Journal of Microscopy, Vol. 130, Pt 1 2008, pp. 160–162 Received 18 October 2007; accepted 14 November 2007 An easily built diffuse illumination system effective at both very low and moderate magnifications, for observing in situ stained slides M . W. F RO H L I C H ∗ & E . M OY RO U D † ∗ Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, U.K. †Laboratoire de Physiologie Cellulaire Vegetale, CEA-Grenoble, France Key words. Diffuse illumination, in situ hybridization, light scatter, low magnification microscopy, microscope illumination. Summary Effective study of in situ stained sections often requires illumination that is difficult to achieve with commonly used research microscopes. One must be able to switch quickly and conveniently from the very lowest to moderate magnifications. At all magnifications contrast due to light scatter must be minimized, so that the weak staining that signifies low gene expression can be observed reliably. For the lowest power objectives (e.g., 1.25× or 2×) many microscopes require that the condenser be removed to illuminate the full field of view. This is not only very inconvenient when switching magnifications, but without a condenser the low numerical aperture of the illuminating light beam results in unwanted contrast due to light scatter. We have devised a simple system that diffusely illuminates the full field of view of the lowest power objective (1.25×) and has high enough numerical aperture for use with the 25× and 40× objectives. A key feature is the use of a large diameter ring light (internal diameter 5.8 cm), placed on the microscope base, to illuminate a large diameter diffuser placed just below the slide. Introduction In situ hybridization is widely used to determine gene expression patterns in plant and animal tissues. Some structures of interest are relatively large (>1 cm), and they often contain smaller structures (<100 µm) that are also of interest, so a range of magnifications from very low to moderate is needed for effective study and for photography. It is most convenient if one can switch through the range of magnifications without major adjustments to the illumination system. Correspondence to: Michael W. Frohlich, Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS. Tel. +44 20 8332 5378; fax. +44 20 8332 5310; e-mail: m.frohlich@kew.org With non-radioactive detection methods the colour signal may be subtle. For effective visualization, one must minimize alternative sources of visible contrast, such as differential light scatter by tissues of different cytoplasmic densities. Contrast due to light scatter is minimized by illuminating the specimen with a large cone of light (Slayter, 1970: 255), at a numerical aperture much larger than the objective lens can accept. This is especially important if the mounting medium for the specimen does not have a high index of refraction, close to that of cytoplasmically dense tissues. We typically view in situ slides in water mounts, which have a substantially lower index than the tissues, so minimizing contrast due to light scatter becomes especially important. Very low power objectives have been manufactured for many microscopes. The widely used Zeiss Universal microscope system has parfocal plan objectives available down to 1.25× magnification (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). The field of view can be further expanded with the 0.63× magnification nosepiece, which more than compensates for the minimal increase in magnification (1.25×) of the ‘optovar’ magnification adjuster in the light path. With a wide-field eyepiece the resulting field of view on the slide is approximately 18 mm across. Microscopes have traditionally been designed to optimize performance at the higher magnifications, not the lowest magnifications. Condensers typically cannot illuminate a very wide field of view. Even condensers with a swing-out top lens are unable to illuminate an 18-mm-wide field of view. Instead, one typically removes the condenser from the light path for the lowest magnification objectives, so illumination comes directly from the exit window of the light source in the microscope base. This does illuminate a very wide field of view. The numerical aperture of illumination is quite small (e.g. less than 0.07 in the Zeiss Universal). This is appropriate for most common applications because the increased contrast resulting from light scatter is desirable. However, this seriously impedes observation of the often subtle colouration of in situ slides.  