From the dawn of science there has been an interest in being able to look at smaller and smaller details of the world around us. Biologists have wanted toexamine the structure of cells, bacteria, viruses, and colloidal particles. Materials scientists have wanted to see inhomogeneities and imperfections in metals, crystals, and ceramics.In geology, the detailed study of rocks, minerals, and fossils on a microscopic scale provides insight into the origins of our planet and its valuable mineral resources.
For example, try looking at a newspaper picture, or one in a magazine, through a magnifying glass. You will see that the image,is actually made up of dots too small and too close together to be,separately resolved by your eye alone. The same phenomenon will be observed on an LCD computer display or flat screen TV when magnified to reveal the individual “pixels” that make up the image
Optical microscopes are the ones most familiar to everyone. They use visible light and transparent lenses to see objects as small as about one micrometer (one millionth of a meter), such as a red blood cell (7 μm) or a human hair (100 μm).
With visible light it was impossible to resolve points in the object that were closer together than a few hundred nanometers
similar to the way glass lenses are used to bend and focus visible light.
In the 1920s, it was discovered that accelerated electrons behave in vacuum much like light. They travel in straight lines and have wavelike properties, with a wavelength that is about 100,000 times shorter than that of visible light. Furthermore, it was found that electric and magnetic fields could be used to shape the paths followed by electrons similar to the way glass lenses are used to bend and focus visible light. Ernst Ruska at the University of Berlin combined these characteristics and built the first transmission electron microscope (TEM) in 1931. For this and subsequent work on the subject, he was awarded the Nobel Prize for Physics in 1986. The first electron microscope used two magnetic lenses, and three years later he added a third lens and demonstrated a resolution of 100 nm, twice as good as that of the light microscope. Today, electron microscopes have reached resolutions of better than 0.05 nm, more than 4000 times better than a typical light microscope and 4,000,000 times better than the unaided eye.
The positively charged protons and neutral neutrons are held tightly together in a central nucleus. Negatively charged electrons surround the nucleus.Normally, the number of protons equals the number of electrons so that the atom as a whole is neutral. When an atom deviates from this normal configuration by losing or gaining electrons, it acquires a net positive or negative charge and is referred to as an ion.
The transmission electron microscope (TEM) was the first type of Electron Microscope to be developedPasses a beam of electrons through the specimen.The electrons detected on a fluorescent screen on which the image is displayed.Thin sections of specimen -as electrons have to pass through the specimen for the image to be produced.This is the most common form of electron microscope and has the best resolution [0.2nm] .
The electron beam from the electron gun can be focussed and defocussed by a series of electro- magnetic lenses. Similar to the light microscope, the "Condenser Lenses" concentrate the beam onto the specimen. Electrons passing through the specimen will be focussed by the "Objective" &"Intermediate" lenses to form an intermediate image.The "Projector lens" enlarges this image into a final image on the fluorescent viewing screen at the bottom of the microscope column.Each lens is basically a circular electro-magnet. A variable electric current through the lens will produce a magnetic field of variable strengths which will deflect or bend the electron beam passing through
caused by collisions between the beam electrons and the atoms of the specimen
Specimen interaction is what makes Electron Microscopy possible. The energetic electrons in the microscope strike the sample and various reactions can occur as shown below. The reactions noted on the top side of the diagram are utilized when examining thick or bulk specimens (SEM) while the reactions on the bottom side are those examined in thin or foil specimens (TEM).
The incident electron is then scattered "backward 180 degrees- backscaterred electron.Sec electron- Caused by an incident electron passing "near" an atom in the specimen, near enough to impart some of its energy to a lower energy electron (usually in the K-shell). This causes a slight energy loss and path change in the incident electron and the ionization of the electron in the specimen atom. This ionized electron then leaves the atom with a very small kinetic energy (5eV) and is then termed a "secondary electron". Each incident electron can produce several secondary electron.Auger Electrons-Caused by the de-energization of the specimen atom after a secondary electron is produced. Since a lower (usually K-shell) electron was emitted from the atom during the secondary electron process an inner (lower energy) shell now has a vacancy. A higher energy electron from the same atom can "fall" to a lower energy, filling the vacancy. This creates and energy surplus in the atom which can be corrected by emitting an outer (lower energy) electron; an Auger Electron.
