Optimized Morphologic Evaluation of Biostructures by Examination in Polarized Light and Differential Interference Contrast Microscopy

Optimized morphologic evaluation of biostructures by examination in polarized light and differential interference contrast microscopy

Elena Patrascu1, Petru Razvan Melinte1, Gheorghe S. Dragoi2,*

______Abstract: Differential interference contrast microscopy (DIC) was possible after the invention of Wollaston prisms that were modified by Nomarski and could duplicate linear polarized light before passing through a specimen (Condenser DIC prism) and to recompose it afterwards (Objective DIC prism), thus creating the phenomenon of interference that depends on the thickness and birefringence of the studied structures. The authors proposed themselves to discuss the performances of this microscope and to widen the area of its usage in the optimized microanatomic analysis of cells and tissues on the native or stained sections. The authors consider it necessary to use DIC microscopy both in forensic medicine (identification of diatoms) as well as in reproduction biology (spermogram) and in early discovery of uterine cervix cancer. Key Words: DIC microscopy, Wollaston prism, Nomarski prism, polarized light, interference.

he progresses achieved in the area of wave (Maxwell, 1864) [2] and has a photogenic Timprovement and diversification of structure (Einstein, 1921) [3]. It consists of an electric optic systems determined the increase of photonic and a magnetic field, orthogonal, which vibrate in microscopes performances regarding the phase contrast, phase, in the direction of propagation. Within an polarized light and not least, differential interference electromagnetic wave, the electric and the magnetic contrast (DIC) examinations.They assure nowadays fields oscillate simultaneously, but in different planes. the optimization of biostructural assessment due to The usage of these characteristics brought to Einstein transformation effects of enlighten electromagnetic the Nobel Prize for Physics in 1922. waves when passing through optic prism systems, The usage of prisms as device for the study of which leads to personalized views. Our aim is to light phenomena - dispersion, refraction, reflection, highlight the performances of these microscopes polarization, diffraction and rays-of-light interference with the purpose of determining their capacity in the made famous various scientists. Newton (1660) [4] optimization of biostructures assessment, in ortology proved that white light, passing thought a triangular as well as in general and forensics pathology. prism, decomposes into “colored rays” (spectrum), which can recombine into white light when passing Stages in the history of practical and theoretical through a second prism. Rasmus Bartholin (1669) [5] acknowledgement of enlighten electromagnetic waves discovered the double refraction (birefringence) of the It is known that light has an undulate light when passing through “Iceland spatula” (Calcite). component (Huygens, 1678) [1]; it is an electromagnetic Louis Malus (1809) [6] established the polarization

1) University of Medicine and Pharmacy of Craiova, Departament of Anatomy, Romania 2) Romanian Academy of Medical Sciences, Bucharest, Romania * Corresponding author: Prof.MD, PhD, E-mail [email protected]

