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Chapter 3 Scanning Electron microscopy of human nail

3.1. Introduction

3.2.Electron microscopy

3.3. Materials and methods

3.4. Results and Discussion

References

3.1. Introduction

Molecular biophysics typically addresses biological questions that are

similar to those in biochemistry and molecular biology, but the questions

are approached quantitatively. The scientists in this field conduct

research concerned with understanding the interactions between the

various systems of a cell, including the interactions between DNA, RNA

and protein biosynthesis, in addition to how these interactions are

regulated. Great varieties of techniques are used to answer these

questions.

Fluorescent technique imaging techniques, as well as electron

microscopy, X-ray crystallography NMR Spectroscopy and atomic force

microscopy are often used to visualize structures of biological

significance.

Proteins can be informally divided into three main classes, which

correlate with typical tertiary structures, globular proteins, membrane

proteins and fibrous proteins. Nearly all globular proteins are soluble

and many are enzymes. The membrane proteins often serve as receptors

or provide channels for polar or charged molecules to pass through the

cell membrane. Fibrous proteins are often structural, such as collagen

65

the major component of connective tissue, or keratin, the protein

component of hair, nails, feathers, hooves and some animal shells

Scanning electron microscopy may be used as a potential tool to

provide valuable information in the study of biological systems. It is very

much helpful in the study of structure of various micro molecular

components and their conformation with in tissue.

Marchisio et al (2001) studied the morphological expression of human

hair and nail invasion in vitro by 31 isolates of nine Scopulariopsis

species was studied by light microscopy on whole material and on semi-

thin sections, as well as by transmission electron microscopy. Some

isolates of Scopulariopsis brumptii, , S. carbonaria S. candida and S.

koningii were keratinolytically active. They came either from nail lesions

or from outdoor aerosols. Most active isolate belonged to S. koningii and

was recovered from a fingernail lesion. Both nail and hair degradation

followed the biochemical and morphogenetic model described by the

authors for other keratinolytic fungi.

The development of the nail apparatus of human foetuses from 6th to

18th weeks of pregnancy was studied by scanning electron microscopy

by Mazzarello et al (1990). The main results can be summarized as

follows: 1) the first structure to appear is the nail fields, defined by

continuous shallow grooves; 2) the shape of the nail field is the first

ovoidal and extends beyond the tip of the finger and later it becomes flat

as in the adult nail plate; 3) the globular blebs of periderm cells

accumulate mostly in the tip of the finger; 4) the surface of the nail field

is first uniform, and then it becomes irregular for increasing the process

of characterization.

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The nail dust particles were analyzed by scanning electron

microscopy for size and topography by Abramson and Wilton (1992).

Percentage of "fines" that could be inhaled and deposited in the alveoli

and bronchioles were determined by quantitative particle size analysis.

The distribution representing the largest total mass was graphed

between 1 and 2 .mu.m. They found that 86% of nail dust would reach

the bronchioles and alveoli, and 31% could be expected to deposit in

these areas.

Garson et al studied (2000) the three layers (characterized by different

orientations of the keratin molecules) from the outer to the inner side of

human nail were observed by synchrotron X-ray micro diffraction. Three

layers are associated with the histological dorsal, intermediate and

ventral plates. Hair-like type alpha-keratin filaments are only present in

the intermediate layer. This "sandwich" structure in the corneocytes and

the strong intercellular junctions, gives the nail high mechanical rigidity

and hardness, both in the curvature direction and in the growth

direction.

Bai and Li (1986) investigated the character and the regulation of

micro-vascular construction in the great toe was studied with scanning

electron microscope. ABS was injected into the popliteal artery of the

lower limb of a dead child. There are only a few vascular loops in the nail

bed of the great toe, and the arrangement of those loops is parallel with

the surface of finger nail. No vessel ball can be seen in the nail bed.

A case of a fully developed hereditary onycho-osteo-arthrodysplasia

(nail-patella syndrome) is presented by Stellamor and Anzboeck (1989).

Typical signs, such as the iliac horns or variations of the knees, cubitals

and nails should be familiar to every radiologist. The associated

nephropathy seems to be caused by typical changes in the glomerular

67

basement membrane seen in electron microscopy. Asymptomatic

proteinuria is found in about 60% of the cases, in 5, 5-8% the disease

leads to the necessity of haemodialysis because of renal insufficiency.

Therefore, early diagnosis is very important.

Schulz-Kiesow et al (1996) described a familial disorder featuring

hystrix-like keratosis, thickened nails and plantar hyperkeratosis. Index

patient, a 10-year-old girl, suffered also from joint laxity and had long

fingers, while in her mother only the typical skin lesions were observed.

