Confocal fluorescence microscopy with the tandem scanning light

microscope

SHIRLEY J. WRIGHT 1 *, JAMES S. WALKER2, HEIDE SCHATTEN1, CALVIN SIMERLY1,

JON J. MCCARTHY2 and GERALD SCHATTEN1

1 Integrated Microscopy Resource for Biomedical Research, University of Wisconsin, 1117 West Johnson Street, Madison, Wisconsin 53706, USAzTracor Northern, Inc., 2551 West Beltline Highivay, Middleton, Wisconsin 53562, USA

* Author for correspondence

Summary

Applications of the tandem scanning confocalmicroscope (TSM) to fluorescence microscopy andits ability to resolve fluorescent biological structuresare described. The TSM, in conjunction with acooled charge-coupled device (cooled CCD) andconventional epifluorescence light source and filtersets, provided high-resolution, confocal data, sothat different fluorescent cellular components weredistinguished in three dimensions within the samecell. One of the unique features of the TSM is theability to image fluorochromes excited by ultra-violet light (e.g. Hoechst, DAPI) in addition tofluorescein and rhodamine. Since the illumination

is dim, photobleaching is insignificant and pro-longed viewing of living specimens is possible.Series of optical sections taken in the Z-axis with theTSM were reproduced as stereo images and three-dimensional reconstructions. These data show thatthe TSM is potentially a powerful tool in fluor-escence microscopy for determining three-dimen-sional relationships of complex structures withincells labeled with multiple fluorochromes.

Key words: tandem scanning light microscope, confocalmicroscopy, fluorescence microscopy.

Introduction

Confocal fluorescence microscopy is rapidly becoming apowerful tool in cell biology for analyzing biologicalstructure and function at the cellular level, since elimin-ation of out-of-focus fluorescence improves clarity ofstructures that previously were unobservable with con-ventional epifluorescence microscopy (Webb, 1986;White et al. 1987; Dixon and Benham, 1987; Shuman etal. 1989; Taylor, 1989; Inoue, 1989). The shallow depthof field of confocal microscopes allows information to begathered only from the optical section thickness(0.5-1.5/im) rather than the entire specimen. Thisresults in a dramatic increase in contrast and effectiveresolution (Wilson and Sheppard, 1984; Wijnaendts vanResandt etal. 1985; Wilson, 1986).

Types of commercially available scanning confocalmicroscopes include: the confocal laser scanning micro-scope (CLSM), which employs a scanning laser beam toview the specimen (Brakenhoff et al. 1979; Wilson andSheppard, 1984; Wijnaendts van Resandt et al. 1985;Brakenhoff et al. 1986; Wilson, 1986; White et al. 1987;Dixon and Benham, 1987; Robert-Nicoud et al. 1989);and the tandem scanning light microscope (TSM), whichuses a multi-aperture, spinning Nipkow disk (Wilson and

Journal of Cell Science 94, 617-624 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

Sheppard, 1984; Boyde, 1986; Petran et al. 1986;McCarthy and Walker, 1989). Stunning confocal fluor-escence images have been obtained with the CLSM(Brakenhoff et al. 1986; White et al. 1987; Dixon andBenham, 1987; Oud et al. 1989); however, there areseveral limitations including photobleaching, damage ofliving specimens due to the intense laser beam, inabilityto image in real time, and lack of fluorescence excitationin the ultraviolet range (however, see Robert-Nicoud etal. 1989). While fluorescent images obtained with theTSM are dim owing to low light illumination, we haveused a highly sensitive imaging device, the cooled charge-coupled device (cooled CCD; Hiraoka et al. 1987;Janesick and Blouke, 1987), and fluorescent probes(Cowden, 1985; DeBiasio et al. 1987) to show that theTSM has the potential to be a powerful, non-invasive toolin fluorescence microscopy for determining three-dimen-sional (3-D) relationships of complex structures withincells labeled with multiple fluorochromes.