Mass absorption contrastOn passing through matter, a beam of electrons is gradually attenuated. The degree of attenuation increases with the thickness of the specimen and its mass, so variations of mass and thickness across the sample give rise to contrast in the image.Diffraction contrastDiffraction of electrons from Bragg planes causes a change in their direction of travel. Hence, contrast can arise between adjacent grains or between different regions near the core of a dislocation.Phase contrastScattering mechanisms often cause a change in the phase of the scattered electrons, as well as a change in direction. Interference between electrons of different phase which are incident on the same part of the image will cause a change in intensity and give rise to contrast.
Biological material (light elements):Only few electrons escape from specimenAlmost no contrast, similar contrast everywhere on specimenBlurred image (electrons from “large” volume)Contrast enhancement important & needed:Localization of the signal to the surfaceCoating of biological specimen with thin heavy metal layer (a few nm)Reducing acceleration voltage
Electron microphotograph showing myelin figure (´ 10,000) and Zebra body-inset (´ 15,000) (Uranyl acetate and lead citrate stain).
he typical ciliary axoneme consists of 2 central microtubules surrounded by 9 microtubular doublets. Each doublet has an A subunit and a B subunit attached as a semicircle. A central sheath envelops the 2 central microtubules, which attach to the outer doublets by radial spokes.The outer doublets are interconnected by nexin links, and each A subunit is attached to 2 dynein arms that contain adenosine triphosphatase; one inner arm and one outer arm. The primary function of the central sheath, radial spokes, and nexin links is to maintain the structural integrity of the cilium, whereas the dynein arms are responsible for ciliary motion.
Mitochondrial myopathy, ultrastructure. A large number of enormous mitochondria can be seen in the intermyofibrillar network of this myofiber. These mitochondria are larger than entire sarcomeres. Normal mitochondria are much smaller than sarcomeres.
The four stages of melanosome development are shown in the upper panels. Note the dense bilayered coat (arrowhead) and intraluminal vesicles (arrow) of stage I melanosomes, the proteinaceous fibrils (arrow) of stage II, and the melanin deposition (black) in stages III and IV.
ELECTRONMICROSCOPY -Dr Ganga H
• Introduction• Transmission EM• Scanning EM• Preparation of tissue for EM• Diagnostic applications
Introduction and History• The word microscope is derived from the Greek mikros (small) and skopeo (look at).• The light microscope -developed from the Galilean telescope during the 17th century.• Dutchman Antony van Leeuwenhoek (1632-1723) – simple microscope.• Discovered protozoa, spermatozoa, and bacteria, and classified red blood cells by shape.
• The limiting factor in Van Leeuwenhoek’s microscope was the single convex lens.• Addition of another lens could magnify the image produced by the first lens.• This compound microscope – consisting of an objective lens and an eyepiece, a mirror or a source of light and a specimen table for holding and positioning the specimen.
Resolution of the Human Eye• In sufficient light, the unaided human eye can distinguish two points 0.2 mm apart.• A lens or an assembly of lenses (a microscope) can be used to magnify this distance and enable the eye to see points even closer together than 0.2 mm.
Types of microscopes• Three basic types:• Optical, charged particle (electron and ion), or scanning probe.• Electron and ion microscopes, use a beam of charged particles, and electromagnetic or electrostatic lenses• They can see features as small a tenth of a nanometer, such as individual atoms.• Scanning probe microscopes use a physical probe (a very small, very sharp needle) which scan over the sample in contact or near-contact with the surface.• These instruments are capable of atomic scale resolution
• A modern light microscope - 1000x .• The resolving power of the microscope limited by the wavelength of the light used for illumination.• Using light with a shorter wavelength--a small improvement.• Using oil --- small improvement, but all together only brought the resolving power of the microscope to just under 100 nm.