Histologic examination of the spiny hyperkeratoses in the index patient

showed parakeratosis with a marked cornoid lamella. On electron

microscopy the keratinocytes exhibited intracellular vacuolization and

aggregated tonofilaments, but no concentric shell formation. They

concluded the striking skin lesions present in the 2 cases can be

distinguished from other forms of hystrix-like hyperkeratoses such as

nevus corniculatus or multiple digitate hyperkeratoses and hence may

represented a new autosomal dominant genodermatosis.

Von-Bierbrauer et al (1996) suggested that Electron microscopy was

the most sensitive method of histological examination, detecting

abnormalities in 87.5% of patients; with light microscopy and

immunohistochemical techniques, abnormalities were revealed less

frequently (83.3% and 75%, respectively). In contrast, normal findings

were observed in most of the healthy controls: capillaroscopy = 90%;

histology = 80%. They concluded that, Microvascular lesions are a

predominant feature in scleroderma and seem to have a central

pathogenetic role in the disease. The capillaroscopy is able to identify

this microangiopathy noninvasively, and capillaroscopically guided nail

fold biopsy can detect the frequency and nature of the underlying

ultrastructural changes. Therefore it may be a useful tool in describing

the pathogenetic role of the microvascular system in scleroderma.

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Nine cases of skin and nail infection due to Fusarium oxysporum,

diagnosed in Tuscany in the period 1985-97, are described by Romano et

al (1998). Seven as onychomycosis and two manifested as interdigital

intertrigo of the feet. They all were diagnosed on the basis of repeated

mycological examination, direct microscope observation and culture, as

well as histological examination of biopsy specimens in two cases of

intertrigo. The fragments of the fungal colonies were examined by

scanning electron microscopy (SEM) for more detailed observation of

fungal morphology. All the patients had normal immune status and a

history of the infection extending several years. The four of the patients

with onychomycosis were treated with oral itraconazole, and clinical and

mycological recovery was achieved in three cases. Two others patients

were treated with cyclopyrox nail lacquer, successfully in one case. One

patient with intertrigo was treated with oral itraconazole and one with

oral terbinafine; both were also treated and with topical drugs, however

clinical recovery was not confirmed by the mycological results.

The nail Pityrosporum yeasts from a patient with onychomycosis was

investigated by Ran et al (1998) by means of culture, pathological and

scanning electron microscopic examination and 20% KOH preparation.

Physical examination showed that each finger and toe nail appeared

brownish-black, rough and thick, some of the fore part of the nail plate

detached from the nail bed. Fingernail specimen's culture results showed

that Trichophyton rubrum grew on Sabouraud's dextrose agar and

Pityrosporum ovale grew on the medium containing rapeseed oil. The

pathological examination revealed P. ovale yeast involvement in the

fissure of the nail plate. Under the scanning electron microscopy, a lot of

P. ovale yeasts with characteristic collarette structure inserted in the nail

tissue was noticed. In the 20% KOH preparations of nail incubated at 56

degree C for 1h and stained with Quink Parker ink, spores and hyphae

were identified morphologically with P. ovale and T. rubrum respectively.

69

The patient received intermittent pulse therapy with itraconazole, the

color of the nails became much brighter 1 to 2 months after the fourth

cycle of therapy, but no further improvement was observed afterwards. P.

ovale and T. rubrum grew again 6 months after treatment when the

clippings of the fingernail were cultured. They conclusion that, this is the

first document of onychomycosis related with P. ovale in China.

Ectopic nail is an extremely rare condition related to acquired or

congenital anomalies were studied by Ena et al (2003). Nearly 40 cases

are reported in the literature, mostly in Japanese patients. Majority of

these patients, ectopic nails developed in the dorsal aspect of the fingers;

they are associated in some cases with acquired or congenital growth

anomalies or to polydactyly. In recent times, they observed two male

adult patients with true ectopic nails of the foot (sole and heel). The both

patients were not affected by ectodermic dysplasia or foot malformations.

The lesion relapsed after surgical excision in one case. The histology

showed features of a well-developed and normal nail plate and matrix.

Transmission electron microscope study was done in one case, showing

typical aspects of onychocytes. The other nail was reproduced by a

silicone replica technique and its superficial texture, shape and

relationship with surrounding tissue were analysed by scanning electron

microscopy.

