4 f 3.4 49.3 2 3.5
0.7 i0.1 89.5 + 3.7 74.6 f 4.0 5 1.3f 4.3
0.3 + 0.1 80.1 f 3.9 70.2 2 3.9 52.2 f 4.4
0.3?0.1 88.3 f 5.0 74.1 2 5.6 57.6 ? 6.2
0.5+0.1
87.3 f 2.3 82.0f5.0 58.6 f 3.4
0.7f0.1 89.0 f4.2 71.053.8 62.9 f 5.8
dextrans of various sizes were allowed to diffuse into them. T h e larger FITCconjugated dextrans were partially excluded from both the cytoplasmic and nuclear compartments of all cell types. I n contrast, the lower molecular weight factors evenly permeated throughout most compartments of all the cell types, achieving 85-90",, of the concentration of fluorophore found outside the cells (Table 1). Reduced staining with carboxyfluorescein and the FITC-conjugated dextrans was most prominent in the nuclei, especially the nucleoli (e.g. Fig. 6A, B) and if quenching was occurring in the cytoplasmic compartments of the cell, it was quite small (e.g. compare the emission from carboxyfluorescein seen in Fig. 6A with the reflectance image showing the extent of the cytoplasmic compartment in Fig. 6C). I n all the cell types examined here, the nucleoli were densely packed with double stranded nucleic acids, visualized by ethidium bromide staining (Kasten, 1989; see Fig. 6E, F) and protein, visualized by the direct conjugation of the sulphonyl chloride, Texas red (e.g. Fig. 6G, H), or the isothiocyanates, F I T C (Fig. 6L) or R I T C (Fig. 6N) to the cells. This raised the possibility that failure to visualize carboxyfluorescein and F I T C conjugated to 4 kDa dextran in these regions was due to physical exclusion of the dye rather than quenching, and this was examined in more detail in one of the cell types, the rhabdomyosarcoma cells. FITC-conjugated dextrans and carboxyfluorescein were ideal for the quenching studies described above as they did not exhibit detectable specific binding to permeabilized cells (Table 1, Fig. 6D). Most other fluorophores were rejected a priori for this type of study as they were expected to exhibit some type of specific binding to permeabilized cells. Of many other fluorophores tested (including eosin, fluorescein and the other vital markers, RITC-conjugated dextrans, Sulphorhodamine 101 and Lucifer yellow) some degree of specific binding to permeabilized cells was detected. T o determine whether exclusion or quenching reduced visualization of carboxyfluorescein in nuclei and nucleoli, a second type of fluorescence ratio method was employed, where the relative quenching of F I T C was compared with a second fluorophore as an internal standard. Cells were fixed and permeabilized with methanol and then F I T C and R I T C directly conjugated to them at concentrations that were Fig. 5 . The rate of fading was even throughout human glioblastoma multiformae cells immunofluorescently labelled for intermediate filaments composed of glia fibrillary acidic protein. Images were collected when (A) O " , , , (D) 20",,, (GI 40",, and (J) 60",, of the original intensity was bleached away. The images were renormalized to give a constant mean value to bright regions: (B) O",,, (E) 20",,, (H) 40",, and (K) 60",, bleaching. These were subtracted from the re-normalized image at O",, bleaching, the pixel values multiplied tenfold and mapped to images where any variation in fading that was 1 1 standard deviation of the background was white and < 1 was black: ( C )O " , , , (F) 20",,, (I) 40",,,and (L) 60",, bleaching. Scale bar = 10 pm and the grey scale shows the relationship between recorded and presented grey level in the mapping function employed in images ( C ) ,(F),(I) and (L).
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previously found to give less than l o , of the maximal binding levels, thus avoiding competitive binding. Images were collected visualizing each stain in turn, the intensities of the two images were re-normalized to equalize the means and the images subtracted from each other. T h e result was then visualized in a form where if the difference between two pixels in the images was >5", of the modal value of the intensity values, they were displayed in white. If they were < 5",, they were displayed in black (Fig. 6J). It can then be seen that the pixels which deviate from the modal value by more than 5 ° , do not exhibit any discernible pattern indicating that if quenching was occurring it was not specifically associated with either the nuclei or the nucleoli of the cells. Additionally, a similar image was generated displaying pixels where the difference deviated from the modal value by greater than loo,, (not illustrated) and less than 1O C l of pixels were found to be outside that range. When cells stained with the appropriate concentration of only one of the fluorophores were examined, the level of F I T C signal found in the R I T C window was negligible (Fig. 6K), as was the level of R I T C signal found in the F I T C window (Fig. 6M). Comparison of F I T C and Texas red staining gave essentially identical results (data not shown). These findings demonstrated that there was no difference betwen the relative levels of quenching between F I T C and the two rhodamine-based fluorophores. Given the differences in structure and absorption spectra of the dyes, these results indicate that no detectable quenching was found in the cells.