Materials and methods

Sea urchin (Strongylocentrotus purpuratus) eggs and embryoswere obtained (Costello et al. 19S7) and stained with: anti-

617

tubulin antibody, fluorescein isothiocyanate (FITC)-labeledsecond antibody, anti-centrosome antibody, rhodamine-labeledsecond antibody (Falkner et al. 1981; Balczon and Schatten,1983; Walter and Biessmann, 1984; Schatten et al. 19886) andHoechst 33258 for DNA (Luttmer and Longo, 1986). Surf clam(Spisula solidissima) eggs and embryos were obtained (Allen,1953; Costello et al. 1957) and stained with: anti-tubulinantibody, FITC-labeled second antibody, anti-Pi antibody,rhodamine-labeled second antibody (Balczon and Schatten,1983; Kuriyama et al. 1983; Chaly et al. 1984) and Hoechst33342 for DNA (Luttmer and Longo, 1986). All secondantibodies were from Cappel. Mouse oocytes were collected(Schatten et al. 1985) and either: (1) processed for immuno-cytochemistry (Schatten et al. 1985, 1988fl) with anti-tubulinantibody YL 1/2 (Kilmartin et al. 1982) and FITC-labeledsecond antibody followed by staining in Hoechst 33258(Luttmer and Longo, 1986); or (2) stained with 100f/M-Fluo-3AM (Minta et al. 1987; Molecular Probes) for 1 h, placedunder a partially sealed coverslip on a glass slide and activatedwith medium containing 1 mM-ionomycin (Calbiochem). Forcontrols, medium without ionomycin was drawn through thecoverslip. Mouse oocytes labeled with Fluo-3AM were imagedfor 30 s at 2-min intervals with a fluorescein filter set in theTSM.

Specimens were examined using a Nikon 40x fluor 1.3 NAoil immersion objective on a Tracor Northern TSM (TracorNorthern, Middleton, WI). The TSM was equipped with a200 W Nikon mercury light source and a Nipkow disk contain-ing 60;l<m square holes that provide 1% light transmission(McCarthy and Walker, 1989). The design of this microscopewas intended for general usage with reflected light. It was notdesigned specifically for fluorescence microscopy in that it useda 50-50 beam splitter and a standard 1 % transmission Nipkowdisk. Further improvements may be gained by using dichroicmirrors and high-transmission Nipkow disks; aspects that arebeing explored. Specimens were imaged with a Photometries200 cooled CCD camera (Photometries, Tucson, AZ) contain-ing a Thompson chip with 384x576 pixels (Hiraoka et al. 1987;Janesick and Blouke, 1987). Exposure times of 30-120s wereemployed depending on the fluorescent staining, size of thefluorescent structure of interest, and desired contrast. Imageswere processed with a TN-8502 image processor (Tracor

Northern). In addition, in cases where scan line artifacts fromthe rotating Nipkow disk were prominent, a fast Fouriertransform was performed with the TN-8502 to remove theartifacts. For conventional fluorescence microscopy, specimenswere viewed using a Zeiss Axiophot microscope fitted with anepifluorescence attachment and a Zeiss 100x Plan-Neofluar 1.3NA oil immersion objective, and photographed using a Nikon35 mm camera and Tri-X film (ASA 1600). Specimens were alsoviewed at a zoom of 2 with a Bio-Rad MRC-500 Laser ScanningConfocal Microscope (White et al. 1987) using a Nikon 60xPlan-Apo 1.4 NA oil immersion objective with a 1.5 neutraldensity filter and the laser on full power. Optical sections(20-40) at increments of 0.5-1.25 ,wm in the Z-axis were used togenerate either stereo images with the TN-8502 or 3-D recon-structions with Vital Images software (Vital Images, Inc.,Fairfield, IA) on a Silicon Graphics computer (SiliconGraphics, Inc., Mountainview, CA). Objective movement forgeneration of stereo images was driven by a Tracor NorthernTSM controller and TN-8502 computer. Images were photo-graphed directly from the monitor using a Nikon 35 mm cameraand Kodak ASA 400 color slide film or Panatomic-X ASA 32film. For demonstration of photobleaching, film and printswere developed and exposed identically for specimens at thebeginning and end of photobleaching.

