Laser scanning fluorescence microscopy

Laser scanning fluorescence microscopy

Johan S. Ploem

Fluorescence laser scanning microscopy (LSM) offers many advantages over conventional fluorescencemicroscopy. Very strong excitation light can be concentrated on small spots (0.5 Atm) of the specimen,enabling the detection of low concentrations of fluorescent substances. The low levels of autofluorescencegenerated in the microscope objective and in the immersion oil in LSM provide images of great contrast, evenwith weakly fluorescent specimens. Confocal LSM permits the visualization of multiple focal layers of thespecimen and 3-D image reconstructions. Combination of images stored in computer memory allow thecomparison of phase contrast and fluorescence images of the same area of the specimen enabling multiparam-eter analysis of cells.

1. Introduction

In optical laser scanning microscopy (LSM) the ob-ject is not illuminated as a whole, but scanned step bystep. From each illuminated point the transmitted,reflected, or emitted light is measured. The image isbuilt up by storing these point-to-point measure-ments, after analog-to-digital conversion, as a matrixin a computer image memory.

Laser scan microscopes have been developed by anumber of workers.'-8 Some of the original versions ofthe instruments were not directed towards fluores-cence applications. However, in the newer designsthey have been included.

Presently there are three main types of laser scan-ning microscope, the ultrafast types,3'5 the fast typesusing laser beam scanning,9 and the slower stage scan-ning types.' 0 "'1 They aim at different applications.The first type is designed for rapid automation ofcytology and hematology specimens. The latter twoare for investigations which require a combination ofmicroscope methods (phase contrast, reflection, fluo-rescence, interference contrast).

II. Components of a Laser Scanning Microscope

Laser light sources generate highly collimated lightwhich can be focused onto extremely small spots with ahigh intensity. For LSM, frequently He-Cd, Ar, andHe-Ne lasers are used. For scanning microscopy twomethods are in general use. Either the microscopic

The author is with University of Leiden, Department of Cyto-chemistry & Cytometry, Wassenaarseweg 72, 2333 AL Leide, TheNetherlands.

Received 13 May 1987.0003-6935/87/163226-06$02.00/0.© 1987 Optical Society of America.

object itself is transported step by step, or the beam ismoved across the resting microscopic object, or a com-bination of both is used.3 7 0 1 2-14 In principle beamscanning can be obtained with (a) servo-controlledgalvanometers, (b) acoustooptical deflectors, (c) reso-nant galvanometers, and (d) fast rotating polygonscanners.

A. Optical Scheme

The laser beam is expanded with a telescope to a sizesuitable for microscope objectives and moved in twoaxes by an x-y scanner unit consisting of two orthogo-nal galvanometer scanners, one for the lines and onefor the scanned spots. Between the scanners relayoptics are placed which image the first mirror into thesecond one. Both mirrors are imaged into the pupil ofthe objective lens. Independent of the scanning angle,the full aperture of the objective is always used. Thebeam is then focused by a diffraction-limited spot onthe object. With transmitted illumination the light iscollected by the microscope condenser and directed toa photomultiplier tube. For fluorescence and reflec-tance microscopy the light retraces the entire beampath backward (including the scanning mirrors) untilit is reflected by a beam splitter onto the photomulti-plier tube.

In the instrument used in our laboratory (Zeiss, F. R.G.) scanning obtained with servo-controlled galva-nometers is used. An advantage of this type of scan-ning is the relatively short scanning time of 2 s/field of512 X 512 scanned spots, and the great flexibility of thescanning process compared with scanning using a step-ping stage. The scanning time per field of 512 X 512pixels can be increased to 8, 32, or 64 s when noisyfluorescent images emitting a relatively low number ofphotons have to be analyzed.