275 Patrascu E. et al Optimized morphologic evaluation of biostructures concept through reflection and refraction. William tube had the highest success. It was able to suppress Nicol (1823) [7] achieved the polarization of light the halo phenomena, inherent to phase contrast. F.H. using a calcite rhomboid crystal (“Iceland spatula”), Smith (1947) [14] was the first to use Wallastone cut under a 68 degrees angle, then sectioned in prisms in order to separate and recombine fascicles diagonal and reattached (glued) with Canada balm. of polarized light. Still, the system achieved by Smith The passing of the light through this prism determined had a major practical inconvenient: the prism used for the appearance of two rays: one that suffers a total fascicles recombination had to be placed in the frontal reflection at the level of Canada balm, called “ordinal plane of the objective, or, more specific, exactly inside ray” and another one which is completely transmitted, the objective - inaccessible places. The prism placement called “extraordinary ray” (Diagram 1B)). This prism problem was solved by Nomarski, using equally the invented by Nicol is a “polarizer”, widely used in Wallastone prisms for the externalization of fringes polarized light microscopy and in the interference and the placement of the prism at a certain distance contrast one (DIC). from the objective, in post-objective microscope’s The polarizer converts an unpolarized wave tower. This system determined with certainty the into linear polarized light, in the case of polarized improvement of interferential contrast microscopy. light microscope and DIC. The analyzer is a device The first microscopes with differential interference identical with the polarizer, but acts at the level of the contrast were called Nomarski microscopes; they were already polarized wave (Diagram 1 D). Wallastone produced by Carl Zeiss society, in 1965. From this (1766-1828) [8] invented a prism that transforms a ray moment on, they have been used in large scale in the of polarized or unpolarized light into two orthogonal scientific laboratories, due to the quality of obtained rays of linear polarized light. This prism is component images. They were also used for reflection system of DIC microscope prisms system (Diagram 1 C). (episcopic enlighten) and for transmission (diascopic Nomarski (1919-1997) [9] invented the differential enlighten). interference contrast microscope (DIC). He modified Nomarski (1919-1997) [9] contributed to the the Wallastone prism in order to lead the linear achievement of differential interference contrast (DIC) polarized rays of light into a focal point, exterior to the microscope, with applications in the microanatomic prism (Diagram 1 E). Diffraction and interference, as research of biostructures. It suppresses the halo markers of undulate characteristics of light, represent phenomena, which appears at the examination with the basis of differential interference contrast (DIU) the contrast phase microscope invented by Zernike microscopy. Experimental evidences have been (1953- Nobel Prize for Physics) [13]. Within the presented by Francois Arago [10] and Fresnel (1819) structure of DIC microscope, there are the following [11], for light diffraction and by Thomas Young (1773- optic systems: polarizer, Nomarski prism inside the 1829) for interference of the light. Huygens principle condenser, Nomarski prism inside the objective and (1678) [1] explains the diffraction phenomena. He the analyzer. The polarizer transforms source light into stated that every point of a wave front can be considered linear polarized light. The condenser prism separates a source of secondary spheroid waves and in a time (t), each ray of polarized light into two rays, perpendicular the new position of the wave front, will be at tangent to each other and separated through a distance lesser surface to the secondary waves. than a micron. Later, the two rays are submitted to Interference of the light is the overlapping a different phase shift, when they cross neighboring phenomena of two or more coherent light waves points of the sample. The prism of the objective, which (rays), in a certain area in space, which determines is placed in reverse to the one inside the condenser, the appearance of a stationary frame with minimal recombines the two phase-shifted rays, which cross and maximal fringes that repeats periodically in the the analyzer, perpendicular oriented to the polarizer same area. In order to obtain a stationary interference (Diagram 2). phenomena, the rays of light must have the same frequency and must be coherent, meaning, they must MATERIALS AND METHODS have a constant phase difference. In 1933, Frederic Zernike (1888-1966) [13], We selected fragments of bone, uterine discovered the contrast phase technique which he cervix and superior sympathetic cervical ganglion applies to optic microscope, in order to observe the neuroganglion and we evaluated them by examining internal structure of uncolored cells, preserving in polarized light and differential interference contrast in this manner, their integrity. The first contrast microscopy.The harvested fragments were fixed in 8% phase microscope (or interferential) was achieved in formaldehyde solution, buffered at 7.8 pH; afterwards 1942. Later, other systems have been developed. The they were embedded in paraffin and sectioned at 5 interferential device, placed inside the microscope’s microns interval. The examination was carried out

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C A

D F

Diagram 1. Transformation of electromagnetic waves inside optic systems: phase rings in phase contrast microscopy (A); Nicol prism in polarized light microscopy (B); Wollaston (W) and Nomarski (N) prisms in differential interference contrast microscopy (C-F). A – location of phase rings; B – transformation of non polarized light wave into a linear polarized wave; C, E, F – duplication of linear polarized light while passing through Wollaston prism (C) and Nomarski (D). (After Charlotte P. – Technique de generation en microscopie) [15].

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Diagram 2. Location of the elements of optic system in differential interference constrat microscopy and transformation of non polarized light into liniar polarized light (Nicol prism), duplication of linear polarized light (Nomarski I prism – Condenser DIC prism) and recombination of out of phase rays (Nomarski II prism – Condenser DIC prism). (After Davidson and Abramowitz – optical microscopy) [ 16]. using Nikon Eclipse 80i equipped with an infinity examination in interferential light and with a system of corrected optical system, with a system of optic Nikon objectives optimized for evaluation in polarized polarized prisms that can turn a light fascicle (natural light.The microanatomic imagery was performed in or artificial) into a linear polarized fascicle of light, Nikon Digital Sight DS-Fi1 High Definition Color with an optical system of prisms that can successively Camera Head using Nis Element Advance Research duplicate linear polarized light (Nomarski I prism) Software. Microphotographs were processed in Adobe and recombine out of phase rays (Nomarski prism II), Photoshop CS5 software. with a Nikon Lambda DIC nanocrystal objectives for

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Figure 1. The evaluation of osteon structure on Hematoxiline Eosine stained serried sections (A, C, E, G) and on non-stained sections (B, D, F, H) using diferential interference contrast microscopy (A-F) and polarized light microscopy (G, H). 1. Osteonic canal; 2. Osteonic concentric lamella; 3. Osteocyte lacuna; 4. Anisotropic lamellas; 5. Isotropic lamellas; 6. Cross of Malta. Paraffin section. Nikon Lamba onjectives DIC and optimized Nikon objectives for polarized light. Microphotographs taken by Digital Sight DS-Fi1 Definition Color Camera Head. 70x (B), 140x(A, D), 280x (C, E-H).