Results

Confocal fluorescence microscopy with the TSM andcooled CCD produced high-resolution images with a highsignal-to-noise ratio. Eggs and embryos were chosen asideal test specimens because of their relatively large size(~100,um) and overlapping features, which contribute toout-of-focus fluorescence when viewed with conventionalepifluorescence microscopy. Very bright fluorescentspecimens could be seen confocally by eye with the TSMand appeared black when not in focus. Specimens with aless-intense fluorescent signal were located with reflectedlight before imaging with the cooled CCD. Imagesobtained with the TSM equipped with a non-dichroic

Fig. 1. Fluorescence micrographs of mitotic sea urchin embryos stained for microtubules and imaged with either a conventional(A), confocal laser scanning (B), or confocal tandem scanning light microscope fitted with a non-dichroic beam splitter (C) or adichroic reflector (D). The dichroic reflector reduced cooled CCD exposure times by 75 %. Cooled CCD exposure times:C60s; D, 15s. Bars. A, 20,um; B, lOftm; C,D, 30,um.

618 S. J. Wright et at.

Fig. 3. Confocal fluorescence image of microtubules (green), centrosonies (yellow) and chromosomes (blue) in a mitotic seaurchin embryo imaged with the TSM.

Fig. 2. Photobleaching of mitotic sea urchin embryos stained for microtubules. Specimens were imaged with either aconventional (A,A'), confoeal laser scanning (B,B'), or tandem scanning light microscope fitted with either a non-dichroic beamsplitter (C,C) or a dichroic reflector (D,D'). A,B,C,D were taken at time 0; A',B' ,C' ,D' were taken at 2, 2 (120 scans), 5, andSmin, respectively, after constant illumination. Bars, 10 fim.

beam splitter and cooled CCD showed a reduction in out-of-focus fluorescence, exhibited an improvement overconventional microscope images, and rivaled imagesgenerated with a CLSM (Fig. 1). To obtain relativelyshort exposure times, the beam splitter of the TSM wassubstituted with a dichroic reflector (Omega OpticalInc., Brattleboro, VT; see also McCarthy and Walker,1989). With this improvement, even dim specimenscould be seen by eye. Although exposure times weresignificantly reduced (Fig. 1), photobleaching became aproblem as in conventional and laser scanning confoealmicroscopes (Fig. 2). Although the dichroic reflectordecreased cooled CCD exposure times, the beam splitterwas used to explore further low-intensity light withoutnoticeable photobleaching. Another study is currentlyunderway to investigate the advantages of the dichroicreflector and higher-transmission Nipkow disks in theTSM.

To determine whether conventional fluorescence filtersets including those for ultraviolet light excitation couldbe used with the TSM, specimens stained with multiplefluorochromes were examined. When mitotic sea urchin

embryos triple-stained for DNA, tubulin and centro-somes were examined with ultraviolet, fluorescein andrhodamine filter sets, respectively, the arrangement ofmicrotubule bundles, compact centrosomes and chromo-somes was revealed (Fig. 3). Images obtained with thedifferent filter sets were apparently in register with nospatial distortion because the same non-dichroic beamsplitter was used for all image acquisition. Photobleach-ing was insignificant after 3 h of constant illumination(data not shown). Although the quantum efficiency(ability to detect photons) of the cooled CCD rapidlydeclines in the blue range (<450 nm; Hiraoka et al. 1987;Janesick and Blouke, 1987), high-fidelity images ofHoechst-stained chromosomes were possible by exposingfor twice as long as for fluorescein or rhodamine.