DISCUSSION
We have analysed the utility of a C S L M for quantitative analysis of fluorescence. Quantification of fluorescence from both uniform films of fluorescein and non-uniform fluorescein distributions on antibody-labelled cells was readily achievable. Specifically, we demonstrate that a CSLM can be used to quantify fluorescence at specimen depths of up to 25 pm without any processing of the image data, apart from subtraction of the mean noise, providing that several conditions are fulfilled. (i) T h e detector must give a signal that is both continuous and linear with respect to the incident illumination (Entwistle & Noble, 1990). (ii) T h e samples should be checked to ensure that the level of fluorophore remains in the range where the relationship between the concentration of fluorophore and the level of fluorescent emission is linear. (iii) Depth-dependent effects like the inner-filter and re-absorption effects should be measured to ensure that they do not significantly affect the results. (iv) Samples should suffer as little fading as possible during the collection of the images and any necessary correction for fading must be straightforward. (v) A given amount of fluorophore must have the same luminosity over the entire field of view (a requirement which can present difficulties in poorly aligned scanning laser systems). (vi) Local quenching, for example due to changes in the
Fig. 6. Penetration and staining of fixed human rhabdomyosarcoma cells by fluorescent dyes. (A) Cells equilibrated with 100 I'M carboxyfluorescein in the mountant. (B) Phase contrast image of (A). (C) Reflectance image of (A). (D) Cells equilibrated with 100 PM carboxyfluorescein, rinsed thoroughly with buffer and remounted in plain mountant. (E) Cells stained with ethidium bromide. (F)Phase contrast image of (E). (G) Cells stained with Texas red. (H) Phase contrast image of (G). (I) Cells stained with F I T C and R I T C visualized through a green bandpass filter. (J) Cells seen in (I) but here an equivalent image collected with a red longpass filter was re-normalized to give the same mean intensity as the image shown in (I) and subtracted from (I). Pixels where the grey value is within 5",, of the modal value of the image shown in (I) are presented in grey. Where the difference was greater than 5 " , , of the mode, pixels were displayed in white, and where the difference was less than 5 " , , of the mode, pixels were displayed in black. (K), (L) Cells stained with F I T C visualizedwith the red longpass filter (K) and the green bandpass filter (L).(MI, (N) Cells stained with R I T C visualized with the green bandpass filter (Mj and the red longpass filter (N). Scale bar = 25 prn.
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hydrophobic/hydrophilic nature of the surrounding tissue, must be measured and appropriate correction factors applied where necessary. Fluorophore concentrations can be examined directly with the CSLM and the underlying relationship between fluorophore concentration and emission can always be found by examining appropriate standards. When distributed evenly, the relationship between fluorescent emission and fluorophore concentration is linear up to concentrations as high as 2 mM with both fluorescein derivatives and other fluorophores (Entwistle et al., 1990). Analysis of fluorophore concentrations above the range of linear response has been discussed in the previous paper (Entwistle & Noble, 1992). When quantifying fluorescence from, for example, immunofluorescently labelled samples, depth-dependent reduction in the apparent levels of fluorescent emission appeared to be negligible. Three different effects can give rise to a depth-dependent reduction in the intensity of detectable fluorescence. Inner-filter and re-absorption effects (Tanke et al., 1982) cause reductions in the levels of fluorescence detected that are both depth and concentration dependent. In addition, depth-dependent reductions can occur due to defocusing, which arises both from the change in refractive index at the glass cover slip-sample mountant interface (Carlsson, 1991) and, especially at greater depths, by absorption and scattering in the sample (Carlsson, 1991; Visser et al., 1991). With the concentrations of F I T C found in almost all immunofluorescently labelled samples the total reduction in fluorescent emission due to inner filtering, re-absorbance and defocusing by the boundary between the cover glass and the sample is less than 10°, up to a depth of 25 pm into the sample. In most quantitative biological studies this is small enough to be ignored and where correction is essential this can be achieved using two parameters that can be determined from the samples (Visser et al., 1991). Additionally, it appears from the results above that immunofluorescently labelled preparations meet a previously described requirement for quantitative fluorescence, that local absorbance of the fluorophore must be low (van Oostveldt & Bauwens, 1989). When absorption or scattering of light by the sample occur this results in a considerable reduction in image quality (Carlsson, 1991; Visser et al., 1991). With the confocal microscope employed here a noticeable reduction in image quality was only apparent at depths of 30-50 pm into samples (e.g. Entwistle et al., 1990) and the effects of absorption and scattering are probably negligible in most immunofluorescently stained biological samples at depths of up to 25 pm. Problems with fading can often be avoided or are easily compensated for with CSLMs. First, fading occurring in the collection of over fifty images, each the average of 100 passes of the laser from FITC-labelled samples, can be reduced to < lo",, where it can often be ignored. Reducing fading to this level requires careful alignment of the confocal optics, optimizing the illumination intensity, mounting the sample in a solution which reduces F I T C fading (Johnson et al., 1982), and ensuring that the exposure of the sample to the laser is kept to the essential minimum. Second, when some fading of the F I T C label is unavoidable, or when small losses must be corrected for, fading is essentially linear for the first 20°,, reduction in intensity and occurs evenly throughout the object. Large numbers of images can therefore easily be corrected on a pixel-by-pixel basis by multiplying each pixel in an image by a single factor. However, it is essential to determine the rate of fading for each experiment since some interexperimental variation can occur. Finally, some additional corrections may be needed if the fading of autofluorescence must also be taken into account; rapid algorithms are available to deal with this problem (Koppel et al., 1989). Working with an object field of even luminosity offers many advantages over a field of apparent uneven luminosity, although the latter condition can be compensated for mathematically. Quantitative data can readily be collected from evenly luminous fields without requirements for any image processing other than subtraction of background
