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Bridging the Resolution Gap: Imaging the Same Transcription Factories in Cryosections by Light and Electron Microscopy
Ana Pomboa, Michael Hollinsheada, and Peter R. Cookaa Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
Correspondence to: Peter R. Cook, Sir William Dunn Schl. of Pathology, U. of Oxford, South Park Road, Oxford OX1 3RE, UK.
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Summary |
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 Top Summary IntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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The resolution of conventional light microscopy is limited to ~200 nm in the x- and y-axes and >500 nm in the z-axis. A simple way of improving z-axis resolution is to analyze thin sections of 100–200 nm. The utility of such an approach is illustrated by reference to transcription sites imaged in cryosections of human nuclei. Cells are permeabilized, allowed to extend nascent transcripts in Br-UTP, fixed, cryosectioned, and Br-RNA-immunolabeled with fluorochromes and gold particles. As expected, physical sectioning improves resolution and brings other advantages. First, sections allow improved antibody access and better immunolabeling. Second, more sites (with a more representative range of intensities) can now be resolved against lower backgrounds, facilitating quantitative analysis. Third, problems associated with chromatic aberration when two differently colored images of the same objects are collected can be sidestepped by refocusing between image collection. Fourth, exactly the same sites can be imaged by light and electron microscopy, allowing direct comparison between the two techniques. Immunogold labeling and electron microscopy provided the most accurate counts of site number. The results confirm that nascent transcripts in the nucleoplasm are confined to several thousand sites, or "factories," with diameters of ~40 nm. (J Histochem Cytochem 47:471–480, 1999)
Key Words: BrUTP, CLSM, cryosection, EM, fluorescent–gold immunoprobe, immunofluorescence, Tokuyasu, transcription factories
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Introduction |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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The resolution of the conventional light microscope is at best ~200 nm in the x- and y-axes, and 500 nm or more in the z-axis (
Sites of transcription in mammalian nuclei have traditionally been imaged after growing cells for short periods in [3H]-U and then localizing the resulting [3H]-RNA by autoradiography. However, it was difficult to resolve individual extranucleolar sites because pulses giving sufficient incorporation for detection often allowed time for completed transcripts to move away from synthetic sites, and the resolution of autoradiography is poor (silver grains can lie >100 nm away from the tritium source)(
Figure 1 illustrates the problems encountered in imaging and then counting objects like these transcription sites. In a conventional light microscope, an object of ~50 nm diameter appears wider (i.e., >200 nm) and more elongated (i.e., >500 nm) than it truly is (Figure 1A). This makes it difficult to distinguish faint objects in the optical section from out-of-focus flare from brighter objects lying above or below, and to resolve an object lying immediately above another in the same optical section. In the confocal microscope, objects appear both narrower and less elongated, and optical sectioning reduces but does not eliminate flare (Figure 1A, confocal). The precise appearance depends on the point-spread functions associated with the different microscopes (
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Sections of the appropriate thickness (i.e., 100–200 nm) can be prepared from whole cells by infiltration with a medium that hardens sufficiently (e.g., by polymerization or freezing) to permit sectioning. We used an approach borrowed from electron microscopy—freezing, cryosectioning, coupled to "dry knife, wet retrieval" (
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Materials and Methods |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Transcription Reactions
HeLa cells were generally grown as suspension cultures (
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Cryosections
Cryosections were prepared using a modification of existing methods (
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Immunolabeling and Imaging
Before addition of antibodies, free aldehyde groups were quenched (20 min) with 25 mM glycine in PBS. Then whole cells on coverslips were treated with 0.5% Triton X-100 in PBS for 20 min and cryosections with 0.1% Triton X-100 in PBS for 2 min. After washing in PBS and incubation (30 min) in PBS + 1% BSA + 0.2% fish gelatin (PBS+), Br-RNA was indirectly immunolabeled (1–2 hr) using a mouse monoclonal anti-Br-dU (Boehringer Mannheim; Lewes, East Sussex, UK) as a primary antibody diluted in PBS+ (pH 8.0) to 1 µg/ml (whole cells) or 5 µg/ml (cryosections), and washed in PBS+ (1 hr). (Antibodies were titrated and the maximal concentration that did not give significant background was used.)