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Figure 2. Optimized evaluation of uterine cervix stroma (A, B), striate muscle fiber fascicles (C, D) and neuronal structures inside superior sympathetic cervical ganglion (E, F) in polarized light (B, D) and differential interference contrast microscopy (E, F). 1. Fascicles of picrofuxinofile connective tissue fibers; 2. Fascicles of birefringent connective tissue fibers; 3. Fascicles of striate muscle fibers; 4. Muscle fiber with isotropic and anisotropic bands; 5. Sympathetic neuron; 6. Perikaryon with powdery granulations; 7. Neurons in apoptosis; 8. Fascicles of neurofibers; 9. Terminal neuronal buttons.Paraffin section. Van Gieson stain (A), Hematoxiline Eosin stain (C), Reduced silver nitrate Nonidez’s block method (E, F). Examination using Nikon Lambda DIC and optimized Nikon objectives for polarized lights. Microphotographs taken by Digital Sight DS-Fi1 Definition Color Camera. 280x (A-C, E, F); 700x (D).

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around structures even when the samples are thicker PERSONAL OBSERVATIONS AND DISCUSSIONS than 5 microns. The performances of differential interference We tested the visualization abilities for contrast microscopy (DIC) are based on coupling biostructure examination using Eclipse 80i microscope different variations of light waves at the level of cell equipped differential interference contrast optical and extracellular environment. A peak of those can be systems (DIC). The serried sections that we studied a prominent structure or an area with a big refraction were obtained from fragments of bone, uterine cervix, index. A depression can be an orifice or a vacuole with striate muscle and superior sympathetic cervical a low refraction index. In the case of thick sections, ganglion. one can notice an increased resolution and in case of The serried sections through bone were thin sections, an increase of contrast.The usage of DIC stained with Hematoxiline Eosine and some of them microscopy can be expanded to visualize the diatoms as were not colored but removed from paraffin and a quality index for water and the eutrophic level through examined after the immersion in Canada balm. When nutrients (especially those containing phosphorus examining in interferential light the serried section and nitrogen) inside flowing waters, and in forensic stained with Hematoxiline Eosine, we easily noticed medicine to identify them inside immersion water as the concentric layered structure of osteons. The system well as inside tissues harvested from cadaver (lung, of lamella inside osteon wall has a heterogeneous kidney, encephalon, bone marrow). In reproduction structure determined by the alternation of isotropic biology, DIC microscopy can offer information on the and anisotropic bands. This type of structure can be mobility, morphology and number of sperm cells. guessed on the osteon sections examined in direct light (Brightfield). When examining in polarized CONCLUSIONS light the serried sections stained with Hematoxiline Eosine, the inter lamella spaces are well visible but Differential interference contrast (DIC) have a thin contour compared to the images obtained microscopy has a major indication for usage in the in interferential light. microanatomic analysis of structures from transparent The examination of uncolored bone sections uncolored samples. It ensures an increased contrast in interferential contrast light confirms the existence and resolution by the difference between trajectories of lamella structure of osteons and achieves a false of two rays induced by duplication of linear polarized landmark appearance. The alternation of osseous light at the level of a Nomarski prism. lamella and inter lamella spaces is better visible. The comparative analysis of serried sections Isotropic lamella appear well contoured (Fig. 1). The through uncolored tissue fragments and those stained study of serried sections through superior sympathetic with Hematoxiline Eosine is a proof for images with cervical ganglion, after Cajal argentic impregnation high contrast and resolution in case of colored samples. allowed us to examine in interferential contrast light The usage of DIC microscopy can be expanded the neuronal structures (Fig. 2, C, D).Our observation in forensic medicine to evaluate the presence and state the informational value of microanatomic morphology of diatoms in cases of drawning, and in examination using differential interference contrast reproduction biology to characterize sperm cells, in microscopy (DIC). It ensures the achievement of high hematology and cyto-diagnostic for early discovery of resolution images, with good contrast, with no halo cancer.

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