Confocal microscopy in pharmacological research

CURRENT

TECHNI

Confocal icroscopy in acological research

point source of light

Roger G. King and Peter M. Delaney

Confocal microscopes produce ‘optical sections’ through translucent tissue (commonly to a maximum depth of 200400 pm), without the need for cutting thin slices. They eliminate blur and flare from out-of-focus planes in an object. The basic prin:iple is that illumination of a single point in a specimen is combined with imaging of this illuminated point by a point detector’. Light from any other region of the specimen is not detected to any significant extent. Two-dimensional (2D) and 3D images can be built up by scanning the illuminated point, for example, by moving the specimen in a scanning pattern (‘object scanning’9 or by scanning of the light beam (‘beam scanning’?! (Fig. 1). Lateral resolution of confocal microscopes is theoretically greater than that of conventional optical microscopes by a factor of 1.4-2.0, and can be less than 0.2i~m. However, axial resolution is dramatically improved (the conventional optical microscope has little depth discrimination) and can be as little as 0.5 brn. Examples of different types of confocal microscopes, including their advantages and disadvantages, and types of confocal imaging are given in Box 1.

Examples of applications of confocal microscopy in pharmacological research lon imaging Using confocal microscopy, Ca? ’ movements within cells can be monitoredJai, thereby allowing examination of the effects of drugs, toxins and electrical stimulation on such movements. Simultaneous electrophysiological analysis of neurones may also be performed. To ascertain whether the fluorescent dyeused for ion imaging has deleterious effects, a physiological parameter such as spontaneous contractility of cardiac myocytes may be monitored”. Ca?+ signals from

minuteiy localized areas of a cell may be visualized confocally with high temporal resolution, for example, in studies of the effects of inositol 1,4,5-trisphosphate within XLW~W oocytesisx, or in studies of spontaneous or stimulated opening of single Ca?+-release channels in the sarcoplasmic reticulum of heart scanning mechanism muscle cells?. For Caz+ imaging with the commonly used argon ion laser, suitable fluorescent dyes include rhod-2, fluo-3, Ca green and fura-red. Because variations in the intracellular concentration of a dye with time may be confused with changes in Ca? +levels, and because the absolute concentration of the dye may be unknown, special techniques are required to quantitate Ca?+ concentrations’, including ratiometric methodW1. Alternatively, a UV-laser-scanning confocal microscope can be used with a fluorescent dye such as indo-l for ratiometric quantification of intracellular Ca?’ concentrations (Ref. 12). High-resolution spatial and temporal mea;urements of H* concenFig.1.A generaked laser-scannmg confocal microscope trations may be made within cells using confocal microscopy and fluorescent pH indicators such as the This latter techniql~e has the advantage that photobleaching does not Snarf and Snafl series. occur, and, therefore, 4D-data sets of living cells can be collected. Such lmmunocytochen~ical skdies Confocal microscopy enables ac- data sets may enable the study of curate localization of antibodies for how the distribution of various immunocytochemical studies with- cellular components (for example, antigens on out the problems of blur and flare immunogold-labelled the surface of rat thymocytcs”) from out-of-focus plar.cs. Relatively thick specimens [for example, up to change with time. A further application is the simul200pm (Ref. 13)1 can be used. To study the distribution of binding of a taneoti= study of the distribution R.G. King. primary antibody to its antigen by of two or more cell types or cellular Si?I/, IPO 1I1IPI ,IM . .. . . P.hl.Dalsney. confocal microscopy, a secondary flu- components usmg double-stammg orescently labclled antibody that rec- procedures. If two or more fluor- H”““‘aryA~“~“r”r *\mr,slP n,.il.irtnll*~ll 111 oyy, MnnarlI ognizes the primary antibody is often escent markers are used, speci- F,,n,n~,,r,, IIII.Y*
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Box 1. Examples of types of confocal -