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noise. I n .contrast, mathematical correction of images for uneven luminosity will generate variations in the dynamic range of the intensities over the field of view, thus distorting the signal-to-noise ratio. I n applications where noise must be minimized, such as estimation of polarization ratios, changes in the signal-to-noise ratios will distort the results. Additionally, any errors made during the calibration of the correction factors will be perpetuated throughout all images and could be seriously misleading when interpreting, for example, co-localization of fluorochromes in cells. Achieving even luminosity is critically dependent upon the precision of the alignment of the optical axes of the various components of the confocal microscope, and loss of alignment is quickly seen as an unevenness of luminosity across the field of view. Non-specific quenching by cells of various types from several different species does not present a problem when quantifying with the confocal microscope. An external standard method indicates that the levels of quenching within the cytoplasm of six different cell types are negligible. However, this method shows significant levels of fluorescence reduction in the nuclei of all the cells. This reduction could have been due to either quenching or dye exclusion. Using R I T C as an internal standard, when the distributions of F I T C and R I T C bound to the high levels of nuclear protein were compared they were virtually identical. Given the different nature of these two fluorophores, it is very unlikely that specific quenching of both fluorophores is occurring consistently in the same places. These results therefore indicate that quenching is not a general problem in the nuclei of cells. This in turn suggests that the lower levels of fluorescent emission seen with carboxyfluorescein and FITC-conjugated dextrans in the nuclei, and especially the nucleoli, were due to dye exclusion. Nevertheless, quenching should always be checked for when quantifying fluorescence in individual applications, and fluorophore ratio methods, especially those using an internal standard, are a simple and normally effective approach. Fluorescence polarization measurements demonstrate when the local concentration of fluorophore has exceeded the range where the relationship between fluorescent emission and fluorophore concentration can be assumed to be linear (Entwistle & Noble, 1992). Due to the high level of fluorophore localization expected with indirect immunofluorescent staining, this type of staining is ideal for examination of the usefulness of fluorescent polarization measurements in determining local fluorophore concentrations. With an excess of both the monoclonal primary antibody to a highly expressed epitope and the FITC-conjugated secondary antibody, the local concentration of fluorophore remained in the range where emission is linear with respect to concentration. I n contrast, in a similar experiment where a polyclonal antibody was employed as the primary antibody, the local concentrations of fluorophore exceeded the h e a r range. This suggests that depolarization of fluorescent emission reflects increasing concentration, since the density of binding of primary polyclonal antibodies is expected to be far higher than that observed with primary monoclonal antibodies. In addition to its role in quantification, analysing the polarization of the emitted fluorescence gives information about antibody distribution that cannot be otherwise obtained. In cells immunofluorescently labelled with polyclonal antibodies reactive with the astrocyte-specific intermediate filament, GFAP, the interior of the cells and ‘%ions of densely packed filaments were less intensely labelled and the polarization of the fluorescence in these regions was increased. Together these results demonstrate that the density of antibody binding along the filaments has been reduced, indicating that either the composition of the internal intermediate filaments is different or the antibodies fail to penetrate into these regions. It should be emphasized that the methods described here can only quantify the level Of fluorescence emitted by a specimen. Where the fluorophore visualized is part of a secondary structure (e.g. a multi-layer antibody system) used to visualize a primary
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component (e.g. an antigen), the absolute level of the primary component cannot be determined unless the stoichiometric relationship between the secondary structure and the primary component is known. Thus, determination of the absolute amount present of any specific molecule of interest requires information not contained within the specimens themselves. However, obtaining an accurate assay of the relative differences in fluorophore concentration between different specimens is straightforward, thereby allowing the ranking of specimens to be done with great accuracy. ACKNOWLEDGMENTS
T h e authors wish to thank K. Bevan for the human glioblastoma multiformae cells, G. Wolswijk for the oligodendrocytes and K. Bhakoo for the bovine endothelial cells. REFERENCES
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Steltzer, E.H.K. & Wijnaendts van Resantdt, R.W. (1987) Nondestructive sectioning of fixed and living specimen using a confocal scanning laser microscope: microtomoscopy. SPIE, 809, 140-137. Tanke, H.J., van Oostveldt, P. & van Duijn, P. (1982) A parameter for the distribution of fluorophores in cells derived from measurements of inner filter effect and reabsorption phenomena. Cytometry, 2,359-369. Visser, T.D., Groen, F.C.A. & Brakenhoff, G.J. (1991) Extinction correction in confocal microscopy. 3. Microsc. 163, 189-200. van der Voort, H.T.M. & Brakenhoff, G.J. (1990) 3-D image formation in high aperture fluorescence confocal microscopy: a numerical analysis. 3. Microsc. 158,43-54. White, J.G., Amos, W.B. & Fordham, M. (1987) An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. 3. Cell Biol. 105,41-48. Wilson, T . & Sheppard, C.J.R. (1984) Theory and Practice of Scanning Optical Microscopy. Academic Press, London. Wolswijk, G. & Noble, M. (1989) Identification of an adult specific glial progenitor cell. Development, 105, 387-400.
The quantification of fluorescent emission from biological samples using analysis of polarization
by A. E N T W I S T Land E M. NOBLE,The Ludwig Institute f o r Cancer Research, Courtauld Building, 91 Riding House Street, London W l P 8 B T , U.K.