• In the 1920- accelerated electrons behave in vacuum much like light.• They travel in straight lines and have wavelike properties.• Wavelength is about 1,00,000 times shorter than that of visible light.• Electric and magnetic fields are used to shape the paths followed by electrons
1931-Ernst Ruska at the University of Berlin built thefirst transmission electron microscope (TEM)1986- awarded the Nobel Prize for Physics
• The first electron microscope used two magnetic lenses, and three years later he added a third lens and demonstrated a resolution of 100 nm, twice as good as that of the light microscope.• Today, electron microscopes have reached resolutions of better than 0.05 nm, more than 4000 times better than a typical light microscope and 4,000,000 times better than the unaided eye.
Resolution of a microscope• Wavelength of the illumination source ( λ )• The numerical aperture of the lens (N.A.) Limit of resolution = 0.61 λ/N.A.• The maximum value of N.A. for light microscope is approx. 1.4. therefore, that even the short blue light ( λ = 436 nm) of the visible spectrum will yield a resolution of only 190 nm.• The wavelength of an electron beam is about 100,000 times less than that of visible light and hence the resolution of an electron microscope is far superior to that of the light microscope.
The Electron• An atom is made up of three kinds of particles – protons, neutrons, and electrons.• The electrons, which are about 1800 times lighter than the nuclear particles, occupy distinct orbits, each of which can accommodate a fixed maximum number of electrons.• When electrons are liberated from the atom-they behave like light.
Types of electron microscope 1. Transmission electron microscopy : 2. Scanning electron microscopy:
Transmission Electron Microscope Principle• TEM is the direct counterpart of Light microscope• Involves passage of high velocity electron beam through specimen, thin enough to transmit 50% of the electrons• Transmitted electrons – focused by lens systems to form a 2 dimensional magnified image
Analogy between LM & TEM• Arrangement & function of their components 1. Illuminating system – source & condensor 2. Imaging system – lenses to produce magnified image – objective & projector 3. Image translating system – Final image is viewed
Electron source• The electron source consists of a cathode and an anode.• The cathode is a tungsten filament which emits electrons when being heated.• A negative cap confines the electrons into a loosely focused beam.• The beam is then accelerated towards the specimen by the positive anode.
Electromagnetic lens system• The system allows electrons within a small energy range to pass through, so the electrons in the electron beam will have a well-defined energy.• 1. Magnetic Lens: Circular electro- magnets capable of generating a precise circular magnetic field. The field acts like an optical lens to focus the electrons.• 2. Aperture: A thin disk with a small (2-100 micrometers) circular through-hole. It is used to restrict the electron beam and filter out unwanted electrons before hitting the specimen.
The Vacuum System• The electron beam must be generated in and traverse through the microscope column under a high vacuum condition.• The presence of air molecules will result in the collision and scattering of the electrons from their path.• In the electron microscope the vacuum is maintained by a series of highly efficient vacuum pumps.• THE VACUUM FACTOR: Biological material must be properly fixed and preserved
Sample holder• The sample holder is a platform equipped with a mechanical arm for holding the specimen and controlling its position.
Imaging system• The imaging system consists of another electromagnetic lens system and a screen.• The electromagnetic lens - two lens, one for refocusing the electrons after they pass through the specimen, and the other for enlarging the image and projecting it onto the screen.• The screen has a phosphorescent plate which glows when being hit by electrons.
Image Formation in the TEM• The basis of image formation in the TEM is the scattering of electrons.• The scattering results in a shadow on the viewing screen or photographic film.• Material with high atomic numbers will cause more scattering and produce a deep shadow. Such material is termed "electron dense" and has high image contrast.• Biological material has low electron density and is known generally as "electron transparent". Hence, an inherent low contrast image is formed.• BIOLOGICAL MATERIAL must, therefore, be STAINED with heavy metal salts.
THE SCANNING ELECTRON MICROSCOPE• To directly visualise the surface topography of solid unsectioned specimens.• Probe scans the specimen in square raster pattern.• The first scanning electron microscope (SEM) debuted in 1938 ( Von Ardenne) with the first commercial instruments around 1965.• Differs from TEM in construction & operational modes• TEM – information is obtained from transmitted electrons• SEM – majority is obtained from secondary, backscattered electrons & from X-rays.