Types of confocal microscopes Confocal microscopes that use single-point scanning and detection require both a bright light source and a means of building up an image from the light detected at the point detector. The increasing availability and affordability of suitable lasers and sufficiently powerful computers have contributed to the development of the laserscanning confocal microscope’-‘. An alternative class of confLval microscopes, instead ‘lses light that of scanning a single illuminated 4 passes through a spinning hlipkow disk, which contains thousands of small apertures arranged in spira!?. A recently developed tvpe of confocal microscope YES, an opiical fibre or fibre bundle through which light passes for both confocal illumination and detection”L12.

end can be -ezdily miniaturized and made portablel+l~. Therefort.!, fibreoptic confocal microscopes may be particular! J suited

Advantages and disadvantages of the above types of confocal microscopes are summarized in the Table. Following excitation of a fluorescent molecule, there is a lag time before emission of light occurs. For conventional laser-scanning confocal microscopes when used in fluorescence mode, this lag time imposes an upper limit to the speed of imaging (for example, 4-16 frames per second at relati\rely low resolutions). Nipkow-disk-based confocal microscopes usual!y use a conventional white light source, and allow direct-view real-time imaging in colour. However, since only (1.25-1’2 of the light of illumination passes through the disk, images may be very dim. Extremely senbzitive detectors, image intensifiers and/or specialized image,processing techniques are often required*. Fibre-optic confocal microscopes are similar in many rtzpects to conventional laser-scanning instruments, and can achieve almost identical resolutionI’. By contrast with the latter instruments, fibre-optic instruments eliminate many of the problems associated with optical alignment of bulky laser, beamsplitting and detector components. Only scanning optics (for example, galvanometer mirror scanners and lenses) are required at the end of a flexible

in the same specimen.

In situ hybridita tiorz histockemisfry la .sihr hybridization histochemistrv allows the detection of the position <),fnucleic acid sequences within cells. In neuropharmacological studies, it is of particu!.>- use for identifying cells that express certain receptor types, neurotransmitter-related enzymes or other relevant proteins or pcptides. Radioactively iabelled oligonu&?tide probes are often used for ia &II hybridization with detection by film or emulsion autoradiographic procedures. However, non-isotopic procedures using

optical fibre, and so the imaging

for imaging of subsurface

structures

irl zviz!cJ.

Types of confocal imaging Fhorescerrce irnngirrg For laser-scanning confocal microscopes the most commonly used lasers are the argon ion laser, the argon-krypton laser, the helium neon la!.er and the helium cadmium laser. Fluorescein and som- related fluorescent dyes are particularly suited for us? with argon ion and argon-krypton lasers. Texas red arid rhodamine can also be used, although impmvrd mo,re laser-compatible fluorescent dyes are being developled 1J4.By the simultaneous use of more than one laser (or more than one line in a single laser) with appropriate detection techniques, two or more fluorescent dyes may be imaged Reflectance irringirlg Nipkow-disk-based confocal microscopes are particularly suited to reflectance imaging, for example, in living animals, since they use white light+. Laser-scanning confocal microscopes c;tn also be used for reflectance imaging, but the image obtained will depend on the wavelength used. They are suitable for reflectance imaging of colloidal gold (which can be used in immuno-cytochemical labelling and irr sitar hybridization), and certain unstained living tissues (for example, rabbit cornea”).

A major advantage of confocal micnJscopy (compared with conventional microscopy) is the relative ease of generation of three-dimensional (3D) images, which may be obtained by performing a series of parallel 2D scans, followed by computerized image-reconstruction techniques. To produce thin optical sections (‘Iorexample, -0.4 km for reflectance imaging) for good 3D image resoluCon,

biotin, digoxigcnin, enzyme-labelled reporters or other types of reporters (‘reporters’ enable localization of oligonucleotide probes) are generally quicker, more convenient and allow greater cellular resolutionI*, particularly if the probes are located by confocal microscopy”‘. A disadvantage of using non-isotopic probes may be a reduced sensitivity of detection compared with isotopically labelled probes. However, with the use of multiple digoxigen-labelled or alkaline phosphatase-labelled probes, sensitivities at least equivalent to isotopic irr sit:! hybridization have been obtained4

There are a number of techniques thaatcan be u;ed for detection of nonisotopically labelled oligonucleotide probes by confocal microscopy?‘. Theseinclude: (l)digoxigenin-labelled probes and anti-digoxigenin antibody’?; (2’14-chloro-2-methylbenzene diazonium salt (fast red TR) in combination with alkaline phosphatascl’; (3) biotin-Melled probes and strerjtavidia-firuorescein-5-isothiocyanate (FITS) (Ref.24);and (4) reflectance confocal microscopy using streptavidin colloidal gold with silver enhance mentl“. Because fluorescent markers an: not used in the latter method, plra tobleaching is not a problem.