KEY
w o R D s. Fluorescence quantification, immunofluorescence, confocal microscope.
SUMMARY
The quantification of fluorescent emission from biological specimens can only be carried out in cellular regions where the relationship between fluorophore concentration and fluorescent emission is linear. Using a confocal scanning laser microscope, we show that quantification of fluorescent emission from biological samples labelled with fluorescein and fluorescein analogues mounted in a viscous medium can be readily achieved. Where the distribution of fluorophore is highly localized, for example in cells labelled for immunofluorescence analysis, we demonstrate that analysis of fluorescence depolarization can identify regions in which fluorophore concentration exceeds the range in which the relationship to fluorescent emission is linear. We also demonstrate that, under the conditions examined, depth-dependent effects, fading and quenching are either small enough to be ignored or can be corrected for mathematically when quantifying fluorescent emission. INTRODUCTION
The widespread use of fluorescent compounds to visualize specific molecular structures, for example within cells, offers the opportunity to use quantitative analysis to obtain information about differences between two (or more) structures which are both fluorescently labelled. The quantification of fluorescence emission with conventional microscopes is frequently beset with problems, such as excessive fading (Johnson et al., 1982; Kasten, 1989), fading at different rates in different parts of the object (Benson et al., 1985), fading whose kinetics necessitates complex mathematical corrections (McKay et al., 1981; Koppel et al., 1989) and apparent local variations in quenching (Shapiro, 1988). Many of these problems arise from, or are exaggerated by, noise and out-of-focus signals collected over the large depth of field presented in conventional microscope images. Moreover, when quantifying fluorescence with conventional microscopes, it is difficult to analyse fluorescence with large volumes of fluorophore at concentrations p 1 0 j i (van ~ Oostveldt & Bauwens, 1989). These difficulties result from inner-filter and re-absorption effects (Tanke et al., 1982) over the large depth of field examined; the inner-filter effect is a progressive reduction in intensity of the stimulating illumination by the fluorophore with increasing depth, and re-absorption is the absorption of fluorescent emission from lower layers by upper layers. As confocal microscopes reject light that does not emanate from, or close to, the plane
84 in white. (D) Original image of cells shown in (C).(E),(F)Cells displayed in (D)where pixel intensity is 11-84 with polarization ratios (E)greater than or equal to 2 mM F I T C or (F)greater than or equal to 2 mM fluorescein, displayed in white and the remainder in black. (G),(H)Cells displayed in (D) where pixel intensity is 2 85 with polarization ratios (G)greater than or equal to 2 mM fluorescein, or (H) greater than or equal to 4 mM fluorescein, displayed in white and the remainder in black. Scale bar = 25 Ltm.
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absorption-purified FITC-conjugated polyclonal anti-mouse Ig antibody, and an excess of both the primary and secondary antibodies was used. A suitable cell was selected (Fig. 3A), pairs of images collected, and a final image was created in which pixels where the polarization ratio was less than expected (i.e. less than the polarization ratio from a 2 mM F I T C standard) were mapped to black and pixels where the polarization was greater than or equal to that expected were mapped to white. Regions where the total intensities indicated that the apparent polarization ratio was significantly affected by noise were mapped to grey (Fig. 3B); the majority of these pixels were clearly not parts of the cell (Fig. 3C). Of the pixels examined in the fainter regions of the cell, almost all ( > 99-9O,) were white, indicating that the polarization ratios were above those of a 2 mM F I T C standard and in the range where the relationship between fluorophore concentration and pixel grey level was linear (Fig. 3C, D). In the more intensely stained regions, a higher proportion of black pixels (%2c)0),where the polarization ratio was less than that of a 2 mM F I T C standard, was observed (Fig. 3C, D, upper left). I n a similar image generated to compare the values observed with those generated from a 2 mM fluorescein standard (not shown) the black pixels virtually disappeared ( < 0.1O 0 ) ; however, this level of polarization was very close to the levels where it was no longer possible to assume that fluorescent emission was linear with respect to fluorophore concentration (Entwistle & Noble, 1992). Unlike the results obtained with anti-GalC antibody, the polarization of the emitted light obtained when a polyclonal primary antibody, anti-GFAP, was used indicated that the pixel grey values observed could no longer be assumed to be linearly related to the concentration of F I T C . However, using the polarization ratio values, it was possible to discriminate between regions where quantification could and could not be carried out. An excess of both anti-GFAP and FITC-conjugated anti-rabbit Ig was used to label methanol-fixed cells derived from a human glioma (specifically, a glioblastoma multiformae). Pairs of images representing the two orientations of polarization were again collected but here the polarization ratios in two regions of different pixel intensity were processed separately. We first examined less intensely stained regions, where the absolute pixel grey level was between 11 and 84 (Fig. 3D, grey pixels). Then examination of the more intensely stained regions, where the grey level was above 84 (Fig. 3D, white pixels), was carried out. With the pixels in the range 11-84, polarization ratios less than those generated from a 2 mM F I T C standard predominated (Fig. 3E, black pixels). Indeed, for large numbers of these pixels the polarization