In the Zeiss LSM the following requirements arefulfilled: (1) the optical resolution and the dimen-

3226 APPLIED OPTICS / Vol. 26, No. 16 / 15 August 1987

sions of the microscope fields are equivalent to conven-tional microscopy; (2) all major types of microscopeobjectives can be used; (3) the microscope can also beused as a conventional fluorescence microscope. Inour laboratory a normal fluorescence epi-illuminatorhas been incorporated into the Zeiss LSM instrument.It can thus also be used as a conventional epi-illumina-tion fluorescence microscope. As photodetector, aphotomultiplier is used because of its sensitivity, rapidrise time, and wide spectral bandwidth. Imaging ofthe object on the detecting photosensitive surface isnot needed as only the light intensity corresponding toone illuminated point is registered.

B. Microelectronics

The electronics of a laser beam scanning microscopeshow similarities with a modern scanning electron mi-croscope. A microprocessor controls the scanningprocess. The signals of the photomultipliers pass ananalog signal processor, where contrast and brightnessof the image are controlled. The resultant signal isdigitized and fed into a frame memory. The readoutof this memory is in video frequency. As a result theobserver sees a stationary image on the video monitor,except when the microscope image changes. Thescanning process is controlled by a digital scan genera-tor. Its output is fed into a zoom unit, permitting thechange of the amplitudes of the galvanometer scan-ners. A continuously variable magnification withoutloss of contrast can thus be obtained. Next to thezoom unit, there are scanner drivers which determinethe speed, linearity, and accuracy of the scan. Con-trasting methods for bright-field, phase-contrast, and

Fig. 1. Cyclic GMP reactivity in the hippocampus of the rat. Thebrain of the rat was fixed with paraformaldehyde. Subsequently 35-,um sections were treated with antibodies to cGMP. Magnification

2.5X. Different magnifications are shown in Figs. 2-5.

differential interference contrast microscopy can beselected.

Ill. Ultrafast Laser Scanning

If very powerful lasers are used with some of theseinstruments they can in principle concentrate an enor-mous amount of energy on each small spot of themicroscope field. With the ultrafast scanning sys-tems,3 5 the dwelling time of the illuminating beam isrelatively short. Consequently problems of destruc-tion of the fluorophore or the biological carrier can bemastered. With this instrumentation very high scan-ning rates can be achieved.3 Real speed limits aredetermined by the mechanical stability and deforma-tion of the polygon scanner when rotating at 24.000-48.000 rpm and the energy of the laser available. Inother words, the signal-to-noise ratio is dependent onthe number of photons that is available per time unit.It can be expected that the instrument using veryrapidly rotating polygon mirrors3 will provide similarspeed, as far as the number of cells analyzed is con-cerned (1.6 billion scanned points in 60 s) than the flowcytometry systems. The data acquisition rates are sohigh that present computers, when based on singlemicroprocessors, cannot process the data. Future de-velopments in image processing architecture, such asparallel processing, are expected to solve the type ofdata rate problem of the high-speed laser scanningmicroscopes.

IV. Laser Scanning for Multiparameter Cell Analysis

In the Zeiss LSM it is possible to combine the epi-fluorescence, or reflectance signal, in a sequential waywith transmitted phase contrast or absorption micros-

Fig. 2. Hippocampus (brain) of the rat. Antibody to cyclic GNP.4X objective.

15 August 1987 / Vol. 26, No. 16 / APPLIED OPTICS 3227

copy. This allows multiparameter analysis of cells.The images of a given microscope field are stored incomputer memory and then compared (added, sub-tracted, etc.) with each other. For example, all lym-phocytes (from human peripheral blood) in a micro-scope field can be counted on the basis of their phase-contrast image and then the percentage of a subset oflymphocytes marked with a fluorescently labeled mon-oclonal antibody can be determined. Another exam-ple consists of the comparison of the contrast in tissuesections obtained with absorption photometry withthe image in fluorescence microscopy of the same field.

V. Use of Low Microscope Magnification inFluorescence Microscopy

With a conventional fluorescence microscope, lowmagnifications are difficult to realize when only con-ventional light sources (mercury and xenon high-pres-sure arc lamps and halogen lamps) are used. Micro-scope objectives with low magnifications (below 16X)also have relatively low numerical apertures, and as aconsequence low fluorescence light-collecting power.This results in insufficient brightness in low magnifi-cations. The LSM concentrates all the light of a laserlight source on only one spot of the specimen before itmoves to the next spot. This very intensive excitationlight enables fluorescence microscopy with a 2.5X dryobjective in specimens stained with an FITC markerenabling examination and measurement of fluores-cence in large tissue areas (Fig. 1).

