Indian Journal of Pure & Applied Physics Vol. 42, March 2004, pp 189-194

Surface morphology of gall bladder stones V R Singh & Palwinder Kaur

Instrumentation and Sensors Group, National Physical Laboratory, New Delhi 110 012

Received 22 July 2003: accepted 13 November 2003

Gall bladder stones are crystalline structures formed by concretion or accretion of normal or abnormal bile constitutents. These stones are normally made of cholesterol, calcium, phosphate, carbonate and proteins. The aetiology of gall bladder stones is multifactoral. Scanning electron microsscopy (SEM) is used here to investigate the surface features of the mechanically fractured part of the stone samples. Micrographs exhibit a non uniform polycrystalline surface without specific grain boundaries with porosity, leading to loose bounding of crystals and hence low specific gravity. These results pertaining to surface characterisation are very useful for understanding the disintegration process of stones inside or outside the human body.

[Keywords: Gall bladder stones, Morphology, Surface morphology, Disintegration process]

IPC Code: G 01N 13/10

1 Introduction

The organs of the digestive system perform a vital process. These organs prepare food for absorption and for use by the millions of the body cells. Gall bladder is the part of the digestive system, which stores the bile that enters it by way of the hepatic and cystic ducts, and during digestion, it ejects the concentrated bile into the duodenum1. Lithiases (the stone formation) is a major disease associated with the human gall bladder. When cholesterol concentration exceeds the solubility limit in the lipid bilayers of miscelles, deposition of cholesterol takes place. This leads to nucleation of crystalline cholesterol. The causative factors for the occurrence of gallbladder stones are hereditary, infection and diet. These stones are frequently twined, cracked, yellowish white or yellowish brown in colour and have fine granular crystalline or non-crystalline-structure2,3. These stones on the basis of their chemical composition are classified as: pure cholesterol, pure calcium and combination. Dielectric properties in gall bladder stones are found due to the presence of silicon dioxide, while the variation in ultrasonic properties is because of their complex structures4-14.

The electron microscopy has become an indispensable tool for research and development and

has a key role to play in the field of environmental and forensic science, industrial development, genetic engineering, medical and biological fields to examine cells, tissues and micro-organisms. Materials science studies on lattice defect, inclusions, precipitates, phase transitions and other technical fields associated with semiconductors also involve the use of electron microscopy. SEM is also commonly used in hospitals and medical centres for clinical diagnostic purposes. This system gives a three-dimensional quality to its pictures, even although its limited resolution is around 100 Å. The whole of the specimen is not illuminated at the same time and the electron beam is focussed to needle sharpness so that it strikes an area of only about 10 nm radius at any instant. Several million electrons strike the given sample in one second and scan the sample.

In the present work, surface characteristics of different gall bladder stones have been studied with scanning electron microscope system. This study gives very useful information of the surface structure of gall bladder stones. This is found to be helpful in understanding the physical mechanisms of the disruption of different stones inside or outside the body.

2 Materials and Method

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In the present study, twenty biological (gall bladder) stones were collected from different hospitals in New Delhi such as Lok Nayak Jai Prakash Narain Hospital, All India Institute of Medical Sciences, and Bara Hindu Rao Hospital. These stones were obtained through surgical operations on different patients. Four stone samples (Pg1, Pg2, Pg3 and Pg4) were considered for investigating the physical properties such as the dimension, shape, finish, porosity, specific gravity, and micro and macro structures with SEM technique. The scanning electron microscope gives useful information about the structure of the specimen at high resolution and has good focus over a wide range of specimen surfaces. The system used produces clear images of the specimens ranging from objects visible with the naked eye to the structures as small as size of several angstroms. Fig. 1 shows the photographs of different gall bladder stones used for SEM analysis.

The samples were fixed on the small circular discs separately. These stone samples were coated with gold layer (100-200 Å) to avoid any charging effect. SEM coating unit (make Poland Equipment Ltd. E 5000) was used here. SEM, JEOL model JSM-840, was used to characterise the surface structure of

samples. In the experiment, electron beam emitted from the electron gun was focussed on to the surface of the specimen by an electron lens. The number of electrons striking on the unit area of the specimen was determined by the diameter of the electron probe. Fig. 2 shows the schematic diagram of the system used. The probe was moved horizontally by supplying current to the horizontal-scanning coil located in the electron beam path. The secondary electrons emitted

Fig. 2 ⎯ Layout of SEM set-up

Fig. 1 ⎯ Photographs of GB stones used in the present investigation

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from various positions of the specimen surface when scanned by the probe were determined by the secondary electron detector and the signal induced in the detector was sent to the video amplifier, where it was amplified and level controlled to obtain an optimum contrast and brightness image on the CRT (Cathode Ray Tube). The video output signal was then supplied to the control grid of the cathode ray tube, which was synchronised with probe scanning. The signal modulates the raster thereby displaying a scanning image on the screen.