ratio was less than those generated from a 2 mM fluorescein standard (Fig. 3F, black pixels), as seen in the diffuse processes close to the exterior of the middle regions to the bottom of the cell in the left of the image and the lower right of the cell in the right of the image. I t can also be seen in these images that the polarization ratios for regions of both diffuse and dense fibres in the interior of the cell were generally equal to or greater than those from a 2 mM fluorescein standard (Fig. 3F, white pixels) and frequently equal to or greater than those from a 2 mM F I T C standard (Fig. 3G, white pixels), suggesting that the local concentration of antibody binding was lower in the interior regions than in the peripheral regions. With pixels where the grey level was > 84 (Fig. 3C, white), many pixels in both of the cells shown in Fig. 3(C) had polarization ratios that were less than a 2 mM fluorescein standard (Fig. 3G, black pixels) and some ratios were less than a 4 mM fluorescein standard (Fig. 3H, black pixels); these results indicated that the local concentration in the very bright regions greatly exceeded the range where a linear response with respect to fluorescent emission can be demonstrated. Fading With many immunohistochemically stained preparations, quantification of large data
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sets was possible without correcting for fading since the loss in intensity was small ( < 10°o)even when a large number of images ( 350), each the average of many scans
(loo), was collected. For example, the complex and delicate network of intermediate filaments in a human glioblastoma multiformae cell was clearly visible in sixty sequential images. Vertical steps of 0.2 pm were made between each image, and each image was generated by averaging the data from scans giving a total laser exposure of ~ 0 . nWs 4 pixel-'. Despite the low laser intensities employed, the collection of highcontrast images where the filamentous nature of the staining was clearly visible was easily achieved (Fig. 4A-G). Images collected at the same plane through the cell ( & 0.2 pm), but before and after a total laser exposure of z 21 nWs pixel- (fifty images), were very similar (Fig. 4A, H) and quantitative measurements indicated that the fading was slight (i.e. < loo,,). Even when data sets large enough to induce obvious fading were required, the fading of FITC: was approximately linear over the first part of the decay process making correction simple. Intensity of emission plotted against the total exposure to the laser gave curves that were fitted by a straight line over the first 20°, of the decay process, in twenty different experiments under various conditions; and in all of these experiments, the correlation coefficient R2 was > 0.98. When correcting for fading, corrections for different rates of fading across the image field were not required as the rate was uniform throughout the object even when preparations with a complex structure were examined. Immunofluorescently stained GFAP filaments in human glioblastoma multiformae cells were again imaged (Fig. 5A) and cells were then exposed to the laser until approximately 80, 60 and 40", of the original F I T C intensity remained (Fig. 5D, G, J, respectively). T h e imaged files were renormalized to a constant mean intensity to permit direct visual comparison (Fig. 5B, E, H, K). T h e pixel values in the re-normalized image files were then subtracted from the pixel values in the equivalent image file at time zero (Fig. 5B). T h e resulting files were mapped to a new image where pixels in the re-normalized images (Fig. 5B, E, H, K) that varied from the expected value by more than 1 standard deviation of the background were mapped to white when above the mean and black when below the mean. Pixels within 2 0.33 of the standard deviation of the background were mapped to grey, and values between 0.33 and 1 standard deviation were scaled appropriately. Pixels where the fluorescence in the initial image (Fig. 5B) was less than three times above the background were also mapped to grey. Although significant variation was found in some pixels, there did not appear to be any underlying pattern to their distribution and their position was not constant from image to image. Where a pattern was evident (e.g. Fig. 51, bottom left) the alternation of black and white regions was characteristic of movement of the object following, for example, slight movement ( < 1 pm) of the stage. T h e complete data set used to generate these images took z 80 min to collect and over this time period slight loss of registration was not surprising.
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Quenchi~g Non-specific quenching of fluorescence was not a general problem when quantifying fluorescent emission with a confocal microscope. T o examine quenching, two different ratio methods had to be employed. T h e first compared the level of fluorescence within a sample with an external standard taken outside the cell (where no sample-induced quenching was present). For example, with the six different cell preparations used here (HeLa cells, rat embryo fibroblasts immortalized with simian virus 40 large-T antigen, human rhabdomyosarcoma cells, primary chick myogenic cultures, bovine endothelial cells and primary cultures of cells derived from optic nerves of 7-day-old rats) a region of medium immediately adjacent to the cell under examination was utilized as an external standard. Cultures were fixed and carboxyfluorescein or FITC-conjugated
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Fig. 4. A human glioblastoma multiformae cell immunofluorescently labelled for intermediate filaments with low laser intensities. Sixty images were collected at 0.2-pm vertical intervals, each the average of 100 laser scans. (A-G) Images collected at 1.6-pm intervals starting from just above the cover slip (A). (H) An image collected at the end of the series at the same level i 0.2 Ltm as image (A). Scale bar = 25 pm.
