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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.
66
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
71
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.
78
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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