VI. Reduction of Autofluorescence of the MicroscopeObjective and Immersion Oil in the LSM

For each microscope step all the light from the laseris concentrated at one moment of time on only one

small scanned spot in the preparation. The image isobtained by sequentially scanning 262,144 of suchspots. An advantage of this procedure is that the loadof exciting light on the optical parts, such as objectiveand immersion oil, during the time that each spot isscanned (<1/131.072 s) is very low. Crudely estimat-ed, the energy load on the entrance pupil of the objec-tive may be <1 mW, while there still is a detectablefluorescence obtained by sequentially scanning andstoring the image in memory in most applications.This relatively small amount of energy (light) reachingthe microscope objective per time unit results in ex-tremely low autofluorescence of optical parts and im-mersion oil. With conventional fluorescence micro-scopes (using an HBO 100 mercury high-pressure arc)the amount of exciting light reaching the entrancepupil of an objective can have an energy of >1 Wduring the 30 s needed for visually inspecting the mi-croscope image. This is necessary to obtain a suffi-cient fluorescence intensity level in the entire micro-scope field simultaneously. The low level ofautofluorescence from optical parts in the Zeiss LSMsignificantly contributes to the extreme sensitivity ofthis type of laser scanning microscope.

VII. Addition of Conventional Epi-Illumination to LSM

The laser scan microscope (LSM) can be used with aconventional light source as well as with a laser. Whena laser is used as the light source, the laser beam isscanned over the probe by means of two vibratingmirrors. The fluorescence signal is then digitized,stored in a digital memory, and visualized on a CRTdisplay. With the laser light source, transmitted lightas well as incident light microscopy are possible. Fortransmitted light microscopy, a standard condenser is

Fig. 3. Hippocampus (brain) of the rat. Antibody to cyclic GNP. Fig. 4. Hippocampus (brain) of the rat. Antibody to cyclic GNP.10X objective. 16X objective.


used to obtain absorbance, phase, and differential in-terference contrast images. Reflection and fluores-cence contrast images can be obtained with incidentlight. Because of the implemented laser, it is notpossible to choose another wavelength for excitation asthe laser and the accompanying optical parts are usedin a monochromatic way (488 nm from an air-cooledargon laser). The fluorescence measurements are per-formed with a photomultiplier. The digitized signalsare stored in computer memory. As a conventionallight source, the instrument only permits the use of atungsten lamp guided by a glass fiber, which does notpermit fluorescence microscopy.

With LSM it is also not possible to observe a double-stained probe in the eyepieces when other wavelengthsfor excitation are to be used. Furthermore, the focus-ing is difficult, since the observer has to wait a secondbefore the image is seen on the CRT screen (a rapidcoarse scanning mode is used for focusing). Prepara-tions consisting of only small objects on a large micro-scope slide may be difficult to locate when cell struc-tures can be recognized only on the basis of theirfluorescence. For these reasons we have added in ourlaboratory a conventional epi-illumination fluores-cence microscope to the Zeiss LSM. This additionconsists of (1) a connection for a mercury lamp, like anHBO-50 or HBO-100, without interfering with laserscan microscopy; (2) a mirror to switch the mercurylamp illumination into the light path; (3) a field dia-phragm to alter the illuminated area; (4) filters forfluorescence microscopy with UV, blue, and green ex-citation; and (5) a filter compartment for the separateanalysis of different wavelengths of emitted light.15

Fig. 5. Hippocampus (brain) of the rat. Antibody to cyclic GNP.40X objective.

Vil. Theoretical Calculation of LSM Sensitivity

Since the total energy of the laser is focused into onesingle small spot in the microscope specimen, it wasexpected that sensitivity would be high. To estimatethe sensitivity more quantitatively, experiments wereperformed with test beads from Becton-Dickinson.These particles have a known amount of FITC mole-cules (diameter in microns: 4.3; number of FITC mol-ecules: 2900). In this way it is possible to extrapolatethe sensitivity of the microscope in terms of detectableamount of FITC molecules.

