NDT.net • June 2006 • Vol. 11 No.6

Noninvasive Color Visualization of Blood Cells

Viktor Krakhmalev, Adkham Paiziev
Institute of Electronics Uzbek Academy of Science, Tashkent. Uzbekistan.
F.Khodjaeva str.33. E-mail: adxam_payziev@rambler.ru


Our understanding of biological organization of a live matter and its cellular process we can mainly get from microscopy. But visualization of a biological structures is based on interaction of electrons and photons with sample what leads to destruction of a sample. In some cases to get color contrast image of separate elements of biological sample need to use a variety of dyes and fluorescent substances but it leads to artificial staining of sample, destructive modification and loss very important structural information its native structure. For instance to get color image of morphological structure of blood smears are usually using staining of living blood smears by dyes. But this method leads to disruption of sample caused by the specimen preparation and viewing conditions [1]. Other ways of generating color image contrast is based on visualization of phase gradients within unstaining specimens, as realized by phase contrast [2] and differential interference contrast [3]. Usually in medical practice conventional bright-field microscopy let us see black and white image of separate morphological elements of blood smears only (Figure 1a).

In present paper we are offering the new nondestructive method of optical microscopy capable of examining the structures of living cells in their natural colors without staining them, using a specially designed substrate for deposition of biological sample and observing native structure in reflected light. This method based on physical phenomena of white light interference reflected from sample surface and special supporter on which this sample is deposited. As distinct from phase contrast [2] or differential interference contrast [3] microscopy there we have interference picture not for passed through sample and transparence object-plate two light rays but for two reflected light rays 1 and 2 on the sample surface and substrate respectively (Figure 2). It allows to occur at the image plane converting previously invisible gradients of refractive index within the specimen in to intensity gradients in the image.


FIGURE1. A, Bright-field image of erythrocytes of healthy individuals. B, Color image same blood sample. C, Erythrocytes of healthy individuals, We can see multilayer structure of interference pattern (1), distinct line of cell shell (2), and platelets (3). Different colors can be seen in certain areas of an individual red blood cell (4). D, E, Living erythrocytes of a patient with diagnosed core rectal cancer. Images show a multi-layered pattern of interference picture. Differences between the coloration in left (D) and right image (E) caused by different experimental conditions. We can see double cells (1) and damaged cells (2), both are pathologic.

Color interference contrast image is achieved due to special condition of experiment is connected with chose of angle of incidental light, wave length of light of reflected ray, chemical composition of sample, thickness of sample, refractive index of sample, refractive index of substrate, chemical composition of substrate.


FIGURE 2. Schematic layout to get interference in reflected light rays 1 and 2 on the transparence sample deposited on the no transparence substrate.

The setup for color reflected interference microscopy was centered around ordinary optical microscope (Carl Zeiss, Germany) equipped for digital photo camera (Sony) and substrate which serve as object-plate for sample and as source of coherent light for scattering on morphological structures of sample. Light from a 100 watt xenon source was directed on to the specimen. Microscopic images were obtained with Zeiss lens and digital camera and recorded on a personal computer using commercially available software. To demonstrate the potential usefulness of this method, we provide qualitative data describing color image of healthy and pathological damaged cells for alive and dry blood smears (Figure 1A-1E).

Comparison Figure1A and Figure1B for same samples but obtained by conventional bright-field microscopy and by using new method correspondently showed distinguishing in color not only separate red blood cells but distinguishing difference parts in area separate erythrocytes too.

Usually for healthily individuals a albuminous aureole around the erythrocytes are mainly white-yellow (Figure 1C), but for cancer cells (core rectal cancer) the aureole color is quite different and reflects significant changes in chemical composition of both internal, and external contents of erythrocytes (Figure 1D, 1E).

Easy detection of organic shells around blood cells in our case is evident. Operations by fixing, smear coloring, prolonged processing, the availability for phase-contrast or interference microscope, special illuminators, radiating the exciting short-wave light beams are not required. Interferometric coloring of blood elements occurs on a surface of specially selected substrate. Corresponding colored images of blood elements are formed due to interference phenomena occurring under interaction of light beams reflected from front and back surfaces of blood elements, smeared on a substrate.

As it is seen from the given micro photos, the character of the colored image is the same, as though they were investigated with phase-contrast or interference universal microscopes.

REFERENCES

  1. Kozlovskaya L.V., Nikolaev A.Yu. Textbook on Clinical Laboratory Methods of Investigations. Moscow. Medicine. 1984. 288p.
  2. Zernike F. Phase contrast: A new method for the observation of transparent objects. Physica 1942; 9: 686-693.
  3. Nomarski G. Microinterferometric differential a on des palarisees. J. Phys. Radium. 1955; 16; S9.

© NDT.net |Top|