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Table 1. The apparent level of fluorophore found inside six different cell types following exposure to fluorophores of different molecular weights. Cell type
Fluorophore Carboxyfluorescein Carboxyfluorescein (post-rinse) FITC-4 kDa dextran FITC-40 kDa dextran FITC-400 kDa dextran
HeLa cells
Rat embryo fibroblasts
Rhabdomyosarcoma cells
Chick myogenic cultures
Bovine endothelial cells
Rat optic nerve cultures
85.7 2 1.6
86.6 + 1.6
81.1 t 1.8
90.9 i 6.0
87.9 f 3.5
89.0 2 3.1
0.2i0.1 75.5 f 8.7 68.4 f 3.4 49.3 2 3.5
0.7 i0.1 89.5 + 3.7 74.6 f 4.0 5 1.3f 4.3
0.3 + 0.1 80.1 f 3.9 70.2 2 3.9 52.2 f 4.4
0.3?0.1 88.3 f 5.0 74.1 2 5.6 57.6 ? 6.2
0.5+0.1
87.3 f 2.3 82.0f5.0 58.6 f 3.4
0.7f0.1 89.0 f4.2 71.053.8 62.9 f 5.8
dextrans of various sizes were allowed to diffuse into them. T h e larger FITCconjugated dextrans were partially excluded from both the cytoplasmic and nuclear compartments of all cell types. I n contrast, the lower molecular weight factors evenly permeated throughout most compartments of all the cell types, achieving 85-90",, of the concentration of fluorophore found outside the cells (Table 1). Reduced staining with carboxyfluorescein and the FITC-conjugated dextrans was most prominent in the nuclei, especially the nucleoli (e.g. Fig. 6A, B) and if quenching was occurring in the cytoplasmic compartments of the cell, it was quite small (e.g. compare the emission from carboxyfluorescein seen in Fig. 6A with the reflectance image showing the extent of the cytoplasmic compartment in Fig. 6C). I n all the cell types examined here, the nucleoli were densely packed with double stranded nucleic acids, visualized by ethidium bromide staining (Kasten, 1989; see Fig. 6E, F) and protein, visualized by the direct conjugation of the sulphonyl chloride, Texas red (e.g. Fig. 6G, H), or the isothiocyanates, F I T C (Fig. 6L) or R I T C (Fig. 6N) to the cells. This raised the possibility that failure to visualize carboxyfluorescein and F I T C conjugated to 4 kDa dextran in these regions was due to physical exclusion of the dye rather than quenching, and this was examined in more detail in one of the cell types, the rhabdomyosarcoma cells. FITC-conjugated dextrans and carboxyfluorescein were ideal for the quenching studies described above as they did not exhibit detectable specific binding to permeabilized cells (Table 1, Fig. 6D). Most other fluorophores were rejected a priori for this type of study as they were expected to exhibit some type of specific binding to permeabilized cells. Of many other fluorophores tested (including eosin, fluorescein and the other vital markers, RITC-conjugated dextrans, Sulphorhodamine 101 and Lucifer yellow) some degree of specific binding to permeabilized cells was detected. T o determine whether exclusion or quenching reduced visualization of carboxyfluorescein in nuclei and nucleoli, a second type of fluorescence ratio method was employed, where the relative quenching of F I T C was compared with a second fluorophore as an internal standard. Cells were fixed and permeabilized with methanol and then F I T C and R I T C directly conjugated to them at concentrations that were Fig. 5 . The rate of fading was even throughout human glioblastoma multiformae cells immunofluorescently labelled for intermediate filaments composed of glia fibrillary acidic protein. Images were collected when (A) O " , , , (D) 20",,, (GI 40",, and (J) 60",, of the original intensity was bleached away. The images were renormalized to give a constant mean value to bright regions: (B) O",,, (E) 20",,, (H) 40",, and (K) 60",, bleaching. These were subtracted from the re-normalized image at O",, bleaching, the pixel values multiplied tenfold and mapped to images where any variation in fading that was 1 1 standard deviation of the background was white and < 1 was black: ( C )O " , , , (F) 20",,, (I) 40",,,and (L) 60",, bleaching. Scale bar = 10 pm and the grey scale shows the relationship between recorded and presented grey level in the mapping function employed in images ( C ) ,(F),(I) and (L).
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previously found to give less than l o , of the maximal binding levels, thus avoiding competitive binding. Images were collected visualizing each stain in turn, the intensities of the two images were re-normalized to equalize the means and the images subtracted from each other. T h e result was then visualized in a form where if the difference between two pixels in the images was >5", of the modal value of the intensity values, they were displayed in white. If they were < 5",, they were displayed in black (Fig. 6J). It can then be seen that the pixels which deviate from the modal value by more than 5 ° , do not exhibit any discernible pattern indicating that if quenching was occurring it was not specifically associated with either the nuclei or the nucleoli of the cells. Additionally, a similar image was generated displaying pixels where the difference deviated from the modal value by greater than loo,, (not illustrated) and less than 1O C l of pixels were found to be outside that range. When cells stained with the appropriate concentration of only one of the fluorophores were examined, the level of F I T C signal found in the R I T C window was negligible (Fig. 6K), as was the level of R I T C signal found in the F I T C window (Fig. 6M). Comparison of F I T C and Texas red staining gave essentially identical results (data not shown). These findings demonstrated that there was no difference betwen the relative levels of quenching between F I T C and the two rhodamine-based fluorophores. Given the differences in structure and absorption spectra of the dyes, these results indicate that no detectable quenching was found in the cells.