The low degree of photobleaching permitted acqui-sition of a series of optical sections taken at successivelydeeper focal planes. As shown in Fig. 4, the series ofoptical sections through the meiotic spindle of an unferti-lized mouse oocyte revealed that the bundles of fluor-escein-immunolabeled microtubules formed a hollowspindle around the chromosomes. In addition to obtain-

Fhtorescence microscopy with the TSM 619

Fig. 4. Serial confocal optical sections through the meioticspindle of an unfertilized mouse oocyte imaged with theTSM. Bundles of microtubules, viewed with a fluoresceinfilter set, formed a hollow spindle around the chromosomes.Optical section increments were 1.25 fim for the first ninesections and 2.5^m for the remainder. Bar, lOfJm.

ingZ-series with the fluorescein filter combination, stacksof optical sections were also produced with rhodamineand ultraviolet (u.v.) filter sets. Optical sections ofmeiotic Spisula oocytes viewed with a rhodamine filterset showed that the karyoskeletal antigen, PI, labels onlythe periphery of the maternal and paternal genomes. The3-D arrangement of the tetrad chromosomes in germinalvesicles of Hoechst-stained Spisula oocytes was identifiedwith the u.v. filter combination. Additionally, imagestaken with all three filter sets at each focal plane in triple-labeled Spisula oocytes permitted determination of thelocation of the maternal and paternal genomes withrespect to the meiotic spindle.

To obtain 3-D information from the specimens, stacksof optical sections were obtained for stereo image pro-duction. Optical sections taken at 2/xm intervals ofdividing sea urchin embryos immunolabeled with anti-tubulin and viewed with a fluorescein filter set were usedto generate stereo images (Fig. 5A). Microtubule bundleswere seen radiating from the two asters of the mitotic seaurchin embryos. A stack of optical sections taken at1.25 ^m increments of a meiotic Spisula oocyte viewedwith a rhodamine filter set revealed that the maternal andpaternal genomes labeled with the karyoskeletal antigen,PI, were in different focal planes (Fig. 5). Additionally,stacks of optical sections were submitted to 3-D recon-struction so that specimens could be viewed interactivelyfrom the top, bottom and sides, and obliquely. Whenoptical sections of eggs and embryos were reconstructed,side and oblique views revealed compressed specimens(data not shown).

Viability of living specimens was maintained afterprolonged viewing with the TSM. When sea urchin eggswere fertilized and allowed to develop under constantillumination (for >24h), fertilization was unaffected anddevelopment proceeded at least until after hatching. Thispermitted observation of living specimens stained with

the calcium ion indicating dye, Fluo-3AM, which wasimaged with the fluorescein filter set. When mouseoocytes preloaded with Fluo-3AM were exposed to freshmedium, no changes were observed. However, when theoocytes were later activated with ionophore, the calciumtransient appeared to spread throughout the entire speci-men (Fig. 6).

Discussion

These results demonstrate that the TSM, together withthe cooled CCD, may provide a convenient way ofdetermining 3-D relationships of complex structureswithin cells labeled with multiple fluorochromes.Although the TSM was designed more than two decadesago (Egger and Petran, 1967; Petran et al. 1968; Minsky,1988), it was slow to develop because of the difficulties inmanufacturing and aligning the Nipkow disk, and be-cause its images are dim. The main application of theTSM has been as a confocal reflected light microscope(Egger and Petran, 1967; Petran et al. 1968, 1986; Boyde,1985, 1986; Paddock, 1989). It has also been used in thefluorescence mode (Boyde and Reid, 1986; Boyde andWatson, 1989). With the advent of new imaging devicesand technological advances, imaging dim specimens is nolonger a problem. Hence, we explored the application ofthe TSM in confocal fluorescence microscopy.

The cooled CCD was chosen as the imaging device forthe TSM because of its high resolution, high sensitivity,high signal-to-noise ratio, high quantum efficiency, widedynamic range and geometric stability (Hiraoka et al.1987; Janesick and Blouke, 1987). Although the cooledCCD provides superior images, it is not without itsdisadvantages when used in conjunction with the TSM.Because of its slow image acquisition time and datatransfer rates with dim specimens, the cooled CCD limitsreal-time observations with the TSM. A silicon intensi-fied target (SIT) camera or intensified (ISIT) detectorprovides real-time imaging, but with a much lowersignal-to-noise ratio (Inoue, 1986). Another disadvantageof the cooled CCD is its decreased sensitivity in the bluerange (<450nm; Hiraoka et al. 1987; Janesick andBlouke, 1987). To overcome this problem, specimensstained with u.v.-excitable dyes were exposed for rela-tively twice as long as for fluorescein or rhodamine.

