Reflection analysis: a new optical method. Evidence of mitotic inter-chromosomal RNA

J. Lejeune, M.C. de Blois, M.O. Rethoré, and A. Ravel

Reflection analysis: evidence of mitotic inter-chromosomal RNA. Cytogenet Cell Genet 41:54-55 (1986)

Résumé :

Abstract. Giemsa-stained chromosomes, epi-illuminated with white light produced a brilliant image. The chromatids were yellow-green and the areas between the chromosomes contained tiny orange particles. This inter-chromosomal network was selectively removed by RNase treatment.


In conventional microscopy, stained and trans-illuminated objects absorb the light at typical wavelengths. In reflection analysis, only the reflected light is used to produce the image (Lejeune, 1985). The necessary epi-illumination is provided by a semi-reflector prism which deflects the white light on the axis of the microscope. Hence the incident beam reaches the slide through the objective. Diaphragms, when positioned correctly, remove most of the stray light, so that only reflected light at optimal wavelengths participates in producing the image. The first device was a modified fluorescence epi-condensor, but a metallographic prism with collimation of the incident beam also gives excellent results (Zeiss).

A standard Giemsa-stained preparation was ob-served with an oil immersion planapochromatic objec-tive (100X). The chromatids reflected very brilliant yellow-green light (roughly the complementary color of the purple seen with conventional transillumination). The image was similar to that of ultraviolet fluorescence after acridine orange-staining, but it was much more brilliant and perfectly stable. Repeated observations were made and photographs were taken without fading of the image.

Careful adjustments of the beam diaphragm and of the field diaphragm produced a quasi-dark-field. Thus a highly reflecting object could be detected as a lumi-nous point even if its size was below the theoretical resolving power. This was especially useful when elon-gated prophase chromatids were analyzed after fluorochrome plus Giemsa staining (Lejeune, 1985).

Staining. Metaphase preparations from 72 h lymphocyte cul-tures were spread on slides by standard techniques. The slides were then stained for 5 min in 3% Giemsa in phosphate buffer, pH 6.5, and gently rinsed with distilled water. Overstaining was avoided because it resulted in green reflected light, less brilliance, and poorly-defined chromosome contours.

Destaining. Removal of immersion oil and complete destaining were obtained by dipping the slides in absolute ethanol for 10-15 min.

RNase. treatment. Boiled ribonuclease (RNase I from bovine pancreas. Sigma) was diluted in distilled water to a concentration of 0.01-10 mg/ml. One drop of this solution was deposited on a slide and protected with a coverslip. Incubation was in a humid chamber at 37 °C for 1 h. The coverslip was gently removed by rinsing with distilled water.

When Giemsa-stained metaphase preparations were viewed with reflected light, then, in addition to the yellow image of the chromatids (Lejeune, 1985), very tiny granules of an orange coloration were visible around the chromatids. These granules occupied the entire space between the chromosomes, but they did not extend outside the nucleoplasm. The granules were organized in a filament-like network which ap-peared to connect the chromosomes. Although a care-ful study of these links was attempted, it is not yet clear whether their spatial distribution is random or has a precise specificity. The appearance of the metaphase spreads was the same when Giemsa-staining was followed with RNase treatment.

On the contrary, when unstained slides were first treated with RNase and then stained, the chromatids appeared yellow, their contours contrasted very sharply with the dark background, and very small, yellow extensions were seen outside the chromatids, but the orange granular material was not detectable. The same results were obtained when Giemsa-stained slides were first photographed, then destained in ethanol, RNased, and stained again.

These experiments, repeated several times with var-ious preparations, lead us to conclude that the orange inter-chromosomal material consists of ribonucleic acid (RNA). The fact that pre-stained slides were RNase-resistant, but became sensitive again after de-staining, suggested that on fixed material, the stain prevented access of the enzyme to the RNA.

Reflection analysis revealed the presence of an RNA network in metaphases. This network seemed to emanate from the chromatids themselves which were surrounded by and connected to the network. We did not determine whether this RNA material was a rem-nant of a messenger RNA synthesized before the start of DNA synthesis, or whether it was a structure of the interphase nucleus, or even an RNA newly syn-thesized during mitosis. Further experimentation should allow a differentiation between an early or a late synthesis of this RNA-network. However, only careful analysis of the network itself will show if there exists a topological specificity. Awaiting these experi-ments it seems already evident that reflection analysis can greatly facilitate some cytogenetic investigations.

We thank Mrs. Gavaini, Maunoury-Burolla. Picq, and Miss Schlienger for their expert technical assistance.

Note added in proof: This optical phenomenon has been discussed precisely by Van der Ploeg M. Van Duijn P: Reflection versus fluorescence. Histochemistry 62:227-232 (1979).


Lejeune J: L'analyse par reflexion. Nouvelle methode d'observation des chromosomes humains. Annls Génét 28:67-68 (1985).