DISCUSSION
We have analysed the utility of a C S L M for quantitative analysis of fluorescence. Quantification of fluorescence from both uniform films of fluorescein and non-uniform fluorescein distributions on antibody-labelled cells was readily achievable. Specifically, we demonstrate that a CSLM can be used to quantify fluorescence at specimen depths of up to 25 pm without any processing of the image data, apart from subtraction of the mean noise, providing that several conditions are fulfilled. (i) T h e detector must give a signal that is both continuous and linear with respect to the incident illumination (Entwistle & Noble, 1990). (ii) T h e samples should be checked to ensure that the level of fluorophore remains in the range where the relationship between the concentration of fluorophore and the level of fluorescent emission is linear. (iii) Depth-dependent effects like the inner-filter and re-absorption effects should be measured to ensure that they do not significantly affect the results. (iv) Samples should suffer as little fading as possible during the collection of the images and any necessary correction for fading must be straightforward. (v) A given amount of fluorophore must have the same luminosity over the entire field of view (a requirement which can present difficulties in poorly aligned scanning laser systems). (vi) Local quenching, for example due to changes in the
Fig. 6. Penetration and staining of fixed human rhabdomyosarcoma cells by fluorescent dyes. (A) Cells equilibrated with 100 I'M carboxyfluorescein in the mountant. (B) Phase contrast image of (A). (C) Reflectance image of (A). (D) Cells equilibrated with 100 PM carboxyfluorescein, rinsed thoroughly with buffer and remounted in plain mountant. (E) Cells stained with ethidium bromide. (F)Phase contrast image of (E). (G) Cells stained with Texas red. (H) Phase contrast image of (G). (I) Cells stained with F I T C and R I T C visualized through a green bandpass filter. (J) Cells seen in (I) but here an equivalent image collected with a red longpass filter was re-normalized to give the same mean intensity as the image shown in (I) and subtracted from (I). Pixels where the grey value is within 5",, of the modal value of the image shown in (I) are presented in grey. Where the difference was greater than 5 " , , of the mode, pixels were displayed in white, and where the difference was less than 5 " , , of the mode, pixels were displayed in black. (K), (L) Cells stained with F I T C visualizedwith the red longpass filter (K) and the green bandpass filter (L).(MI, (N) Cells stained with R I T C visualized with the green bandpass filter (Mj and the red longpass filter (N). Scale bar = 25 prn.
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hydrophobic/hydrophilic nature of the surrounding tissue, must be measured and appropriate correction factors applied where necessary. Fluorophore concentrations can be examined directly with the CSLM and the underlying relationship between fluorophore concentration and emission can always be found by examining appropriate standards. When distributed evenly, the relationship between fluorescent emission and fluorophore concentration is linear up to concentrations as high as 2 mM with both fluorescein derivatives and other fluorophores (Entwistle et al., 1990). Analysis of fluorophore concentrations above the range of linear response has been discussed in the previous paper (Entwistle & Noble, 1992). When quantifying fluorescence from, for example, immunofluorescently labelled samples, depth-dependent reduction in the apparent levels of fluorescent emission appeared to be negligible. Three different effects can give rise to a depth-dependent reduction in the intensity of detectable fluorescence. Inner-filter and re-absorption effects (Tanke et al., 1982) cause reductions in the levels of fluorescence detected that are both depth and concentration dependent. In addition, depth-dependent reductions can occur due to defocusing, which arises both from the change in refractive index at the glass cover slip-sample mountant interface (Carlsson, 1991) and, especially at greater depths, by absorption and scattering in the sample (Carlsson, 1991; Visser et al., 1991). With the concentrations of F I T C found in almost all immunofluorescently labelled samples the total reduction in fluorescent emission due to inner filtering, re-absorbance and defocusing by the boundary between the cover glass and the sample is less than 10°, up to a depth of 25 pm into the sample. In most quantitative biological studies this is small enough to be ignored and where correction is essential this can be achieved using two parameters that can be determined from the samples (Visser et al., 1991). Additionally, it appears from the results above that immunofluorescently labelled preparations meet a previously described requirement for quantitative fluorescence, that local absorbance of the fluorophore must be low (van Oostveldt & Bauwens, 1989). When absorption or scattering of light by the sample occur this results in a considerable reduction in image quality (Carlsson, 1991; Visser et al., 1991). With the confocal microscope employed here a noticeable reduction in image quality was only apparent at depths of 30-50 pm into samples (e.g. Entwistle et al., 1990) and the effects of absorption and scattering are probably negligible in most immunofluorescently stained biological samples at depths of up to 25 pm. Problems with fading can often be avoided or are easily compensated for with CSLMs. First, fading occurring in the collection of over fifty images, each the average of 100 passes of the laser from FITC-labelled samples, can be reduced to < lo",, where it can often be ignored. Reducing fading to this level requires careful alignment of the confocal optics, optimizing the illumination intensity, mounting the sample in a solution which reduces F I T C fading (Johnson et al., 1982), and ensuring that the exposure of the sample to the laser is kept to the essential minimum. Second, when some fading of the F I T C label is unavoidable, or when small losses must be corrected for, fading is essentially linear for the first 20°,, reduction in intensity and occurs evenly throughout the object. Large numbers of images can therefore easily be corrected on a pixel-by-pixel basis by multiplying each pixel in an image by a single factor. However, it is essential to determine the rate of fading for each experiment since some interexperimental variation can occur. Finally, some additional corrections may be needed if the fading of autofluorescence must also be taken into account; rapid algorithms are available to deal with this problem (Koppel et al., 1989). Working with an object field of even luminosity offers many advantages over a field of apparent uneven luminosity, although the latter condition can be compensated for mathematically. Quantitative data can readily be collected from evenly luminous fields without requirements for any image processing other than subtraction of background