The fluorescence sensitivity of the Zeiss LSMamounted to -10 FITC molecules/,gm2. In compari-son, a conventional microscope would need -100 timesmore FITC molecules to obtain an image on a highlysensitive photo film. The use of image-intensifyingTV cameras does not ensure an increase in the sensitiv-ity of conventional microscopes, since the autofluores-cence of the microscope objective and the immersionoil determine the lower detection limit. The fluores-cence of the latter is much lower in an LSM than in aconventional microscope.

IX. Applications

A. Central Nervous System Tissue Research

A very low magnification (2.5X dry objective) can beused for the detection of immunofluorescence in speci-mens treated for histochemical evaluation of largeparts of the brain. With this low magnification, theenergy of the laser is simply concentrated in a largerspot of the specimen resulting in a sufficiently strongfluorescence. The area under investigation using a2.5X objective is 3.75 X 3.75 mm (Fig. 1). The lowmagnification in laser scan microscopes means thatinterrelation of nervous elements spread over wideareas of, for example, the brain, can be detected. Us-ing high magnification objectives a whole series ofmagnifications from the same area of the brain can beobtained (Figs. 1-5).

With specific monoclonal antibodies against neuro-transmitters or their precursors, nervous transmissionin the brain and peripheral nervous tissue can be stud-ied in a quantitative way. In applications where suit-able quantitative cytochemical methods are available,quantitative analysis of neurotransmission is in princi-ple possible since all the fluorescence signals can bestored in computer memory. In the stored digitizedfluorescence image, segmentation based on contrast-ing algorithms can be used to isolate nerve fibers fromthe background. This permits an integrated measure-ment of the total neurotransmitter-based fluorescencein a nervous network or in a group of cells. The iris ofthe rat was studied after paraformaldehyde vaportreatment yielding catecholamine fluorescence (FIF).The catecholamine fluorescence has an excitationpeak at 410 nm, but due to the very low backgroundfluorescence, the reaction products could even be ex-cited in a very inefficient way at 488 nm.


B. Studies of Very Weak FITC Fluorescence

The very dark background of the LSM results in ahigh image contrast of immunofluorescently stainedDNA adducts, which requires a very sensitive fluores-cence microscope. This can be applied to, for exam-ple, environmental research, where it is important todemonstrate very low concentrations of drugs or chem-ical noxes that may bind to DNA. With the LSM, cellscould be observed which could not be identified with aconventional fluorescence microscope.

C. Ultrathin Sections for Electron Microscopy

Examination of ultrathin (20-50-A) sections as theyare routinely cut with a glass knife for electron micros-copy is not possible with the conventional light micro-scope if no special contrasting stains (in addition tothose used for EM studies only) are used. For a rapidorientation (and statistical analysis) light microscopi-cal examination of ultrathin sections would, however,be very desirable, especially if no special extra stainingtechniques for light microscopy would be necessary.Using LSM in a reflection mode, very detailed imagesof these ultrathin sections could be obtained, while noextra staining was necessary. Spread polytene chro-mosomes also could be very well observed in LSM,which has been reported by Kalisch et al.' 6

X. Discussion and Conclusions

The principal advantages of laser scanning micros-copy are: (1) The pointwise illumination in LSM.Thereby most of the stray light is avoided that normal-ly occurs in conventional microscopy when an entiremicroscope field is illuminated and which is the resultof interfering lightwaves scattered from neighboringparts of the microscope specimen.'7 (2) The possibili-ty for electronic variation of brightness and contrast.Very weak contrasts can be enhanced, the only limitingfactor being their signal-to-noise ratio. (3) The feasi-bility of overmagnification realized by the electroniczoom facility, while maintaining the same optical reso-lution. This may improve the recognition of very finestructures. (4) The excitation of fluorochromes withhigh-intensity laser light. This often results in a con-siderable increase in sensitivity in comparison withmicroscopy using conventional light sources. Thestrong laser light can be concentrated onto very smallspots of the specimen, resulting in near-maximal exci-tation of the fluorochrome. Intensive excitation cancause fading of the fluorochrome. The fading can,however, be limited by very short exposure times ofeach spot. In the normal scanning mode of the ZeissLSM, each point is exposed only during <1/128.000 ofa second, whereafter the fluorescence emission fromeach scanned point is immediately stored in computermemory. This procedure, however, demands priorfocusing. This is often quite time consuming and canthus cause considerable fading of the fluorescence.The focusing procedure should, therefore, preferablybe carried out with weak exciting light obtained from,for example, a halogen lamp or from a mercury orxenon lamp with a low transmission fluorescence exci-

