Electron microscopy

1. ELECTRON MICROSCOPY -Dr Ganga H

2. Introduction Transmission EM Scanning EM Preparation of tissue for EM Diagnostic applications

3. 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. Introduction and History

4. 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.

5. 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.

6. Types of microscopes Three basic types : O ptical , 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

7. 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.

8. 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

9. 1931-Ernst Ruska at the University of Berlin built the first transmission electron microscope (TEM) 1986- awarded the Nobel Prize for Physics

10. 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.

11. 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.

12. 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 .

13. Types of electron microscope 1. Transmission electron microscopy : 2. Scanning electron microscopy:

14. SEM

15. Normal cell under EM

16. 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

17. Analogy between LM & TEM Arrangement & function of their components Illuminating system – source & condensor Imaging system – lenses to produce magnified image – objective & projector Image translating system – Final image is viewed

18. LM EM

19. THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE Vacuum Air-filled Interior Magnets Glass Lenses High voltage (50kV) tungsten filament Tungsten or quartz halogen lamp Radiation source X 5,00,000 x1000 – x1500 Maximum magnification 0.14nm Fine detail app. 200nm or 0.2micron Maximum resolving power Electrons app. 4nm Visible light 390nm (red) – 760nm Electromagnetic spectrum used ELECTRON MICROSCOPE LIGHT MICROSCOPE FEATURE © 2007 Paul Billiet ODWS Focus Lens is movable Rigidly fixed, adjust lens currents

20. THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE Copper grid Glass slide Support Heavy metals Water soluble dyes Stains Ultramicrotome Slices - 50nm Parts of cells visible Microtome slices - 20 000nm Whole cells visible Sectioning Resin Wax Embedding Glutaraldehyde,OsO 4 formaldehyde Fixation ELECTRON MICROSCOPE LIGHT MICROSCOPE FEATURE © 2007 Paul Billiet ODWS Focussing screen Human eye (retina), photographic film Fluorescent screen, photographic film

21. ELECTRON SOURCE ELECTROMAGNETIC LENS SYSTEM SAMPLE HOLDER IMAGING SYSTEM.

22. 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.

23. 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.

24. TEM9.swf

25. The Vacuum System T he 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

26. Sample holder The sample holder is a platform equipped with a mechanical arm for holding the specimen and controlling its position.

27. 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.

28. 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.

29. Scanning electron microscopy

30. 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.

31. 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.

32. 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.

33. Inelastic scattering: Primary electrons hit electrons of the specimen atom Energy is transferred from the primary electron to the specimen Emission of electrons and radiation

34. Electron specimen interaction

35. 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).

36. 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.

37. The focused electron beam is moved from one pixel to another. At every pixel, the beam stays for a defined time and generates a signal ( e.g.secondary electrons) which are detected, amplified and displayed on a computer screen

38. A smaller area is scanned with the same number of pixels. The scanned pixels are smaller Image magnification in SEM

39. TEM vs SEM TEM SEM 6 lenses – C1, C2, objective, 3 projector 3 lenses – 2 condensor , 1 objective High accelerating voltage - penetration low accelerating voltage Not complicated Specimen Stage – complicated X & y axis X,Y,Z-axis, tilting, rotating

40. Contrast formation in TEM Absorption of electrons Scattering of electrons Diffraction and phase contrast

41. 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

42. Thin section of alga stained with heavy metals (Ur, Pb )

43. 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

44. Uniform layer of heavy metal on specimen surface Primary electron beam Platinum

45. 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.

46. Specimen preparation for electron microscopy Steps include Specimen procurement Fixation Tissue processing and sectioning Staining

47. SPECIMEN PROCUREMENT Tissue preserved in glutaraldehyde . Tissue must be representative of the disease. Areas that show - degeneration, necrosis, haemorrhage must be avoided.

48. 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.

49. 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.

50. 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.

51. Processing schedule Fixation : 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.

52. Dehydration 50% alcohol 5-15 min 70% alcohol 5-15 min 90% alcohol 5-15 min Absolute alcohol 5-15 min Absolute alcohol 5-15 min Absolute alcohol 5-15 min Clearing Propylene oxide 15 min Propylene oxide 15 min Impregnation Epoxy resin 45-60min Polymerization at 60°C 24hrs

53. 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.

