Original Paper

Re¯ection contrast microscopy (RCM): a forgottentechnique?

T. J. Filler* and E. T. PeukerInstitute of Anatomy, Westphalian Wilhelms-University, Vesaliusweg 2±4, D 48149 Muenster, Germany

*Correspondence to:Dr T. J. Filler, Institute of Anatomy,Westphalian Wilhelms-University,Vesaliusweg 2±4, D 48149Muenster, Germany.E-mail: ®ller@uni-muenster.de

Received: 1 March 1999

Revised: 12 August 1999

Accepted: 26 October 1999

Abstract

Re¯ection contrast microscopy (RCM), which utilizes the optical phenomena caused by oblique

epi-illumination in combination with a speci®c optical apparatus, provides an approach for

exploring biological phenomena in greater detail. The lack of stray re¯ection makes it superior to

other microscopes. It bridges light and electron microscopic capabilities by allowing the analysis

of ultrathin sections beyond the usual light microscopic magni®cation. By using consecutive image

analysis, quantitation can be achieved. The wide range of applications of RCM can be combined

with most microscopical techniques, so extending the spectrum of information that can be

gathered. Twenty-®ve years after the development of RCM, there is still scope for its application

in modern cell biology. Copyright # 2000 John Wiley & Sons, Ltd.

Keywords: epi-illumination; interference; re¯ection contrast; microscopy

Re¯ection contrast microscopy (RCM) was originally

developed for epi-microscopy in mineralogy. After its

initial application in cell biology by Curtis in 1964,

following the studies of van den Tempel in 1958 and

Vasicek in 1960, and despite improvements in the

apparatus in 1975, RCM has found little application in

the ®elds of biology and medicine [1]. RCM ®rst

started to thrive between 1973 and 1976, with the

publications of Lochner and Izzard. Patzelt developed

the ®nal technique, up to the production stage,

together with Ploem in 1976 [2±4]. Although publica-

tions concerning RCM have always appeared in

reputable journals, up to the 1990s only a mere ®ve

workshops had been devoted to this subject. At the

same time, Gingell had developed a similar procedure

using the confocal laser scanning microscope [5].

During the last few years, studies have been directed

increasingly towards the application-orientated use of

RCM, with less of the methodical orientation

expounded in earlier publications.The initial discussions regarding `best re¯ection

contrast microscopy' led to irregularities in de®nitions.

BA

Figure 1. (A) Re¯ection contrast microscope (Leitz DIAVERT): (1) beam-splitter; (2) analyser; (3) polarizer; (4) ®eld diaphragm; (5)central stops according to Stach; (6) ®lters; (7) high-pressure Hg lamp for epi-illumination; (8) re¯ection contrast objective withrotatable lambda quarter plate on top of the front lens; (9) specimen; (10) trans-illumination. (B) Optical path for suppression ofstray light: (a) polarizer; (b) lambda quarter wave plate for elliptical polarization; (c) analyser; (1) incoming light (e.g. 546 nmwavelength); (2) linearly polarized light; (3) elliptically (circularly) polarized light; (4) re¯ecting structure; (5) re¯ected light; (6)realigned split waveparts; (7) linearly polarized light perpendicular to the incoming beam; (8) outgoing light without stray re¯ectionsfrom the optical apparatus

Journal of PathologyJ Pathol 2000; 190: 635±638.

Copyright # 2000 John Wiley & Sons, Ltd.

Different nomenclature was used in publications,involving terms such as interference contrast, inter-

ference re¯ection contrast, re¯ection interference con-

trast, surface re¯ection interference, or surface contrast

microscopy. The three relevant phenomena, epi-illumination, re¯ection, and interference, were thus

given a different signi®cance by the various groups,mostly American authors employing Zeiss apparatus.