Zuppan et al (2003) investigated a history of longstanding hematuria

and non-nephrotic proteinuria without renal insufficiency, for which

renal biopsy was performed. They found by routine light microscopy and

direct immunofluorescence study was mild and nonspecific. Electron

microscopy, though, demonstrated the unexpected finding of distinct

collagen fibrils within capillary wall basement membranes, typical of the

nail-patella syndrome. Repeat physical examination following the biopsy

confirmed the presence of normal nails and patellae, and radiographs of

70

the knees were also normal. The boy's renal disease was stable at last

follow-up. They briefly discuss the differential diagnosis, and suggest

that this case represents an unusual manifestation of the nail-patella

syndrome, in which the glomerular changes are present in the absence of

the usual associated constitutional abnormalities.

Rifkin et al (1969) made electron microscopic studies on the

constituents of the encapsulating cysts in the American oyster

,crassostrea virginica, formed in the response to tylocephalum

metacestodes and reviled three types of extra cellular fibers embedded in

homogenious matrix of medium electron density. They reported that the

special relation ship between the extra cellular fiber and the cellular

constitunts suggest that extra cellular fibrillogenesis is infulensed by

fibro blast like cells, leukocytes and brown cells.

Repka Michael et al (2002) investigation the morphology of the human

nail treated with chemical penetration enhancers (CPE), bioadhesives

and surface modifiers for assessment of topical treatment modalities for

onychomycosis. Chemical penetration enhancers CPEs, including

dimethyl sulfoxide (DMSO) and urea were applied to human nail

samples. Further samples were treated with surface modifiers, tartaric

acid (TTA) and phosphoric acid gel (PA). The other nail specimens were

subjected to the bioadhesive polymers Carbopol 971P and Klucel MF.

Scanning electron microscopy (SEM), atomic force microscopy (AFM) and

polarized light microscopy (PLM) were utilized to visualize nail

morphology and topographical changes of the human nail samples

subjected to the various chemical agents. SEM, AFM. and PLM

micrographs revealed changes in topography to the dorsal layer when

CPEs and surface modifiers were applied. The roughness scores as

determined by NANOSCOPE IIIA software indicated a 2-fold increase

when the dorsal nail layer was subjected to PA versus the control (147.8

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vs. 85.0 nm, respectively). When carbomer 971P was applied to the

dorsal surface roughness scores decreased significantly (44.6 vs. 85.0

nm, respectively). SEM, AFM and PLM studies of the human nail

subjected to various chemical agents may be useful in the design and

formulation of novel drug delivery systems for the topical treatment of

onychomycosis. AFM studies offer both a qualitative and quantitative

assessment for nail treatment opportunities.

A scanning electron microscopic (SEM) study made by B. Forslind

and N. Thyresson (1974) of cut surfaces in normal human nails have

confirmed the previous description of nail structure, that is hard dorsal

nail plate supported by the plastic intermediate nail plate.

By a cast method (silicone resins) which allows a form-fitting reprint

of nail-plate relief are made scanning electron microscopic studies of

healthy and sick nails made by Pfister and Neukirchner (1980). In this

method a form-fitting restitution of the surface-relief is guaranteed up to

magnifications of 5000:1. Therefore it is possible to get a better

impression of the affected nail-structure. The accuracy of this nail-

structure-representation cannot be reached by normal light microscopy.

This method is very simple and quickly to use. Nail-changes in

onychoschizia, subungual hematoma and in senile nails are shown.

3.2 Electron Microscopy

Scanning electron microscope is an instrument that emits electron beam

to observe objects in superior size. It gives the information about the

surface of specimen „how it looks‟ means consistency and properties

such as reflectivity, hardness etc. This area of research is called as

topography. The shape and size information is known as morphology.

This gives the specimen properties and structure such as strength,

ductility etc. Moreover it gives the information about element

72

composition and properties such as melting point etc. SEM gives the

information about the arrangement of atoms.

Electron Microscopes were developed due to the limitations of Light

Microscopes which are limited by the physics of light to 500x or 1000x

magnification and a resolution of 0.2 micrometers. First Scanning

Electron Microscope (SEM) debuted in 1942 with the first commercial

instruments around 1965. Its late development was due to the

electronics involved in “scanning” the beam of electrons across the

sample.

Working of Electron Microscopes

Electron Microscopes function exactly as their optical counterparts

except that they use a focused beam of electrons instead of light to

“image” the specimen and gain information as to its structure and

composition.

The basic steps are as follows:

The stream of electrons produced by electron source is accelerated by

using positive potential. Electron stream is focused on specimen using

magnetic lens and metal apertures. Inside the sample, the interaction

takes place which is detected and converted into the image.

For the formation of image, just like in television the electron beam

scan the sample in series of lines. Fig.3.1 shows the schematic diagram

of SEM.