tation filter. The low-intensity fluorescence imagethus obtained can often not be seen by the eye. Inmost instances the specimen can, however, be observedwith an image intensifier (SIT or ISIT TV camera)which can intensify the very weak fluorescence signals.A particularly sensitive instrument has been devel-oped by Hamamatsu.' 8 ,'9 (5) Low level of autofluores-cence of objective and immersion oil, especially in com-parison to a conventional fluorescence microscope.The photons of the exciting light passing through theobjective in LSM are only those needed to excite maxi-mal fluorescence in only one of the of 256,000 spots inthe microscope field. In conventional fluorescenceepi-illumination microscopy, all the photons needed toexcite the entire microscope field must pass the objec-tive simultaneously, thereby causing relatively strongautofluorescence of optical parts and immersion oil.(6) Consecutive scanning of several layers of the micro-scopic object, provided a confocal optical arrangementis used. The out-of-focus fluorescence in confocalLSM interferes only in a very limited way with thefluorescence image in one thin layer of the object. Inconventional fluorescence microscopy, on the con-trary, out-of-focus fluorescence gives a relativelystrong interference with fluorescence from the speci-men layer in focus. (7) Ready availability of all mor-phological information in a quantified 2- or 3-D mode.In addition, image enhancing and image analysis tech-niques can be applied directly to such images stored incomputer memory.

We want to thank H. W. M. Steinbusch for makingavailable to us the sections that were photographed forFig. 1.

References1. C. Cremer and T. Cremer, "Considerations on a Laser-Scanning

Microscope with High Resolution and Depth of Field," Microsc.Acta 81, 31 (1978).

2. M. Achats, R. Beck, C. Seger, and G. von Sengbusch, "Laser-scanning Technologie zur Bildanalyse vpon Zellen," Acta Histo-chem. Suppl. 21, 147 (1980).

3. P. H. Bartels et al., "Ultrafast Laser Scanner Microscope,"Anal. Quant. Cytol. 3, 55 (1981).

4. W. Deinet, M. Linke, R. Mueller, and I. Sander, "Laser-Scan-ning-Mikroskop Mit Automatischer Fokussierung," Microsc.Acta 87, 129 (1983).

5. R. L. Shoemaker, P. H. Bartels, D. W. Hillman, J. Jonas, D.Kessler, R. V. Shack, and D. Vukobratovich, "An UltrafastLaser Scanner Microscope for Digital Image Analysis," IEEETrans. Biomed. Eng. BME-29, 82 (1982).

6. V. Wilke, "Laser Scanning in Microscopy," Proc. R. Microsc.Soc. 17, 21 (1982).

7. V. Wilke, "Laser Scanning in Microscopy," Proc. Soc. Photo-Opt. Instrum. Eng. 396, 164 (1983).

8. R. Mueller, "Scanning Laser Microscope for Inspection of Mi-croelectronic Devices," Siemens Forsch. Entwicklungsber. 13, 9(1984).

9. V. Wilke, "Optical Scanning Microscopy-The Laser Scan Mi-croscope," Scanning 7, 88 (1985).

10. G. J. Brakenhoff, P. Blom, and P. J. Barends, "Confocal Scan-ning Light Microscopy with High Aperture Immersion Lenses,"J. Microsc. 117, 219 (1979).


11. H. T. M. van der Voort, G. J. Brakenhoff, J. A. C. Valkenburg,and N. Nanninga, "Design and Use of a Computer ControlledConfocal Microscope for Biological Applications," Scanning 6,66 (1985).

