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
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).