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Fig.3.1. Schematic diagram of scanning electron microscope

The secondary electrons are selectively attracted to a grid held 50 volt

positive potential. The disc, which is behind the grid, is kept about 10 kV

positive potential with difference to the sample. Emission of light changes

the photons of light into voltage by striking the disc by secondary

electrons through grid.

Number of secondary electrons striking the disc decides the voltage

strength. The secondary electrons ejected from sample give voltage signal

of desired strength, given to electronic console where it amplified and

given to CRT or Monitor. The SEM does not have any objective, lenses as

in optical microscope for magnification. The magnification is increased by

scanning over a small area of the sample.

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Specimen Preparation

There are two basic types of SEMs. The normal SEM, requires a

conductive sample. The environmental SEM can be used to examine a

non- conductive sample without coating it with a conductive material.

Three requirements for preparing samples for a regular SEM such as

remove all water, solvents, or other materials that could vaporize while in

the vacuum. Firmly mount all the samples. Non-metallic samples such

as bugs, plants fingernails and ceramics should be coated so they are

electrically conductive. Metallic samples can be placed directly into the

SEM.

3.3. Materials and methods

For the study of scanning electron microscopy of the human nail,

different age male and female volunteers were selected. The finger nails

are allowed to grow up to 12 weeks i.e. 84 days in female volunteers and

nearly 11 weeks i.e. 75 days in male volunteers. The grown free edge

nails (whitish grown part from the tip of nail) were cut smoothly and nail

samples collected were washed and used to study the scanning electron

microscope. For the scanning electron microscopy of both the specimens,

samples were prepared by cutting them in flat rectangular shape which

is suitable for the SEM instrument. (Plate 3.1)

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Plate 3.1.Human nail cut into flat rectangular shape

Scanning electron microscope (Model ISI – 100A) working at 10 KV

was used to study the distribution and morphology of keratinised hard

tissue the human nail. Suitable specimens were prepared and coated

with a conductive material for the scanning. The scanning was done at

three layers such as outer surface, middle layer, bottom surface and edge

of the specimens at different magnification. Scanning electron

micrographs were taken for the analysis (Fig.3.3. to 3.6)

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3.4. Results and Discussion

Fig.3.2.Nail matrix and direction of movement of keratinised cells in nail

plate.

The important aspect related to keratanised hard biomaterials tissue

is the morphology of the human nail. Scanning electron microscopy is

the best technique to study the structure and arrangement of proteins in

the human nail. This technique gives the qualitative information rather

than the quantitative. One can visualise the happenings in the tissues.

Fig.3.2. shows the nail matrix and direction of movement of keratinised

cells in nail plate.

Fig.3.3. show the scanning electron micrograph of edge portion of male

and female human nail specimens belonging to different ages. Scanning

electron microscopy of the edge of human fingernail clearly reveals the

three nail layers, upper layer known as dorsal nail plate, middle layer

77

known as intermediate nail plate and bottom layer known as ventral nail

plate (from nail bed matrix) differed markedly in structure.

Fig.3.4. show the scanning electron micrograph of upper portion i.e.,

dorsal layer of male and female human nail specimens belonging to

different ages. The dorsal layer appeared to be composed of flat,

overlapping slate-like sheets, which were oriented in the plane of the nail.

In contrast the thick intermediate layer was more fibrous fig.3.5. The

thick intermediate layer composed of long narrow cells, which are

orientated laterally, parallel to the free edge and lunula of finger nail.

Fig.3.6. show the scanning electron micrograph of lower layer i.e., ventral

layer of male and female human nail specimens belonging to different

ages. The thin ventral layer was more similar to the dorsal layer. The only

difference is, in their structure. The outer surface was smooth relatively

to the intermediate layer. Dorsal and ventral layers that are composed of

tile-like cells with randomly oriented keratin fibres, particularly towards

the edge of the nail where they become relatively thicker.

The arrangement of fibres in the outer layers into tile-like cells also

has a further advantage that they provide a smooth waterproof covering,

which can protect the fibrous intermediate layer. Finally, there is one

extra level of sophistication in the design. The dorsal and ventral layers

wrap around the lateral edge of the nail, producing a smooth covering

that prevents potentially dangerous cracks from forming there.

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Fig.3.3. Scanning electron micrographs of human nail edge surface

79

Fig. Scanning electron micrographs of human nail edge surface

80

Fig.3.4. Scanning electron micrographs of human nail outer surface

81

Fig. Scanning electron micrographs of human nail outer surface

82

Fig.3.5. Scanning electron micrographs of human nail middle surface

83

Fig. Scanning electron micrographs of human nail middle surface

84

Fig.3.6. Scanning electron micrographs of human nail lower surface

85

Fig. Scanning electron micrographs of human nail lower surface

86

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