The TSM may offer several advantages over commer-cially available CLSMs. One of the main advantages ofthe TSM is the ability to image fluorochromes excited byultraviolet light (e.g. DNA fluorochromes and ion indi-cating dyes) in addition to fluorescein and rhodamine.This permits examination of several structures labeledwith different fluorochromes within the same cell.Another advantage of the TSM is that for very brightfluorescent specimens, real colors of the image can beviewed directly by eye. When a non-dichroic beamsplitter was used in the TSM, specimens were dim andphotobleaching was insignificant. In contrast, the inser-tion of a dichroic reflector into the TSM increased thefluorescent light intensity and reduced cooled CCDexposure times; however, photobleaching became severe

620 S. jf. Wright et al.

Fig. 5. Confocal stereomicrographs produced with theTSM by pixel shifting.A. Mitotic sea urchin embryorevealing the three-dimensionalorganization of the spindle.B. Meiotic Spisula oocytestained for the karyoskeletonshowing the paternal genomeabove the focal plane of thematernal chromosomes. Bars,

as with conventional and laser scanning confocal micro-scopes. We have not explored whether placement of thenon-dichroic beam splitter in the CLSM would yieldsimilar results of insignificant photobleaching, decreasedspecimen damage and dim specimens. The use of a non-dichroic beam splitter in the TSM permitted prolongedviewing of both fixed and living specimens. This facili-tates acquisition of stacks of optical images for Z-series.The TSM was non-invasive in living specimens, presum-ably because the design of the TSM provides low-lightillumination and the ability to filter out harmful ir-radiation. This holds promise for confocal studies ofliving cells stained with supravital dyes.

Confocal microscopy may facilitate the full use of ionindicating dyes, since information is obtained only fromthe region of interest. At fertilization the eggs of a

number of different species undergo a transient increasein intracellular calcium, which triggers various cellularprocesses including cortical granule exocytosis (see Epel,1978; Jaffe, 1983, 1985). However, since information isgathered from the entire cell with conventional micro-scopes, it is not understood whether the rapid burst offree calcium occurs as a wave only in the egg cortex(Hafner et al. 1988) or throughout all the egg cytoplasm(Miyazaki et al. 1986). The results on mouse oocytespresented here indicate the potential use of the TSM toexamine ion changes in living cells in 3-D and resolve the3-D spatial pattern of calcium ions in the cortex andcytoplasm of cells.

Stereo images of fluorescent structures can be gener-ated with the TSM in a process different from that ofCLSM stereo production. The CLSM obtains stereo-

Fluorescence microscopy with the TSM 621

Fig. 6. Free calcium in an unfertilized mouse oocyte imagedwith the TSM. The top three panels show no change whenfresh medium was drawn under the coverslip; however, whenmedium containing 1 mM-ionomycin was drawn through, aburst in free calcium was seen (middle left panel) and later,free calcium levels dropped below resting levels (subsequentpanels). Bar, 30/im.

scopic images by digital manipulation (pixel shifting) of astack of optical sections taken in the Z-axis. In addition tothis method as shown in Fig. 5, the TSM can also movethe objective in both the X- and Z-axes within the samefocal plane to produce a right and left image, each takendirectly from the specimen without digital manipulation(Boyde, 1985). This provides a separate stack of right andleft images, which, when compiled, produces a stereoimage through two different volumes. This method,however, is not without its disadvantages. (1) Specimensare exposed twice, once for each stereo half, before astereo pair can be made. (2) Care must be taken toprevent mechanical drift of the specimens, which cancause registration problems in the left and right stereoimages.

With the TSM, Z-series stacks may be obtained asdescribed above for stereo image production with lateralmovement of the objective in the A'-axis, or for 3-Dreconstruction in which the objective is stationary in theA' and Y directions. The finding that confocal, through-focus series can be obtained with the TSM may provide aquick, convenient means of obtaining data sets for 3-Dreconstructions so that different views of the specimen(top, lateral, side, etc.) may be studied interactively. Inthis regard, improved methods of specimen preparationand maintenance are necessary, since current preparativemethods for conventional epifluorescence microscopy,although excellent for preserving specimens in two di-mensions, generate compressed specimens in the Z-axisdirection analogous to a 'road kill'.