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noise. I n .contrast, mathematical correction of images for uneven luminosity will generate variations in the dynamic range of the intensities over the field of view, thus distorting the signal-to-noise ratio. I n applications where noise must be minimized, such as estimation of polarization ratios, changes in the signal-to-noise ratios will distort the results. Additionally, any errors made during the calibration of the correction factors will be perpetuated throughout all images and could be seriously misleading when interpreting, for example, co-localization of fluorochromes in cells. Achieving even luminosity is critically dependent upon the precision of the alignment of the optical axes of the various components of the confocal microscope, and loss of alignment is quickly seen as an unevenness of luminosity across the field of view. Non-specific quenching by cells of various types from several different species does not present a problem when quantifying with the confocal microscope. An external standard method indicates that the levels of quenching within the cytoplasm of six different cell types are negligible. However, this method shows significant levels of fluorescence reduction in the nuclei of all the cells. This reduction could have been due to either quenching or dye exclusion. Using R I T C as an internal standard, when the distributions of F I T C and R I T C bound to the high levels of nuclear protein were compared they were virtually identical. Given the different nature of these two fluorophores, it is very unlikely that specific quenching of both fluorophores is occurring consistently in the same places. These results therefore indicate that quenching is not a general problem in the nuclei of cells. This in turn suggests that the lower levels of fluorescent emission seen with carboxyfluorescein and FITC-conjugated dextrans in the nuclei, and especially the nucleoli, were due to dye exclusion. Nevertheless, quenching should always be checked for when quantifying fluorescence in individual applications, and fluorophore ratio methods, especially those using an internal standard, are a simple and normally effective approach. Fluorescence polarization measurements demonstrate when the local concentration of fluorophore has exceeded the range where the relationship between fluorescent emission and fluorophore concentration can be assumed to be linear (Entwistle & Noble, 1992). Due to the high level of fluorophore localization expected with indirect immunofluorescent staining, this type of staining is ideal for examination of the usefulness of fluorescent polarization measurements in determining local fluorophore concentrations. With an excess of both the monoclonal primary antibody to a highly expressed epitope and the FITC-conjugated secondary antibody, the local concentration of fluorophore remained in the range where emission is linear with respect to concentration. I n contrast, in a similar experiment where a polyclonal antibody was employed as the primary antibody, the local concentrations of fluorophore exceeded the h e a r range. This suggests that depolarization of fluorescent emission reflects increasing concentration, since the density of binding of primary polyclonal antibodies is expected to be far higher than that observed with primary monoclonal antibodies. In addition to its role in quantification, analysing the polarization of the emitted fluorescence gives information about antibody distribution that cannot be otherwise obtained. In cells immunofluorescently labelled with polyclonal antibodies reactive with the astrocyte-specific intermediate filament, GFAP, the interior of the cells and ‘%ions of densely packed filaments were less intensely labelled and the polarization of the fluorescence in these regions was increased. Together these results demonstrate that the density of antibody binding along the filaments has been reduced, indicating that either the composition of the internal intermediate filaments is different or the antibodies fail to penetrate into these regions. It should be emphasized that the methods described here can only quantify the level Of fluorescence emitted by a specimen. Where the fluorophore visualized is part of a secondary structure (e.g. a multi-layer antibody system) used to visualize a primary
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component (e.g. an antigen), the absolute level of the primary component cannot be determined unless the stoichiometric relationship between the secondary structure and the primary component is known. Thus, determination of the absolute amount present of any specific molecule of interest requires information not contained within the specimens themselves. However, obtaining an accurate assay of the relative differences in fluorophore concentration between different specimens is straightforward, thereby allowing the ranking of specimens to be done with great accuracy. ACKNOWLEDGMENTS
T h e authors wish to thank K. Bevan for the human glioblastoma multiformae cells, G. Wolswijk for the oligodendrocytes and K. Bhakoo for the bovine endothelial cells. REFERENCES
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Steltzer, E.H.K. & Wijnaendts van Resantdt, R.W. (1987) Nondestructive sectioning of fixed and living specimen using a confocal scanning laser microscope: microtomoscopy. SPIE, 809, 140-137. Tanke, H.J., van Oostveldt, P. & van Duijn, P. (1982) A parameter for the distribution of fluorophores in cells derived from measurements of inner filter effect and reabsorption phenomena. Cytometry, 2,359-369. Visser, T.D., Groen, F.C.A. & Brakenhoff, G.J. (1991) Extinction correction in confocal microscopy. 3. Microsc. 163, 189-200. van der Voort, H.T.M. & Brakenhoff, G.J. (1990) 3-D image formation in high aperture fluorescence confocal microscopy: a numerical analysis. 3. Microsc. 158,43-54. White, J.G., Amos, W.B. & Fordham, M. (1987) An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. 3. Cell Biol. 105,41-48. Wilson, T . & Sheppard, C.J.R. (1984) Theory and Practice of Scanning Optical Microscopy. Academic Press, London. Wolswijk, G. & Noble, M. (1989) Identification of an adult specific glial progenitor cell. Development, 105, 387-400.