Thin Specimen Interactions• Incident electrons which are transmitted through the thin specimen without any interaction occurring inside the specimen- Unscattered Electrons.• The transmission of unscattered electrons is inversely proportional to the specimen thickness.• Areas of the specimen that are thicker will have fewer transmitted unscattered electrons and so will appear darker, conversely the thinner areas will have more transmitted and thus will appear lighter.
Elastic Interactions• No energy is transferred from the electron to the sample. The electron either passes without any interaction or is scattered by electrostatic with the positive potential inside the electron cloud.• These signals are mainly exploited in TEM and electron diffraction.
Inelastic scattering:Primary electrons hit electrons ofthe specimen atomEnergy is transferred from theprimary electron to the specimenEmission of electrons and radiation
• After the impingement of the primary electrons on the specimens, secondary electrons as well as other forms of radiation are emitted.• But only the secondary electrons will be collected by the signal detector.• In the detector these electrons strike a scintillator and the light produced is converted to electric signals by a photomultiplier.• The electric signal is then amplified and displayed on the cathode ray tube (CRT).
• In the SEM the electron beam is rapidly scanned back and forth in an orderly pattern across the specimen surface.• It is a composite of many individual image spots similar to the image formed on the TV screen.• The SEM has a specimen stage that allows the specimen to move freely so that the surface of the specimen can be viewed from all angles.
The focused electron beam is moved from one pixel to another.At every pixel, the beam stays for a defined time and generatesa signal (e.g.secondary electrons) which are detected, amplifiedand displayed on a computer screen
Image magnification in SEM– A smaller area is scanned with the same number of pixels.– The scanned pixels are smaller
TEM vs SEM TEM SEM6 lenses – C1, C2, objective, 3 3 lenses – 2 condensor, 1projector objectiveHigh accelerating voltage - low accelerating voltagepenetrationNot complicated Specimen Stage – complicatedX & y axis X,Y,Z-axis, tilting, rotating
Contrast formation in TEMAbsorption of electronsScattering of electronsDiffraction and phase contrast
Contrast formation in TEM• Biological specimen consist of light elements: Absorption contrast weak Scattering contrast weak LOW CONTRAST Phase contrast weak• Contrast enhancement required: – Treatment with heavy metals (Ur, Pb, Os)! – Heavy metals attach differently to different components
Thin section of alga stained with heavy metals (Ur, Pb)
Contrast formation in SEM (using SE and BSE)• Different number of electrons from different spots of the specimen – Based on topography of the specimen – Based on composition of the specimen
Uniform layer of heavy metal on specimen surface Primary electron beamPlatinum
SCANNING TRANSMISSION ELECTRON MICROSCOPY (STEM)• This is a recent technological advance in the field of Electron Microscopy.• The beam of electrons scans the specimen, as it does in scanning electron microscopy.• However, it is the transmitted electrons that are collected and amplified and form an image on a cathode ray tube.• The small spot size of the beam allows different areas of the specimen to be discriminated and analyzed.• A major use of STEM is in X-ray analysis which allows the elemental composition of the specimen to be mapped.
Specimen preparation for electron microscopySteps include Specimen procurement Fixation Tissue processing and sectioning Staining
SPECIMEN PROCUREMENTTissue preserved in glutaraldehyde.Tissue must be representative of the disease.Areas that show - degeneration, necrosis, haemorrhage must be avoided.
Drying of the surface must be avoided.Tissue must be properly fixed.The suitability of the tissue can be confirmed by a frozen section or touch preparation.
• Fixation : most commonly used are osmium tetroxide, glutaraldehyde and paraformaldehyde.• Dehydration : acetone or ascending concentration of alcohol, 5-15 min in each concentration.• Use of dimethyoxypropane for rapid dehydration.• Clearing agent: propylene oxide.
• Embedding media : methacrylate and epoxy resins• These medias infiltrate well and help in thin sectioning• Blocks are transferred to suitable capsule containing fresh resin and these capsules are transferred to incubator for polymerization.