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microscopes and imaging Table. Advantages and disadvantages of particular types of confocal microscopes Type of confocal microscope

Advantages

Disadvantages

Conventional scanning

Good confocal sectioning and resolution Good sensitivity

Limit to speed of fluorescence imaging Type of laser limits dp selection’ Photobleaching of fluorescent dyes”

Nipkow-disk-based

Direct view real-time imaging in colour Use with full range of fluorescent dyes (little or no photobleaching]

Images are dim Optical sectioning may be compromised owing to relatively large holes in Nipkow disk (e.g. 20-60 km)

Fibre-optic scanning

As for conventional laser scanning Imaging end miniaturizable and portable Fewer requirements for precision engineering

As for conventtonal

laser-

laser-

laser scanning

,lMany mstruments not sulted to Imaging UV-excitable dyes such as furad aThis may be at least partly overcome by using a combination of low laser power and sensltlve detectton systems

lenses of high numerical aperture are required. The majority of these lenses have a close working distance, which may limit the maximum depth of the 3D image obtainable. In addition, since light is absorbed and scattered when passing through layers of the specimen above the focal plane, appropriate corrections may be required, because optical sections obtained deep within a specimen will be darker and of lower resolution than surface sections. For 3D fluorescent imaging, antioxidants can be used to reduce photobleaching, which may be a problem during acquisition of large data set+. References 1 Carlsso~~, K.

I

cf RI. (1985) O/t/. Lctf. 10,5.3--55 2 Wilke, V. (1985) Scmrrrin,q 7,88-96 3 Carlsson, K. and Aslund, N. (1987) AI@. Op. 26,3232-3238 4 White, j. G., Amos, W. B. and Fwdham, M. (1987) 1. Cc/l RioI 105, 41-48

Confocal imaging in

living

animals Examination

of

the

effects

of

drugs and toxins at a microscopic level in subsurface tissues of living animals or humans could potentially provide significant new pharmacological and toxicological insights. For example, the effects of therapeutic strategies on subsurface microscopic pathology could be monitored irt zGzw. Confocal microscopy (particularly using optical fibres) has t!lc potential to provide such new insights?5-?H. The fixation, sectioning and processing procedures involved in

5 Egger, M. D. and P&an, M. (1967) Scimc 157,305307 6 Petran, M., Hadravsky, M., Egger. M. D. and Galamhos, R. (196X) /. 0/1t. Soi. AN 58, 661-664 7 Boyde. A. (1985) Sc%*rw230, 1270-1272 6 Jester. .I. V., Andrews, I’. M., Petroll, W. M., Lemp, M. A. and Cavanagh, H. D. (1991) 1. El~ctnm Mic-rose. Tt*~~rr~iq~rc~ 18, SO-60 9 Xiao, G. Q~, Cork, T. R. and Kino, G. S. (1987) Proc. 4r. Pln~fo-Op. hslr. EII~. 809,107-113 10 Delaney, P. M., Harris, M. R. and King, R. G. (1993) C/in. E.Y~.P/mrrmcol.P/y~iol. 20, 197-l 98 11 Delaney, I’. M., Harris, M. R. and King, R. G. (1993) AppI. Op!ics 33, 573-w 12 D&mey, I’. M., Harris, M. R. and King. R. G. (1993) Clijr. E.y. P/larumt.ol. Phy5iol. (SuppI.) 1, 20

