Reflection contrast was developed by the Ernst Leitz Wetzlar Company in the seventies. It
is a microscopic technique which ensures optimal contrast on visualizing cell structures by means of reflected light.
When transparent specimens are examined in thin-layer preparations typical interference patterns occur in reflection contrast. Thus, erythrocytes show characteristic annular shaped interference lines consisting of
alternating maxima and minima. The bright maxima and dark minima mark cell regions of constant thickness in a manner similar to cartographic altitude lines or isohypses. When the examinations are carried out in
monochromatic green light, the difference in thickness between maximum and adjacent minimum lines is about 113 nm. Based on this, differences in local thickness are measurable. With the help of drawing modes used in
cartography, three dimensional reconstructions of cell reliefs can be carried out.
The interference lines differ in their position when specimens are illuminated in monochrome light with different (long and short) wave lengths. In this case, the absolute local thickness of transparent cells can be
calculated by specific interferometric formulas. When the path of the illuminating light beam is appropriately modified, the three dimensional reliefs of cells are also visible in
live microscopy. The resulting
three dimensional images are similar to interference contrast or scanning electron microscopy.
It can be considered to be an advantage of reflection contrast, that this method can be combined with all examination modes based on transmitted light (bright- and darkfield, phase and interference contrast,
flourescence microscopy), suitable for examinations of living specimens, too.
The measurements and three dimensional reconstructions mentioned above were carried out with normal erythrocytes and several pathological erythrocytes from patients affected with hereditary spherocytosis, sickle cell
anemia and thalassemia.
All methods for experimental cytometric measurements were elaborated in cooperation with the Institute for Anatomy, University of Bonn, section for light microscopy (head: Prof. Dr. Franz Pera) in 1978-1982.
The methods were carried out in clinical practice combined with systems for
automatic image analyses in cooperation with the section for angiology, University of Bonn (head: Prof. Dr. Truebestein), based on cases
with occlusive arterial disease, pernicious anemia and Raynaud's syndrome.
Erythrocyte in a stained blood smear, reflection contrast,
objective oil 100x, monochromatic green light (546 nm)
Stained erythrocytes in modified reflection contrast
objective oil 100x, monochromatic green light
Three dimensional reconstructions of normocytes and pathological erythrocytes,
phase contrast (left), reflection contrast (center), cartographic reconsructions (right),
unstained normocyte (above),
stained sphaerocyte, hereditary spherocytosis (central)
Target cell, thalassemia major (below)
Pera, F., Piper, J.: Quantitative morphological analyses of erythrocytes by reflection contrast microscopy.
Blut 41, 377-386,1980
Piper, J., Pera, F.: Reconstructions of the surface reliefs of erythrocytes with help of the Leitz equipment for reflection contrast.microscopy (in German)
Leitz-Mitt.-Wiss. u. Techn., Bd. VII, Nr. 7, 230-234, 1980
Pera, F., Piper, J., Kunde, V.: Morphometrc examinations of blood cell alterations induced by preparations in smears
Verh. Anat. Ges. 76, 147-148, 1982
Wilgalis, M., Pera, F., Trübestein, G., Ludwig, M., Piper, J.:
Morphological variability of erythrocytes in patients affected with occlusive arterial disease, pernicious anemia and Raynaud's
syndrome - microscopic examinations in reflection contrast using systems for semiautomatic image analyses (in German)
German Society of Angiology, 1982
Piper, J.: Qualitative and quantitative morphological analyses of normal and pathological erythrocytes by reflection contrast microscopy (dissertation, in German), 1983
Copyright: Joerg Piper, Bad Bertrich, Germany, 2010