The TSM is not without its limitations. The relativelylarge aperture size (60 fim) of the holes in the Nipkowdisk of the TSM as compared to that of CLSMs

compromises optical sectioning and, as a result, the limitof resolution and image quality may be reduced due to anincrease in out-of-focus fluorescence. Optical sectionthickness of the TSM is approximately 1 jum with theobjectives and 60 /.im Nipkow disk used (McCarthy andWalker, 1989) rather than the 0.7 jum for the CLSM usedin the present study. However, image quality can beimproved with the use of an image processor to removeout-of-focus fluorescence, and digitally enhance the im-ages. Since image quality (resolution, contrast andbrightness) is related to aperture size and population,Nipkow disks containing smaller apertures (20 fim) withdifferent populations and placements of holes are cur-rently being tested in the TSM for greater light efficiencyand improved image quality without photobleaching andscan line artifacts. To produce a Nipkow disk relativelyfree from imperfections, such as scan line artifacts andspatial non-homogeneity, a high degree of precision in thesize, shape and arrangement of the apertures is required.Advances in new technologies have produced a durableand nearly defect-free Nipkow disk for the TSM used inthe present study (for details, see McCarthy and Walker,1989). Any remaining scan line artifacts are readilyremoved with image processing (for the study presentedhere, only Fig. 3 was processed to remove scan-lineartifacts). Another limitation of the TSM is that thefluorescence is dim and, thus, difficult to image. We haveavoided this problem by using the cooled CCD camera;however, real time imaging was compromised. For fixedspecimen studies, the combination of the TSM andcooled CCD provided high-resolution images. In con-trast, high-resolution real-time imaging with the cooledCCD was not possible with living specimens under thecurrent conditions, due to the long acquisition times anddata transfer rates of the cooled CCD. Real-time fluor-escence imaging is possible with the TSM when either aSIT or ISIT camera is used; however, the image quality(signal to noise ratio) is drastically reduced (unpublishedobservations). Future improvements in both the TSMand cooled CCD are designed to address these problems.

In summary, confocal microscopy with the TSM andcooled CCD may offer a convenient means of examiningboth living and fixed specimens labeled with multiplefluorochromes (including u.v.-excitable fluorochromes)for prolonged periods to determine the 3-D organizationof different cellular components. The capabilities of theTSM in fluorescence microscopy are proving invaluablein studying fluorescent structures of thick specimens inthree dimensions. The TSM may well find a uniqueenvironment in which it complements the well-developedlaser scanning confocal microscopes and offers the advan-tage of excitation with u.v. light. In addition, the dimillumination of the TSM and an attenuated laser beam inCLSMs would provide insignificant photobleaching, anda non-invasive method of viewing living specimens.Future improvements in confocal microscopy including:greater light efficiency, modifications in Nipkow diskdesign and dichroic reflectors to shorten exposure times,and advances in cooled CCD data transfer rates willprovide a means of imaging dim specimens rapidly andmay make imaging with the TSM more attractive.

622 S. Jf. Wright et al.

Perhaps future developments will produce an optimizedconfocal microscope that has the advantages of both laserand tandem scanning microscopes.

We gratefully acknowledge the skilled efforts of Jim Ashbach,Dave Kinser and Ammar Rabbat, of Tracor Northern. Thegenerosity of Vital Images and Silicon Graphics in making theirequipment available and the resourcefulness of Drs VincentArgiro and William Van Zandt of Vital Images are gratefullyacknowledged. We thank Drs H. Biessmann, N. Chaly, J.Kilmartin and M. Walter for their generous donations ofantibodies. We also thank Drs Jim Pawley and Steve Paddock,and Peter DeVries of the Madison Integrated MicroscopyResource, for their helpful discussions. This research wassupported by research grants from the NIH and the MadisonI MR is supported as an NIH Biomedical Research TechnologyResource.

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