Clrcwicxb(5th edn), Molecular Probes 15 Masters, 8. R. and Paddock, S. (1990) 1. Mirnac. lS8,267-274 16 Ciloh, H. and Sedat, J. W. (1982) Sci~*nn~217,1252-1255

preparing samples for conventional light and electron microscopy often produceunwanted or even unknown artefacts. In addition, it is difficult to gain an appreciation of the dynamic nature of cellular and subcellular processes using these techniques. Confocal microscopy is particularly suited to ia z~izw imaging, although relatively few studies have been performed in living animals. One prob lem encountered is the movement of tissues in animals resulting from respiration, cardiovascular pulsations and other muscular movements. ‘Real-time’ imaging (2 25 frames per second) using Nipkow-disk-based

confocal microscopes can capture images without movement-induced artefacts, although it may be necessary to perform frame-averaging of selected stationary frames to reduce noise and improve resolution. Laser-scanning confocal microscnpy of living tissues is also possible. However, because living cells are prone to light-induced damage, particularly if labelled with fluorescent marker+!, care must be taken to prevent damage that might occur with prolonged exposure to scanning by light from a laser used at a high power setting. Following intravenous injection of fluorescein, 3D

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bl5min

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fig.2 Fibre-optrc,confocai imaging of the subsurfacemicrovasculatureof the gingiva of an anaesthetized rat in viva at the times indicated following i.v. administration Of fluorescein-5isothiocyanate(FITC)-dextran1150klla). Each Image represents a full three-dimensionalview of the microvasculatureand was constructed from 20 individualscans (each having 1Opm depth of field) spannmg1ODpm depth beneath the surface cf !he gingiva. Endothelin 1 (3 pg kg I i.v.1 was administered at t = 19min (i.e. immediately after obtaming Image c). 8: b: and c: little change in blood-vessel diameter was observed at times up :o 19 min after administration diameter were observed to constnct visibly fallowmg adminrstratlon of endothelin 1

background

of FITC-dextran

d: e: and 1: Vessels of 20-70 km

noise

Position fig.3.Image analvslspermits quantification

of vascular responses. a: Bysetting a threshold mtensrty value above the background noise ir a three-dimensional (30) image of a region contarnmg a vessel oi specrfred srze. and assummg lntensitres above this value to be attnburable to intravascular dye. the proporbon of the area of the image that is intravascular Irepresentedm b bythe red reyon In the green box) can be calculated The relative change In this parameter provides a useful Index of vascular response [e g. 46.6 + 14.5% change for vessels of 20-90 pm drameter approxrmately 15mln after endothehn 1 13 kg kg ’ I v ) n = 41. Such analysrs is performed only on vessels not at the edges of the 3D image.

reconstruction of the rat brain cortical microcirculation has been achieved using Alorescence-iaserscanning confocal microscopy through a glass window created in the skulP. These studies have since been extended by measurement not only of microvascular morphology and capillary haemodynamics, but also of leukocyte behaviour”,J2 Huorescence-scanning confocal mi-

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croscopy with 3D reconstruction has also been used to assess the dynamics of the terminal arboriz-, ations of individual retinal ganglion PIIS (for up to five days) in develop ing larvae of the frog Xer~yrrs”3. Fibre-optic confocal microscopy irr iriilo may be used for fluorescence imaging of blood vessels in the anaesthetized rat following intravenous injection of FITC-conjugated dextran

(Figs 2 and 3). This technique permits imaging of microvascular structure up to 300 Frn beneath the tissue surface?g. It can also be used for fluorescence imaging of the colonic mucous glands in the anaesthetized rat following topical application of fluorescein?“, eosin or mercurochrome”J. Furthermore, a miniaturized fibreoptic-bundle confocal microscope has been used for imaging of human

CURRENT tongue and skin in zliz~following topical application of fluorescein and/or eosi+. In these studies, movement artefacts have been minimized by gentle apposition of the moistened tissue onto a warmed glass surface at the imaging end of the fibre-optic instrument.