There is no strict requirement for this nomenclature,

because in this device interference depends on re¯ec-

tion. The use of the word interference sometimes leadsto confusion with procedures such as DIC (differential

Figure 2. (a) Complete reconstruction from RCM images. Rat sciatic nerve, 2.3% glutaraldehyde, OsO4, Richardson staining,semithin sections. (b) Part of a; bar=3 mm. (c) Myelin sheath identi®cation by image analysis of b. (d) Axon identi®cation by imageanalysis of b. (e) Subsequent ultrathin section of b; TEM

636 T. J. Filler and E. T. Peuker

Copyright # 2000 John Wiley & Sons, Ltd. J Pathol 2000; 190: 635±638.

interference contrast, as in Nomarski optics), TSLRM(tandem scanning light re¯ecting microscopy), andTIRC (total internal re¯ection contrast).

Fundamental to RCM is the suppression of strayre¯ections, which cannot be found to such a degree inany other microscope and by which the optical effect iscreated. By these means, the images received can beinterpreted as being exclusively from the section. Forthis purpose, it is possible to utilize monochromaticlight (in the RCM, usually 546 nm) to create quanti®-able interference. The idea of an annular light source(central stop, according to Stach), which, however,blocks out up to 90% of the light and therefore needsvery strong sources, originates from the re¯ectancemicroscopy of coal [6]. The application of a polarizercombined with a lambda quarter wave plate forelliptical polarization and re-polarization of the frontlens, as well as a suitably aligned analyser, allows onlythe re¯ections from the section to reach the eyepiece(Figure 1). The re¯ections are created with 45u epi-illumination; this combination is described by Leica asthe Ploemopak epi-illuminator [7]. The conical, annu-lar illumination combines the characteristics of dark-ground illumination. By using RCM, it is possible toportray the structure of ultrathin sections by re¯ectionbeyond the useful range of magni®cation, down to5 nm [8,9]. For this reason, the technique provides abridge between light and electron microscopy.

RCM is best known for its use in in situ hybridiza-tion and immunocytochemistry [10]. With a penetra-tion depth of up to 30 mm and an extremely narrowdepth of focus in object space, there is the possibility of3D reconstruction from thick sections. In additionto the speci®c image of ®xed and stained material,un®xed cells show selective re¯ections of sub-cellularcomponents, so that it is possible to carry out imagequantitation as well as functional experiments(Figure 2) [11±13].

The re¯ection of border surfaces allows the visuali-zation and quantitation of adhesions through theformation of interferences of zero order. Interferencesare also created by re¯ections on surfaces inclinedtowards the optical plane. The isohypsis of the maximaand minima represented in such a way allows anevaluation in the third dimension. The interpretationof interferences which develop on neighbouring sur-faces, such as opposite cell membranes on extremelythin cell outlets, is much more time-consuming. This isof particular interest concerning unstained native cells.Due to the fact that other illuminative microscopictechniques may be combined with RCM simulta-neously and serially, the spectrum of informationrelevant to any particular question can be extended;for example, ¯uorescence microscopy concerning topo-graphical and functional aspects. This has in particularbeen implemented on chromosomes, and in the initialstages of the analysis of focal contacts (Figure 3) [14].

There are many further possibilities beyond theapplications mentioned above. Elliptical polarizationhas so far not been utilized for experiments regarding

the optical quality of structures and stains. In thecontemporary generation of microscopes, the rotationof the lambda quarter wave plate for analysis is nolonger implemented. Manoeuvrable analyses are onlyconducted sporadically. The analysis of cell substratespaces has been almost exclusively researched bytheoretical methods. Important improvements tomicroscopes during the last decade have been madeprimarily to the optics, the correction to in®nity and inoperator convenience. The latest trend in the develop-ment of the microscope lies in the combination ofdifferent techniques in order to have access to moreinformation.

From its beginning, the re¯ection contrast micro-scope was designed to be used in parallel with otherilluminative microscopic techniques [15]. Twenty-®veyears after the development of the RCM, there are stillquestions which could bene®t from its application.

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