3 Results and Discussions Table 1 presents the physical observations of

different gall bladder stone samples. These stones are found to be earthy (dull) lustre but differing in colouring in colour, steak and hardness. All the samples are soft because of their low specific gravity and hardness. Their hardness on mho’s scale is around 2.

Table 1 ⎯ Physical properties of gall bladder stones Sample No. Form Colour Lustre Steak Hardness on

mho’s scale Observed specific

gravity Pg1 Amorphous Yellowish Resinous/Silky Yellow 3.0 Low Pg2 Amorphous

(Nodular) Brownish black Earthy Yellow 2.5 Low

Pg3 Crystalline Brownish white Earthy White 2.0 Low Pg4 Amorphous Yellowish Brown Earthy Yellow 1.5 Low

Fig. 3 ⎯ Micrographs of GB sample Pg1 at different magnifications

Table 2 ⎯ SEM data for gallbladder stone samples (Pg1, Pg2, Pg3 and Pg4) at an accelerating voltage of 10 kV

Sample Magnification Particle size (Range) (μm)

Working distance(WD) (mm)

Pg1 ×12 .

×2000

×5000

1000 10 1

34 34 34

Pg2 ×16 .

×2000

×5000

1000 10 1

32 32 32

Pg3 ×12 .

×1000

×2500

1000 10 10

31 32 31

Pg4 ×12 .

×2000

×5000

1000 10 1

31 31 31

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SEM experiments were performed at an accelerating voltage of 10 kV and probe current of 10-11 Ampre and resolution of the order of 60 Å. The micrographs depict the deposition of crystals, which form the irregular and unoriented stone. Table 2 shows the observations of gall bladder stones with SEM at different magnifications with working distance (focussing distance) and depth of field. Micrographs of different magnifications give the surface features of the samples used, which are helpful in breaking these stones. The surface

morphology of specimen Pg1 (gall bladder stone) is given in Fig. 3(a,b,c). Its surface appears slightly smooth and all crystals are more or less of the same shape but size of crystals is large. There are cavities on its surface, which result in a non uniform discontinuous rough surface as shown at magnification×5000. Fig. 4(a,b,c) shows the surface structure of the sample Pg2. It possesses polycrystalline uneven surface and at magnifica-

tion×2000, a number of pits were observed. Crystals inside the body are without any grain boundaries,

Fig. 4 ⎯ SEM pictures of internal structure of GB sample Pg2 at different intensities

Fig. 5 ⎯ Micrographs of Pg3 sample of GB at different magnifications

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which causes heterogeneous nature. At magnification ×5000, some crystals appear in stick type growth while others have no definite shape. The microstructure of sample Pg3 is given in Fig. 5(a,b,c). Its surface appears flat with black spots on it. At magnification ×1000, there are cracks on the surface, which exhibits an amorphous type of pattern with

many islands. These islands are formed by small polycrystalline grains. Fig. 6 (a,b,c) shows the surface morphology of stone sample Pg4. It gives better surface texture as observed at magnification ×12. All crystals are of different sizes and shapes with cavities without any definite pattern because deposition of these stones also depends upon the diet intake of the patient. These results of the physical properties and SEM analysis confirm that the gall bladder stones can be broken inside or outside the human body.

4 Conclusions Surface structure study of different gall bladder

stones proves that gall bladder stones are soft and smooth with large cavities on them. Different crystals normally are of different sizes and shapes and cavities on them form loose bonding which helps crushing these stones. The present work would be helpful in understanding better the mechanisms involved in the disintegration of gall bladder stones, after correlating the micrographs with the nature of particular stone samples.

Acknowledgement We are thankful to Dr. Kusum Gupta, Bara Hindu

Rao Hospital, Delhi, for fruitful suggestions in the paper and for the help in the procurement of the stone samples used in the study.

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