Other examples of confocal imaging suitable for pharmacological research Following intracellular recording from neurones in brain or spinal cord, the individual neurone can be mapped in 30 by confocal microscopy after injection of markers such as the fluorescent dye lucifer yellow’2 or horseradish peroxidase followed by staining with nickel-intensified diaminobenzidine (reflection imagingWh. Confocal microscopy has been used to identify atypical 3D structure of pyramidal cells in epileptic human cortex”‘. Various secretory processes have been examined by confocal microscopy. For example, studies have been made of exocytosis in living salivary gland tissue?“, of microtubule dynamics in cultured cells injected with rhodamine-labelled tubulin3y, and of the uptake and of photosensitizing localization porphyrins in cerebra! glioma turnours”‘.

Possible future applications in pharmacology To date, confocal microscopy has been relatively under-used as a methodology in pharmacological research. This may be due in part to the expense of conventional laserconfocal microscopes. scanning However, new fibre-optic systems are now somewhat more affordable than conventional ones, and the falling prices of computer and laser hardware may also help reduce the cost of confocal instruments. Developments in instrumentation may open up new applications for confocal microscopy. For example, by extending the spectral range of laserscanning instruments to UV waveIengthsJl-J”, a greater selection of fluorescent dyes can be used, resolution

TECHNlQuES is improved, and, because many biological molecules are themselves fluorescent under UV light, autofluorescence imagin; could yield valuable information. Laser-scanning instruments that are partially confocal and are suitable for reai-time fluorescence imaging have been developed”Q”. In addition, technical advances may lead to improvements in resolution of confocal microscopy-‘“,a7.The use of confocal microscopy may allow further research on how 3D structure is correlated with function, for example, in the study of neurones in epilepsy and the effects of antiepileptic drugs. Further use of confocal imaging in living animals is a particularly exciting prospect, and may enable visualization of previously unobserved biological phenomena. Fibre-optic confocal endo-microscopes may in the future facilitate the diagnosis of subsurface pathology irr ZCO, and allow the monitoring of disease processes and therapeutic interventions. The results obtained may be of greater biological significance than many conventional microscopic techniques in that imaging is possible in three or four rather than two dimensions. Selected references 1 Minsky,M.(1961) US Pnfc~rt3.013.467 2 Brakenhoff, G. j., Blom, I’. and Bar~nds, I’. (1979) J. Miuux. 117,219-232 3 Davidovits, P. and Egger, M. D. (lY71) /l\@. C$t. 10, 1615-1619 4 Williams, D. A. (1990) Cdl Cdci77777 11, 589-597 5 Williams, D. A., Delbridge, I.. M., Cody, 5. H., Harris, P. J. and Morgan,T. 0.11YY2) A717.1. Pllysiof. 262, C731-C742 6 Delbridge, L. M., Harris, I’. j. and Morgan, T. 0. (1989) Cli~r.Exp Plysiol. Pl7flr777ml.16, 179-184 7 Parker, I. and Ivorra, I. (1990) Scic71cr 250, 977-Y79 8 Parker, I. and Ivorra, I. (lYY3) J.PIpsid. 461, 133-165 9 Cheng. H., Lederer, W. J. and Canncll, M. 8. (1993) Scicrra 262,740-744 10 Lipp, I’. and Niggli, E. (1993) 0II Cokin~ri 14,359-372 11 Lipp, P. and Niggli, E. (1993) Biopk!/.~. J.65, 2272-2276 12 Niggli, E. ct nf. (1994) Artr. 1. Pkysit7l.266, c30.3-C31u 13 Carlsson. K., Wall+n, I’. and Brodin, L. (19X9) /. Mirr77sr. 155, 15-26 14 White, N. S., Lackie, P. M. and Shottun, D. M. (1989) Cl-I/ Biol. ht. RIP/I.13,941-94X 15 Shotton, D. and White, N. (lW9) Trt*rids