C 2008 The Authors C 2008 The Royal Microscopical Society Journal compilation  D I F F U S E I L L U M I NAT I O N S YS T E M 161 Fig. 1. Illumination system. (a) Ring light placed on microscope base. Plastic bag diffuser is taped over the bottom of the paper cup, shown lying beside the ring light. (b) Illumination system in operation. Furthermore, the need to remove and reinsert the condenser when switching between the lowest and higher magnifications is most inconvenient. Materials and methods We have devised a simple method that fully and evenly illuminates an 18-mm-wide field of view on the slide, and does so at high enough numerical aperture (NA approximately 0.9), for this illumination system to be fully effective with objectives up to 0.65 NA, such as the Zeiss 25× planapo and 40× planacromat (NA = 0.65). With the 40× dry planapo (NA = 0.95) contrast due to light scatter is not suppressed, and illumination is appropriate for ordinary stained slides. Our system uses two layers of diffuser placed immediately below the mechanical stage. The diffuser is not illuminated by the microscope’s light source, but rather by a fiber optic ring light, intended for a dissecting scope, placed on the microscope base and pointed upward. The ring light surrounds the raised exit window on the microscope base, which holds the ring light in place (Fig. 1a). We use a 150-W light source at 3000 K. The most effective diffuser we have found is two layers of an ordinary opaque white plastic bag, of the sort provided at stores to hold purchases. This diffuser is taped over the cut-out bottom of a paper drinking cup that is mounted upside down on the ring light (Fig. 1b) and held in place by more tape. The cup is trimmed so the diffuser is positioned immediately beneath the stage. This illumination method has proved far superior to any of the Zeiss condensers we have tried, even the pancratic condenser; with top lens removed it illuminates the full field of the 1.25× objective, but does not have a high enough NA to minimize contrast due to light scatter by the specimen. The relatively large NA (0.9) of the illuminating light results from the broad exit angle of the plastic bag diffuser (much broader than other diffusers tested), from the 6 cm diameter  C 2008 The Authors C 2008 The Royal Microscopical Society, Journal of Microscopy, 130, 160–162 Journal compilation  of the diffuser and from its placement only 1.4 cm below the slide. The built-in microscope light cannot illuminate the full width of the diffuser, requiring alternative illumination. Axial illumination of the diffuser is not effective because some light passes straight through the plastic; one can look through the diffuser at a light source and see a dull red image of the light source. With axial illumination this reddish, directly transmitted light comprises too much of the illumination for the low-magnification objectives with small NA, resulting in an unacceptable colour shift compared with the higherpower objectives. With illumination by ring light, this directly transmitted light forms a cone at NA 0.28, so it does not enter the low-power objectives. This directly transmitted light is collected by the 10× planapo (NA 0.32) and by higher NA objectives, but they accept such a large cone of scattered light from the diffuser that the directly transmitted light has little effect on the colour balance. For photography, we use a Zeiss 6.3× photo eyepiece and an old Nikon PMF system with a ground glass focussing screen, due to the difficulty of focussing low-magnification images with a focussing tube. Our Nikon D100 (Nikon, Tokyo, Japan) has a CCD 23.7 × 15.6 mm, resulting in photographic fields of view 7.5 and 3.6 mm wide for the 1.25× and 2.5× objectives. Numerical apertures were calculated by dividing radius of illuminating aperture by hypotenuse of the right triangle with apex at objective lens and right angle at the centre of the illuminating aperture. Results and discussion The system provides comfortable and adequately bright viewing and reduced contrast due to light scatter with all objectives from 1.25× through 25× planapo. It is effective even 162 M . W. F RO H L I C H A N D E . M OY RO U D with the 40× dry planapo, though without reducing contrast due to light scatter. The ability to switch among all these objectives without adjusting the light source greatly facilitates study of our specimens. By minimizing contrast due to light scatter, we are better able to observe the signal from our nonradioactive in situs. Photographic exposure is the same for objectives 1.25× up to 10×, but the 25×, requires double the exposure. Illumination is uniform in brightness and in colour both visually and in photographs. Figure 2 shows two uncropped images of the same slide, using the 1.25× and 25× objectives. (The overstaining of this slide simplifies interpretation of the structure and the difference in magnification.) The system may be useful for observing other types of preparations as well; in particular, if one needs to switch rapidly between very low and moderate magnifications, or if contrast due to light scatter distracts from more informative contrast due to light absorption. Thin sections (or acetate peels) of permineralized palaeobotanical specimens are one such example. Acknowledgement We thank P. Webber and P. York for comments on the manuscript. Reference Fig. 2. Two photos of the same specimen (Welwitschia male cone in situ) made without altering the illumination set up. (a) 1.25× objective. Strong expression signal is visible in developing microsporangia with weaker expression in lateral meristems. Signal in young pollen is an artefact. Unstained tissue shows little contrast. (b) 25× objective. Apical meristem shows expression in incipient lateral meristems. Scale bar represents 1 mm for (a), 50 µm for (b). Slayter, E. (1970) Optical Methods in Biology. Wiley-Interscience, New York, London.  C 2008 The Authors C 2008 The Royal Microscopical Society, Journal of Microscopy, 130, 160–162 Journal compilation