Processing scheduleFixation:• Glutaraldehyde 2.5% at 4° C for 1-4 hrs.• Wash in buffer.Post fixation treatment:• 1% osmium tetroxide at 4°C for 1 hr.• Wash in water.
When formalin fixed tissue used – area that is likely to be fixed from outer surface to be chosen. Paraffin blocks-the corresponding light microscopic section should be examined so that best portion of the tissue can be mapped. However paraffin embedded tissue is never satisfying for an electron microscopist because of considerable distortion.
Certain types specimens require special processing unlike surgical specimens.These include-1. Percutaneous renal biopsies- 1-2mm pieces from both the ends of the core are fixed to ensure cortical glomeruli are represented in tissue .
Aspirate directly expressed into glutaraldehyde with gentle agitation and kept for fixation2.FNAB Filtration of the fixed specimen through 20µm mesh screen Cells washed with pelleted buffer and processed as for solid tissue
3.Bone marrow aspirate Centifugation of Gentle Layering Disk is gently heparinized of fixative on the transferred and aspirate in buffy coat and further processed haematocrit tube fixation
4.Core biopsies of boneChallenging as decalcification causes severe damage to cells. Fixation in glutaraldehyde Soft marrow dislodged with fine needle under a dissecting microscope Processed in routine fashion
5.Body fluids Non • Centrifugation& • Fixation in glutaraldehydehemorrhagic fluidsHemorrhagic • Erythrocytes removed fluids with brief hemolysis. • Rinsing in buffer and fixation
TISSUE SECTIONINGPreparation of thick or semithin sections:After the tissue has been embedded in plastic resinThe blocks are embedded into sections at a thickness of approximately 1µm.
And these are stained with methylene blue or toulidine blue andExamined to verify that blocks selected are representative of the disease process
Thin sections are used for ultrastructural study-50nm thickness.These very thin sections are necessary-poor penetrating properties of electron beam.Ultramicrotomes are used for thin sectioning
STAINING• Staining done using heavy metals such as uranium and lead
Diagnostic applications As a rule the pathologist performing the EM should come to presumptive diagnosis from Clinical history and light microscopic findings before performing the ultra structural studies.
Electron microscopyUltrastructural diagnosis of Ultrastructure of non tumour tumors biopsy
Renal biopsies Aided in classification of renal disease inparticular & better understanding of thepathogenesis of glomerular disease
a. Detailed study of glomerulus- epithelial cell, endothelial cells, basement membrane & mesangium.b. Best method - to evaluate the thickness and the structure of glomerular basement membrane.c. Aids in identifying the exact location of immune- complex deposits within glomerulus.
Renal B opsy D agnosi s U i i sual l y R equi r i ng El ect r on M cr oscopy iMinimal change nephropathyPost-infectious glomerulonephritisMembranoproliferative glomerulonephritisMembranous nephropathyDense deposit diseaseDiabetic nephropathy—early morphological changes (GBM thickening)Fibrillary glomerulonephritisFocal-segmental glomerulosclerosis—early recurrence in renal allograft
Algorithm of Interpretation of Ultrastructural Findings: Discrete Immune-Type Electron-Dense Deposits Present COMBINED INTRAMEMBRANOUS SUBENDOTHELIAL, (Usually Combined SUBEPITHELIAL, SUBEPITHELIAL with Mesangial) SUBENDOTHELIAL MESANGIAL AND MESANGIALMembranous GN Dense deposit disease MPGN IgA Lupus (WHO nephropathy classes III and IV)Lupus (WHO class V) GN related to Lupus (WHO class Henoch- MPGN type III endocarditis, deep- III and IV) Schönlein seated abscesses purpuraPostinfectious GN Cryoglobulinemic Lupus (WHO GN related to GN (microtubular class II) endocarditis, substructure) C1q deep-seated nephropathy abscesses Rare other forms of mesangioprolif erative GN
Algorithm of Interpretation of Ultrastructural Findings: No Discrete Immune-Type Electron-Dense Deposits Present Subendothelial Fluffy Electron- Finely Granular Fibrillary/MicrotNormal GBM Diffusely Abnormal GBM Lucent Material Deposits ubular DepositsMinimal change Diffuse thinning All forms of Monoclonal Amyloidosis,disease, FSGS thrombotic immunoglobuli fibrillary GN, Thin GBM disease, early microangiopathies, n deposition cryoglobulinemic including malignantdisease GN, diabetic hypertension glomeruloscleros Alport syndrome is, collagen type Diffuse thickening III glomerulopathy Diabetes, hypertension, long-standing ischemia (also wrinkling) Diffuse lamellation /splitting Alport syndrome
Minimal change diseaseExtensive foot process effacement.