54. Certain types specimens require special processing unlike surgical specimens. These include- Percutaneous renal biopsies- 1-2mm pieces from both the ends of the core are fixed to ensure cortical glomeruli are represented in tissue .

55. 2.FNAB

56. 3.Bone marrow aspirate

57. 4.Core biopsies of bone Challenging as decalcification causes severe damage to cells.

58. 5.Body fluids

59. TISSUE SECTIONING Preparation of thick or semithin sections: After the tissue has been embedded in plastic resin The blocks are embedded into sections at a thickness of approximately 1µm.

60. And these are stained with methylene blue or toulidine blue and Examined to verify that blocks selected are representative of the disease process

61. 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

62. STAINING Staining done using heavy metals such as uranium and lead

63. 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.

64. Electron microscopy Ultrastructural diagnosis of non tumour biopsy Ultrastructure of tumors

65. Diseases of kidney Metabolic storage diseases Respiratory tract biopsies Skeletal muscle diseases Infectious agents Cutaneous diseases Peripheral nerve biopsies Epithelial tumors Mesothelioma Melanoma Hematopoietic and lymphopoietic tumors Soft-tissue tumors Central nervous system tumors Small round cell tumors Non tumor biopsies Tumor diagnosis

66. . NON TUMOR BIOPSIES

67. Renal biopsies Aided in classification of renal disease in particular & better understanding of the pathogenesis of glomerular disease

68. 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.

69. Renal Biopsy Diagnosis Usually Requiring Electron Microscopy Minimal change nephropathy Post-infectious glomerulonephritis Dense deposit disease Diabetic nephropathy—early morphological changes (GBM thickening) Fibrillary glomerulonephritis Focal-segmental glomerulosclerosis—early recurrence in renal allograft Membranoproliferative glomerulonephritis Membranous nephropathy

70. SUBEPITHELIAL INTRAMEMBRANOUS (Usually Combined with Mesangial) SUBENDOTHELIAL MESANGIAL COMBINED SUBENDOTHELIAL, SUBEPITHELIAL, AND MESANGIAL Membranous GN Dense deposit disease MPGN IgA nephropathy Lupus (WHO classes III and IV) Lupus (WHO class V) GN related to endocarditis, deep-seated abscesses Lupus (WHO class III and IV) Henoch-Schönlein purpura MPGN type III Postinfectious GN Cryoglobulinemic GN (microtubular substructure) Lupus (WHO class II) C1q nephropathy Rare other forms of mesangioproliferative GN GN related to endocarditis, deep-seated abscesses Algorithm of Interpretation of Ultrastructural Findings: Discrete Immune-Type Electron-Dense Deposits Present

71. Normal GBM Diffusely Abnormal GBM Subendothelial Fluffy Electron-Lucent Material Finely Granular Deposits Fibrillary/Microtubular Deposits Minimal change disease, FSGS Diffuse thinning All forms of thrombotic microangiopathies, including malignant hypertension Monoclonal immunoglobulin deposition disease Amyloidosis, fibrillary GN, cryoglobulinemic GN, diabetic glomerulosclerosis, collagen type III glomerulopathy Thin GBM disease, early Alport syndrome Diffuse thickening Diabetes, hypertension, long-standing ischemia (also wrinkling) Diffuse lamellation / splitting Alport syndrome Algorithm of Interpretation of Ultrastructural Findings: No Discrete Immune-Type Electron-Dense Deposits Present

72. Minimal change disease Extensive foot process effacement. edema) and as well as steroids to halt the disease process.

73. Post streptococcal glomerulonephritis Subepithelial humps of IgG & C 3

74. Membranous glomerulopathy

75. Membranoproliferative glomerulonephritis TYPE 1 TYPE 2

76. 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.

77. Gaucher disease involving the bone marrow A, Gaucher cells with abundant lipid-laden granular cytoplasm. B, Electron micrograph of Gaucher cells with elongated distended lysosomes.

78. 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 with whorled configurations

79. 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

80. Viral & other infections Body fluids, skin blister fluid, curetting from warty skin lesions, surgically resected, PM specimens Size 20-300nm Negative staining- 4% PTA

81. A, Adenovirus, an icosahedral nonenveloped DNA virus with fibers. B, Epstein-Barr virus, an icosahedral 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 disrupted virus

82. Electron microscopic picture of HSV

83. Whipple’s disease

84. 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.