For Figure 2, samples were incubated (1–2 hr) with a donkey anti-mouse IgG conjugated with FITC (fluorescein isothiocyanate, 10 µg/ml; multiple-labeling grade from Jackson ImmunoResearch Laboratories, West Grove, PA), washed (1 hr) in PBS+ and then in PBS supplemented with 0.1% Tween 20, incubated in TOTO-3 (20 µM in PBS/Tween for 20 min; Molecular Probes, Eugene, OR), washed successively in PBS/Tween and PBS, and mounted in Vectashield (Vector Laboratories; Peterborough, UK) on an England finder (BDH; Poole, Dorset, UK). Images shown in Figure 2A and Figure 2C were collected using a CCD camera (Hamamatsu Photonics C4742; Hamamatsu City, Japan; 1000 x 1018 pixels, 10-bit images, pixel size 12 µm), run under Image v1.41 (NIH, Bethesda, MD), and a x 63 Plan-Neofluar objective (NA 1.4) on a Zeiss Axioplan microscope (Carl Zeiss-Oberkochen; Welwyn Garden City, Herts, UK). Images shown in Figure 2B and Figure 2D were collected using a confocal microscope (hybrid BioRad MRC-1000/1024; Hemel Hempstead, Herts, UK) running under Comos software (v. 7.0a), and fitted with an argon/krypton laser, a x 60 PlanApo objective (NA 1.4), and a Nikon Diaphot 200 inverted microscope. Images were collected with a closed iris (to obtain best resolution), Kalman filtering (n = 6–10), and the minimal laser power that filled the whole gray scale in the low scan/low signal mode.
For Figure 3, immunolabeling was as above, except that two secondary antibodies were used together—the IgG–FITC described above and a donkey anti-mouse IgG conjugated with Cy3 (iodocarbocyanine, 1 µg/ml; multiple-labeling grade from Jackson ImmunoResearch)—and images were collected sequentially on the confocal microscope (as above).
For Figure 4, Br-RNA was indirectly immunolabeled using the mouse anti-Br-dU antibody, followed by rabbit anti-mouse IgG (1 hr, 1 µg/ml; Cappel Laboratories, supplied through Organon Teknika, Turnhout, Belgium) and then FITC–protein A adsorbed to 6-nm gold particles (3 hr, 1:100 dilution); the conjugate was made as described by
For Figure 5, Br-RNA on grids was immunolabeled and treated with glutaraldehyde as for Figure 4. Then grids were washed eight times in water, incubated (4C, 10 min) with 0.3% uranyl acetate in 2% methylcellulose, captured on a wire loop, excess methylcellulose blotted onto a filter paper, recovered, and imaged in the electron microscope.
Quantitative Analysis
For light microscopy, images were transferred to Adobe Photoshop (Adobe Systems; Edinburgh, UK), contrast-stretched without processing in any other way, nucleoplasmic regions identified by reference to TOTO-3 staining, and foci within them counted as described by
For electron microscopy, a cluster of gold particles was defined as one containing >1 particle lying within 40 nm of another (center-to-center distance; 
(xy). The average diameter, D, of the underlying sites marked by these clusters was determined using the sequential-subtraction method (
r (i.e., 5 nm) with a maximal radius, ri (i.e., 5 nm, 10 nm, 15 nm). The frequency (f) with which circles with different radii are seen depends on section thickness (T), sphere radius, and bin size. For a population of single-sized spheres, the frequency of complete spheres and caps in each bin is determined from the following expressions. When ri = R,
When ri < R,
The mean diameter of a population of spheres with differing radii is deduced by sequential subtraction. Starting with the largest bin, the contribution of polar caps arising from spheres with this diameter is calculated and subtracted from the smaller categories; then the next largest bin is treated in the same way, and so on.