Post streptococcal glomerulonephritisSubepithelial humps of IgG & C 3
Membranoproliferative glomerulonephritisTYPE 1 TYPE 2
Storage disorders Deposition - lipid and glycogen can be visualized in biopsies of skin, brain, rectum, muscle, nerve, spleen, lymph nodes , bone marrow, heart and kidney. Gaucher’s disease- abnormal glucocerebroside accumulation in reticuloendothelial cell of liver,spleen,lymph nodes, and bone marrow.
Gaucher disease involving the bone marrowA, Gaucher cells with abundant lipid-laden granular cytoplasm.B, Electron micrograph of Gaucher cells with elongated distended lysosomes.
Ganglion cells in Tay-Sachs disease.A, Under the light microscope, a large neuron has obvious lipid vacuolation.B, A portion of a neuron under the electron microscope shows prominent lysosomes withwhorled configurations
Niemann -Pick disease• Accumulation of sphingomyelin in lysosomes..• Electron microscopy- engorged secondary lysosomes contain membranous cytoplasmic bodies resembling concentric lamellated myelin figures called zebra bodies
Viral & other infections• Body fluids, skin blister fluid, curetting from warty skin lesions, surgically resected, PM specimens• Size 20-300nm• Negative staining- 4% PTA
A, Adenovirus, an icosahedral nonenveloped DNA virus with fibers. B, Epstein-Barr virus, anicosahedral enveloped DNA virus. C, Rotavirus, a nonenveloped, wheel-like, RNA virus. D,Paramyxovirus, a spherical enveloped RNA virus. RNA is seen spilling out of the disruptedvirus
Respiratory Tract Biopsies EM helps in studying several abnormalities of ciliary structure. That is abnormalities in structure, number and pattern of microtubules that compose the axoneme of the cilium.
• Abnormal fine structure of cilia is seen in ciliary dysfunction, such as immotile cilia syndrome
Skeletal Muscle Biopsies Alterations that can be studied under EM are relatively non specific. Inclusions within myofibrils - lysosomal and non lysosomal storage disorders.*Fabry’s,Pompe’s+ Congenital multicore disease - disaaray of myofibrils
Fabry’s Disease Deficiency of alfa galactosidase and accumulation of glycosphingolipids Concentric intracytoplasmic inclusions
Ultrastructure of Tumors• Electron microscopy is an useful adjuvant techniques in the diagnosis and understanding of neoplasms.• Electron microscopy along with immunohistochemistry is more helpful than EM alone.
Indications• Confirming the light microscopic diagnosis of a neoplasm.• Differentiating primary neoplasms from metastatic neoplasms.• Evaluating metastatic tumors of unknown primary origin.• Evaluating histologically undifferentiated malignant neoplasms.
• Subtyping sarcomas• Subtyping lymphomas and leukemias• Evaluating neoplasms with unusual features such as crystalloid inclusions.
Squamous cell carcinoma Well differentiated squamous cell carcinoma Abundant cytokeratin filaments. Frequent desmosomes between cells.
Well differentiated squamous cellcarcinoma- frequent desmosomes
Poorly differentiated squamous cell carcinomasReduction in cytokeratin filamentsReduction in desmosomesDiminution in organellesLoss of basal lamina
AdenocarcinomaMicrovilli – short, stubby, prominent microfilament,& glycocalcyeal vesicles.Few desmosomes and cytokeratin filaments.Intracytoplasmic mucin or glycogen depositsTight junctional complexes
Adenocarcinoma small intestine– demonstrates tight junctional complex, mucin granules and luminal microvilli are typical .