Hil~c/7r77l. Sii. 14, 435439 16 Brodin, I.. cl ,,I. (19RR)Cqr. HnrrrrRri 73, 441-446 I7 Schweitzer, E. S. and Paddock, S. (1990) 1. &,I/ Sci. 96, 375-381 18 Emson, P. C. (1993) Tnwf~ N[*II~IIXI. 16. Y-16 19 van den Brulc, A. J. C. 1-fof. (1991) A777.1. Pl7’/7Of. 1.79.1037-l 045 20 Emsun. I’. C., Heppelmann, 8. and Augood, S. J. (19941in It! Sllrr Hylvi1l7~1~i~711 (Eberwine, J. H., ed). Oxford University Press 21 Ballard, S. G. (1990) Pmr. Sot. Pkofo-Opf. Inst. Eq. 1205,2-10 22 Cripe, L., Morris, E.and Fulton, A. B. (1993) Proc.NotI Ad. Sri. USA 90,2724-2728 23 Sprel, E. J., Schutte, B., Wiegant, J., Ramaekers,F. C. and Hopman, A. H. (lY92) 1.Hishlrlrnn.Cytod~rr~~. 4b, 12Y9-l ?OA 24 Bauman, J. G. J., Bayer, J. A. and van Dekken, H. (1990) J.Microsc.157.73-81 25 Delaney. I’. M., Harris, M. R. and King, R. G. (1993) Cli77.ESJI. IJ/mr777m7/. P/~,~/t;iol. 20, 197-198 26 Delaney, P. M., Harris, M. R. and King, R. G. (1993) Cfb~. EXJI.Plmrtlmrc~l. Plr,~/siol, ISuppl.) 1,20 27 Delaney, I’. M., Harris, M. R. and King, R. G. (1994) A,@. OPlirs 33,573-577 28 Delaney, P. M., King, R. G., Lambert, J. R. lnd Harris, M. R. (1994) /. Armh~rrry184. 157-160 29 Vigers. G. I’. A., Coue, M. and McIntosh, I R. (1988) J. ClslfE&l. 107,1011-1024 Bloo11 FkIit 30 Dirnagl, U. cfnf. (I 991) J.C~*rcl~ml Mrtnb. 11,353_3Mi 31 Dirnagl, U., Villringer, A. and Einhtiupl, K. M. (1992) 1.Miirosc. 165, 147-157 32 Villringer, A. ct 711.(1991) Micror’~lsi.Rcs.42, 305-315 33 Fraser, S. E. and O’Rourke, N. A. (1990) 1. Eql. Biol. 153, 61-70 34 Papworth, G. D., Delaney, I’. M. and King, R. G. (1YY3) Clia. C.~I~Pliriri~l~i~~tJ. i’i7,7~str7l. fSuppl.) 1,55 35 l&itch, J.S., Smith, K. L., Swarm. J. W. and Turner, J. N. (1990) /. Mkro~r. 160.26%278 36 Deitch, J. S., Smith, K. L.,Swann, J. W. and Turner, J. N. (1991) /. Ekcfrorr Misrosz.Tcclr777077118, R?-90 37 Bdichencko, P. c’l711. (lY92) i%~rmx‘7wrf 3, 765-768 38 Segawa, A., Tcrakawa, S., Yamashina. S. and Hopkins, C. R. (1991) EII~. J. Ccl1Viol. 54, X2-330 39 Mcrdes, A., Strlzer, E. H. K. and dc Mey, J. (1991) 1. EkH,ntrMirror. Il;rl~r~rqr~c 18, 61-73 Hill, J. S. 1.1nl. (lYY2) Pnti. Nl7fI AiflnB.!?i. USA 89,1785-17X9 Amdt-Jovin, D. J., Robert-Nimud, M. and Jovin, T. M. (1990) J, Micros.. 157, hi-72 Montar. M. (‘1 (11.(lY91) 1. Miuw. 163, 201-2lii Ulfhake, B., Carlsson, K.. Mossberg, K.. Arvidsson. U. and Helm, P. I. (1991) 1. N~~rrn7~ri.‘M~~l/rcIds 41, 394 44 Lichtman, J. W., Sunderland, W. J. and Wilkinson, R. S. (19X9) NrrclRioI. 1, 7.5-82 45 Bcnedetti, P. A., Evangelista,V..Cuidarini, D. and Vestri, S. (1992) J. Micnai. 165, 119-129 46 McCallum, B. C. and Rudenburg, J. M. (1992) Ullmr77ir~naq7!( 45, 371-3Hll 47 Young, M. R. 1stII/. tlYO2) J. Mirnl\c, 165. 131-13x

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