Numbers of sites in 3-D space in a nucleus were calculated as follows (
where T is the section thickness, and (c) the number of sites per nucleus calculated with knowledge of nucleoplasmic volume. The volume of nucleoplasm (i.e., 554 ± 163 µm3; range 420–921; n = 11) in permeabilized HeLa cells was determined using Adobe Photoshop from serial optical sections collected at nominal 0.4-µm intervals through whole cells stained with TOTO-3. Each voxel had x and y dimensions of 45 nm (calibrated according to BioRad's instructions), and a z dimension of 0.39 µm and not 0.4 µm (determined by reference to 10-µm latex Nile Red FluoroSpheres from Molecular Probes, as described by
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Results |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Improving Z-axis Resolution of the Light Microscope
The improved resolution given by sections is illustrated in Figure 2. HeLa cells were permeabilized in a physiological buffer, allowed to extend nascent transcripts by ~1100 nucleotides in the presence of Br-UTP, fixed, and Br-RNA indirectly immunolabeled with FITC. All four cellular RNA polymerases incorporate the analogue (unpublished data), so Br-RNA is found in nucleoli (made by polymerase I), the nucleoplasm (made by polymerases II and III), and the cytoplasm (made by the mitochondrial polymerase). When a whole cell is viewed with a conventional microscope, fluorescence (marking Br-RNA) appears distributed throughout the cell in a complex pattern. Bright foci in the nucleolus and nucleoplasm are seen against a diffuse nucleoplasmic signal, and these are surrounded by a few mitochondrial foci (Figure 2A). Here (and in all other fluorescence images shown), the primary image has been stretched to fill the gray scale but not treated in any other way. As a result, foci appear more blurred than those presented after subtracting "background" (e.g.,
Minimizing Chromatic Aberrations when Doubly-labeled sites Are Imaged
Multiple fluorochromes are often used to localize different objects within cells. The approach requires that there is little misregistration or difference in magnification between the two resulting images. This is difficult to achieve in practice because fluorochromes emit light of different wavelengths, which is diffracted to different degrees (
Br-RNA in cryosections was labeled with both red and green fluorochromes. After focusing on sites through a red filter, an image was collected (Figure 3A). If the same sites are then imaged using the green filter, they appear more blurred (Figure 3B). They become sharper after refocusing (Figure 3C), and changing back to the red filter then defocuses them again (not shown). We compared foci in Figure 3A–C to gauge how great the differences in misregistration, magnification, and brightness were in these thin sections. Such comparison is complicated by the fact that antibodies bind to different sites in differing ratios; some in-focus foci appear slightly brighter when viewed through the green filter (e.g., focus 1), others through the red filter (e.g., focus 2). (This also indicates that sites are detected with similar sensitivities using the two fluorochromes.) Switching filters leads to some increase in the size of sites, with adjacent sites apparently fusing (e.g., adjacent foci 3); some faint sites may even be lost (e.g., focus 4). A combination of both effects leads to a ~20% reduction in the number of sites counted after switching filters without refocusing. Roughly equal numbers are seen with refocusing.
It remained formally possible that starting and stopping scans, and/or the z-axis movements required for refocusing, might alter x–y positions, so we tested whether this was so. First, we established how x–y position varied after collecting a series of images like those in Figure 3A without any refocusing. After contrast-stretching to fill the gray scale and subtraction of background noise (the average intensity outside cells), the center of gravity of the remaining foci (defined as 
2 touching pixels that contained any intensity) was determined (not shown). The centers of gravity of only 8% foci moved by >1 pixel from scan to scan, and this movement was not in any one direction. After refocusing between scans (as in Figure 3A and Figure 3C), no further movement was seen (i.e., the center of gravity of only 6% foci moved by >1 pixel). These results show that refocusing has no detectable effect on x–y registration in our system and that this approach minimizes the chromatic aberrations associated with imaging multiply-labeled sites.
Imaging the Same Sites by Light and Electron Microscopy
Because the cryosectioning technique was borrowed from electron microscopy, we investigated whether it was possible to image exactly the same sites by both light and electron microscopy. For this purpose, we prepared a probe labeled with both a fluorochrome and gold particle (see Materials and Methods). This reagent allows direct correlation without the need for amplification. Because large gold probes apparently do not penetrate efficiently into cryosections (
10 nm penetrate as efficiently as immunoglobulin–fluorochrome conjugates (see Materials and Methods). Sections containing Br-RNA were prepared as before, incubated with FITC–protein A adsorbed onto 6-nm gold particles, and fluorescence images collected on a confocal microscope (Figure 4A–C). Next, the embedding resin used for electron microscopy was poured onto the section, allowed to set, and the resulting block re-sectioned so that the first section (~150 nm) contained the original cryosection. Finally, the same area was identified and an image was collected using the electron microscope (Figure 4D and Figure 4E). Transcription sites were seen either as fluorescent foci or as clusters of gold particles. These clusters appear in stereo views to be spread throughout the thickness of the cryosection (not shown). Foci and clusters co-localize almost perfectly (e.g., Figure 4F). Moreover, lone gold particles did not always give foci visible by light microscopy, and two gold particles gave visible foci if they lay within 100 nm of each other (not shown). (Note that even if gold particles quench fluorescence, there remains sufficient to enable all clusters of gold particles to be detected by light microscopy.)