85. Abnormal fine structure of cilia is seen in ciliary dysfunction, such as immotile cilia syndrome

86. 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

87. Mitochondrial myopathy

88. Fabry’s Disease Deficiency of alfa galactosidase and accumulation of glycosphingolipids Concentric intracytoplasmic inclusions

89. 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.

90. 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.

91. Subtyping sarcomas Subtyping lymphomas and leukemias Evaluating neoplasms with unusual features such as crystalloid inclusions.

92. Squamous cell carcinoma Well differentiated squamous cell carcinoma Abundant cytokeratin filaments. Frequent desmosomes between cells.

93. Well differentiated squamous cell carcinoma- frequent desmosomes

94. Poorly differentiated squamous cell carcinomas Reduction in cytokeratin filaments Reduction in desmosomes Diminution in organelles Loss of basal lamina

95. Adenocarcinoma Microvilli – short, stubby, prominent microfilament,& glycocalcyeal vesicles. Few desmosomes and cytokeratin filaments. Intracytoplasmic mucin or glycogen deposits Tight junctional complexes

96. Tight junctional complex

97. microvilli

98. Adenocarcinoma small intestine – demonstrates tight junctional complex, mucin granules and luminal microvilli are typical .

99. Adenocarcinoma of colon- shows micrvilli , dense core rootlets below and rounded glycocalyceal bodies in the villi

100. Ultastructure Of Mesotheliomas EM helps in differentiating mesotheliomas from adenocarcinoma.

101. 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.

102. Electron microphotograph of malignant mesothelioma-long thin non intestinal type of microvilli devoid of glycocalyx and actinic rootlets.

103. Ultrastructure of Melanoma Helpful 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.

104. Melanosomes – cytoplasmic organelles where melanin is produced. There are four stages in development.

105. Hematopoietic And Lymphocytic Tumors

106. Immunostaining remains the main stay. However EM is helpful – immunostaining is equivocal or negative.

107. 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

108. 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.

109. Burkitt's lymphoma Numerous nuclear projections ( np ), polar aggregation of mitochondria (m), sparse endoplasmic reticulum ( er )

110. Histiocytosis X Mononuclear langerhans cells with curved nucleus & an abundant cytoplasm Birbeck granules in the cytoplasm

111. Spindle cell tumors Fibrosarcomas - abundant rER , collagen formation Leiomyosarcomas – myofibrils & focal densities Spindle cell SCC - tonofibrils & occasional desmosomes

112. Fibrosarcomas - abundant rER , collagen formation

113. Smooth muscle cell tumours - poorly developed ER , myofibrillary filaments attached to focal densities

114. Small round cell tumors

115. 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

116. Ewing’s sarcoma Prominent lakes of glycogen

117. Embryonal RMS – cytoplasm showing haphazardly arranged abortive cross striations

118. Neuroblastoma Cytoplasmic processes wrapping around a neuroblastoma cell

119. 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

120. Central Nervous System Neoplasms

121. 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.

122. Meningioma Long,interdigitating cellular processes Numerous cytoplasmic filaments Prominent desmosomes

123. Filgree pattern of the curving strands of attenuated cells- extracranial meningioma

124. Disadvantages EM is not economical - stable high voltage supply, vaccum system etc Findings unlikely to influence treatment, IHC n LM together are confirmatory . Tissue preparation is tough Only a small proportion of neoplasm can be studied Misinterpretation of non- neoplastic elements belonging to the tumor

125. Conclusion Currently the use of EM is limited for the expense and lack of surgical pathologists to interpret EM findings . Still it provides unique insight into the structure of some tumors and renal pathologies. So better to use it selectively in study and diagnosis of human diseases and research areas and correlating the findings with LM findings and IHC results.

126. 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.

127. “ 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." Hammar [2002] has succinctly summarized the current diagnostic status for IHC and EM :

128. References Theory and practices of histopathological techniques, John D Bancroft, 4 th edition;pg 585-639. Robbins and Cotran, PATHOLOGIC BASIS OF DISEASE, 8 th edition . Cellular pathology technique, C.F.A. Culling, 4 th edition;pg 603-620. Various internet sources.

129. Thank you