Adenocarcinoma of colon- shows micrvilli, dense core rootlets below and rounded glycocalyceal bodies in the villi
Ultastructure Of MesotheliomasEM helps in differentiating mesotheliomas from adenocarcinoma.
Mesothelioma and Adenocarcinoma• Mesotheliomas are characterized by having long, narrow, branching microvilli with a length to width ratio of around 10-16:1 ,-on free surfaces of cells.• By contrast, adenocarcinomas have short, stubby microvilli with core rootlets.
Electron microphotograph of malignant mesothelioma-long thin non intestinal type ofmicrovilli devoid of glycocalyx and actinic rootlets.
Ultrastructure of MelanomaHelpful in diagnosing melanomas -not express anti-S-100 or the HMB-45 monoclonal antibody.In such cases identification of premelanosomes or melanosomes hallmarks the diagnosis of melanoma.
Melanosomes –cytoplasmic organelles where melanin is produced. There are four stages in development.
• Immunostaining remains the main stay.• However EM is helpful –immunostaining is equivocal or negative.
• Ultrastructural findings of leukemias and lymphomas 1. Nuclear pockets 2. Absence of intercellular attachment 3. Lack of endoplasmic reticulum 4. Cytoplasm filled with free ribosomes 5. Sparse mitochondria 6. Lipid droplets in large cell lymphomas
7. Immunoblastic transformation- the cisternae of the endoplasmic reticulum become abundant and organised.8.Birbeck granules in Langerhans cells- are endocytic organelles that transport antigens from receptors on cell surface to interior to fuse with saccules of the golgi complex producing racket like configuration.
Smooth muscle cell tumours- poorly developed ER , myofibrillary filaments attached to focal densities
• Diffuse sheets not classified by other means• Myofilaments of skeletal muscle in embryonal or alveolar rhabdomyosarcoma• Lakes of glycogen in Ewing’s tumour• Distinctive cytoplasmic processes in neuroblastoma
Neuroblastoma Cytoplasmic processes wrapping around a neuroblastoma cell
Neuroendocrine tumours• Neuro secretory vacuoles in cytoplasm• Spherical , ovoid• Electron dense centre surrounded by a clear lucent halo enclosed in distinct membrane.• Carcinoids, APUD, chemodactoma, medullary ca thyroid
• Classification of various cellular conformations of the CNS is difficult when trying to distinguish glial and neuronal elements using only light microscopy and immunohistochemistry.• Electron microscopy often plays a pivotal role in diagnosis because it can provide accurate diagnosis when immunohistochemical studies are equivocal or negative.
Filgree pattern of the curving strands of attenuated cells- extracranialmeningioma
Disadvantages1. EM is not economical- stable high voltage supply, vaccum system etc2. Findings unlikely to influence treatment, IHC n LM together are confirmatory .3. Tissue preparation is tough4. Only a small proportion of neoplasm can be studied5. Misinterpretation of non- neoplastic elements belonging to the tumor
ConclusionCurrently the use of EM is limited for theexpense and lack of surgical pathologists tointerpret EM findings .Still it provides unique insight into thestructure of some tumors and renalpathologies.So better to use it selectively in study anddiagnosis of human diseases and researchareas and correlating the findings with LMfindings and IHC results.
Recent advances• One of the latest developments in electron microscopy is the environmental scanning electron microscope (ESEM), which enables soft, moist and/or electrically insulating materials to be viewed without pre-treatment.
Hammar  has succinctlysummarized the currentdiagnostic status for IHC and EM: “ There are no immunohistochemical features that are absolutely specific 100% at this time in diagnosing a neoplasm. There are a number of ultra structural features of neoplasm that are 100% or nearly 100% specific in diagnosing certain neoplasms."
References• Theory and practices of histopathological techniques, John D Bancroft, 4th edition;pg 585- 639.• Robbins and Cotran, PATHOLOGIC BASIS OF DISEASE, 8th edition.• Cellular pathology technique, C.F.A. Culling, 4th edition;pg 603-620.• Various internet sources.