We next compared the strengths and weaknesses of the different approaches used to analyze light and electron micrographs. Consider, for example, how the number of transcription sites in a nucleus would be determined. In analyzing fluorescence images like that in Figure 4C, we are confronted with the problem of how to define a focus. We must make (often arbitrary) decisions on how many contiguous pixels are needed to constitute a focus, where to draw the threshold between signal and noise, and whether two peaks of intensity are part of one extended focus. In practice, most observers agree that the image contains the five foci shown by the ovals in Figure 4F, largely because their eyes and brains are so well adapted to analyze complex images (1 particle/µm2, representing 6% total number of particles) is found in controls incubated without Br-UTP and outside of cells (not shown). Using this simple but rigorous definition of what constitutes a cluster, Figure 4E is found to contain six clusters, which can be seen more clearly in Figure 4F. Not surprisingly, the electron microscope resolves some foci into more than one cluster (e.g., two clusters are contained within the large focus at the top center in Figure 4C). Further analysis of 48 foci showed that 39 contained one cluster of particles, eight contained two, and one contained three (not shown). As a result, the number of sites in such samples is only underestimated by ~17% in the light microscope (not shown). The density of sites seen by electron microscopy (i.e., 1.6 cluster/µm2) corresponds to 6500 extranucleolar sites/nucleus, taking into account the average size of clusters (see below) and nucleoplasmic volume (see Materials and Methods).
These results demonstrate that the same objects can be imaged by both light and electron microscopy without amplification. They also show that electron microscopy can yield the most accurate counts.
Diameter of Transcription Sites Determined by Electron Microscopy
The diameter of transcription sites, seen as clusters of gold particles in cryosections, was determined using the sequential-subtraction method (
The sequential-subtraction method also provides information on sensitivity of detection; undetected sites and caps would give negative frequencies in Figure 5. Because none are seen here, the method must be very sensitive. Moreover, detected sites range in volume from 1/373 to 9 times the average, so Br-RNA can be detected over a ~3000-fold range. However, sites soon become saturated with gold particles (i.e., 35 at most with the immunolabeling procedure described here; not shown), so the number of gold particles per site is a poor measure of Br-RNA content.
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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The Use of Cryosections Improves Resolution of Light Microscopy
The resolution of the conventional light microscope is limited to ~200 nm in the x- and y-axes, and at least 500 nm in the z-axis (
The use of thin cryosections confers several other advantages. For example, two different fluorochromes are often used to mark two different antigens (e.g., a nascent transcript and a polymerase or transcription factor) to see if they co-localize (e.g.,
Cryosections also facilitate imaging of the same objects by light and electron microscopy (Figure 4). This was demonstrated by indirectly immunolabeling sites containing Br-RNA with FITC–protein A adsorbed on to 6-nm gold particles. After collecting a fluorescent image on the confocal microscope, the same section was transferred to the electron microscope so that the gold particles could be seen. This enables comparison of the sensitivities of the two methods and assessment of the strengths and weaknesses of the different approaches used for analysis. The two methods had similar sensitivities, but electron microscopy gave the highest resolution. For example, the number of sites was underestimated by ~17% by light microscopy, largely because several small sites that some contained could not be resolved. Of course, electron microscopy is required to determine precisely the diameters of small objects.
The use of cryosections to analyze spherical structures, not much smaller than the thickness of the section, has one major disadvantage: some spheres are cut nonequatorially to give polar caps, which may be missed because they are too small to be detected. [For example, with spheres of 40-nm diameter, ~7% of the profiles seen in sections 150 nm thick will be nonequatorial.] However, the sequential-subtraction method of
Nascent Transcripts are Concentrated in Discrete Factories
The total number of nucleoplasmic transcription sites in 3-D space can be calculated (using standard stereological procedures) from the numbers and diameters of clusters seen in 2-D sections, with knowledge of nucleoplasmic volume. The density of sites seen here (i.e., 1.6 cluster/µm2) corresponds to 6500 extranucleolar sites/nucleus (see also
Until recently, it had been assumed that active polymerases were spread randomly throughout euchromatin, so that only a few would appear to form local clusters. Then, using an insensitive detection method for detecting nascent RNA, we might observe only the few clustered transcripts, while missing the majority. However, the results described above make this possibility unlikely. First, the two detection methods (involving light and electron microscopy), which would be expected to have different thresholds of detection, image roughly the same number of sites (Figure 4). Second, no signal above background is detected between sites by either method (Figure 4). This is so, despite one method proving sufficiently sensitive to detect Br-RNA over a 3000-fold range (see above). Moreover, similar results are obtained with intact and permeabilized cells (
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Acknowledgments |
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Supported by the Junta Nacional de Investigação Científica e Tecnológica of Portugal and the Wellcome Trust.
We thank Drs D. Vaux, T. Wilson, and N. White for their help.
Received for publication July 21, 1998; accepted November 10, 1998.
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