12. C. J. R. Sheppard and A. Choudhury, "Image Formation in theScanning Microscope," Opt. Acta 24, 1051 (1977).

13. C. J. R. Sheppard and T. Wilson, "Image Formation in theScanning Microscope," Opt. Acta 25, 315 (1978).

14. T. Wilson and C. Sheppard, Theory and Practice of ScanningOptical Microscopy (Academic, London, 1984).

15. J. S. Ploem, "Organic and Biological Surfaces: FluorescenceMicroscopy," in Analysis of Organic and Biological Surfaces, P.Echlin, Ed. (Wiley, New York, 1984), pp. 609-628.

16. W. E. Kalish, T. Whitmore, and A. Siegel, "Laser ScanningMicroscopy of Surface Spread Polytene Chromosomes, J. Mi-crosc 117, 217 (1985).

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BibliographyH. Hartmann, A. Hund, S. H. Moll, and A. Thaer, "Attachment for

Combined Scanning Electron and Light Microscopical Examina-tions," Beitr. Electronenmikroskip Direksabb. Oberfl. 11, 381(1978).

G. R. Hillman, D. Johnston, S. W. Kwan, D. Carney, and G. Childa,"Histochemical Applications of Image Analysis Techniques," inProceedings, International Symposium on Medical Imaging andImage Interpretation (IEEE, New York, 1972).

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1987 NEWPORT AWARDS

The Newport Corporation and the Optical Society of Ameri-ca have announced the funding of three Newport ResearchAwards for doctoral candidates working on research projectsthat advance laser and electrooptics technology. In addition,funding has been renewed for last year's recipients. Awardsof $16,000 each are being made to the following Ph.D. candi-

dates:Myra M. Boenke, a student in electrical engineering and

computer sciences at the University of California, Berkeley.Boenke will conduct research on focused ion beam implanteddoping gratings for InGaAsP/lnP distributed feedback lasers.

Chung Ki Hong, a student in the physics and astronomy

department of the University of Rochester. Hong's two-partproject will investigate photon correlation effects.

Michael Mittelstein, a student in applied physics andelectrical engineering at the California Institute of Technology.His research will focus on multiple-quantum-well unstableresonator semiconductor lasers and quantum-well broadbandtunable semiconductor lasers.

Newport Research Awards were renewed for the following

1986 winners:Seng-Tiong Ho, a student in electrical engineering and

computer sciences at the Massachusetts Institute of Technol-ogy. He will continue his research on the generation of

squeezed-state light.Nan M. Jokerst, a student in electrical engineering/elec-

trophysics at the University of Southern California. Jokerst isworking to develop a laser in the 3-Mm region.

Kurt J. Weingarten, a student in electrical engineering atStanford University. He will continue his efforts to develop and

demonstrate electrooptic sampling for picosecond time resolu-tion, internal-node voltage measurements of GaAs integrated

circuits.Frank W. Wise, a student in applied physics at Cornell

University. Wise is studying the relaxation of hot carriers insemiconductors.

The Newport Corporation of Fountain Valley, CA-a manu-facturer serving the laser and electro-optics industry-beganfunding awards for doctoral research in 1984. Each $16,000award includes a $12,000 stipend for the student plus $4,000 tothe academic department supervising the research.

The awards are administered by the Optical Society ofAmerica, which designated the selection committee. Mem-bers of the 1987 committee included:

Arthur L. Smirl. (chair 1987) of North Texas State Univer-

sity.Richard A. Elliott of Oregon Graduate Center.Jerald R. lzatt of the University of Alabama.Charles K. Rhodes of the University of Illinois.David G. Seiler of North Texas State University.John J. Wharton, Jr. of the Defense Advanced Research

Projects Agency.Shaoul Ezekiel (past chair) of the Massachusetts Institute of

Technology.

The Optical Society of America is a nonprofit educational

and scientific society of scientists and engineers devoted to theadvancement of optics, pure and applied. Founded in 1916,

the Society is headquartered in Washington, D.C.


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Laser scanning fluorescence microscopy