STUDY OF ELECTRON MICROGRAPHS �OF DIFFERENT COMPONENTS �OF ANIMAL CELL

STUDY OF ELECTRON MICROGRAPHS �OF DIFFERENT COMPONENTS �OF ANIMAL CELL

1. STUDY OF ELECTRON MICROGRAPHS OF DIFFERENT COMPONENTS OF ANIMAL CELL

2. Components of a TEM Some what similar to a Light Microscope (LM) Electron gun produces electron beam and magnetic lenses, stacked vertically to form a lens column 2) I llumination system the electron gun, and 2 or more condenser lenses Specimen stage assemble/remove the specimen; hold/move specimen Imaging system three lenses that together produce a magnified image on the monitor screen

3. TEM images are always monochromatic / gray scale (black and white) false colors may be added to the images with image processing software

4. Sample preparation for transmission electron microscopy Primary Fixation : Glutaraldehyde 2.5% + 2-4% Paraformaldehyde Secondary Fixation : Osmium tetroxide, electron dense, fixative and a stain Dehydration : in acetone or ethanol Infiltration : with resins Embedding : Epoxy resin Sectioning : Semi-thin sections (0.5-1.0 um) stained with toluidine blue for selection of the target areas Ultrathin sections (60-90 nm) are cut and collected onto grids air dried for 2 h – overnight Staining : uranyl acetate or lead citrate

5. STUDY OF ELECTRON MICROGRAPHS OF DIFFERENT COMPONENTS OF ANIMAL CELL Cell Membrane low/medium magnification, appears as an electron­dense line high magnification, appears a bilayer 7 to 11 nm thick membrane called trilaminar membrane, has three visible lines two electron dense lines separated by one electron translucent line because the lines are parallel, it is called ‘ railroad track ”.

6. mitochondria spherical or cup­shaped , rod shaped or irregular an outer, and an inner mitochondrial membranes parallel to the outer membrane Inner membrane continues to form the cristae mitochondrial (dense bodies) matrix is internal called matrix granules that are calcium salts lamellar (sheet-like) or tubular; branching and anastomosing cristae may be observed

7. Rough endoplasmic reticulum ( r ER ) part of the flattended endo -membranous components (cristae) found in the cytoplasm and are studded with ribosomes Ribosomes are seen on the external surface cisternae are interconnected, the lumen is dense occassionally , seen in continuity with nuclear envelope The rER and smooth endoplasmic reticulum ( sER ) are commonly found to be continuous

8. Smooth endoplasmic reticulum ( sER ) no ribosomes composed of anastomosing tubules (100 nm dia.) irregular shape and diameter appears as a ''contorted" network of smooth membranous elements tubules form a continuous membrane system that weaves in and out of the plane of section

9. Golgi apparatus appears as a group of stack membrane cisternae (Golgi stack or dictyosome ) cisternae form a slightly curved or U­ shaped structure generally located near the nucleus stacks contain a number of cisternae , less than ten convex face: forming or cis face concave face is known as the maturing or trans face vacuoles containing dense material seen near the trans face

10. Peroxisomes & lysosomes Peroxisomes ( microbodies ) displays a finely granular matrix lysosome is more densely granular and contain ingested material

11. Centrioles short, cylindrical structures seen in pairs called diplosomes near the nucleus close by the Golgi apparatus composed of microtubular elements, fused as triplets . Nine sets of triplets are arranged in a ‘pinwheel fashion’.

12. Endomembrane system of cell

13. The Nucleus . spherical, ellipsoidal, flattened, lobed, or highly infolded Nuclear features include the nuclear envelope, the nuclear lamina, and the nucleolus

14. The Nuclear Envelope and Nuclear Lamina Nuclear envelope present in eukaryotes a double membrane: outer and inner membranes that encompass the nucleoplasm space between the two membranes is called the perinuclear space rER is often in direct continuity with the nuclear envelope at intervals, the two membranes participate in forming nuclear pores Nuclear lamina : a uniform layer of dense material along the inner aspect of the nuclear envelope

15. Chromatin Heterochromatin condensed or visible chromatin, is stable (inactive) Euchromatin uncondensed form of chromatin are relatively clear areas of the nuclear matrix; 10% available for transcription

16. The Nucleolus number and size variable occupy only a small portion of the nucleus has three structural components: the pars fibrosa ; fibrillar centers contain DNA and RNA synthesis centers the pars granulosa , is an aggregation of the ribosomes RNA synthesis takes place in the nucleolus.

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18. Cell surface The Glycocalyx fuzzy coat external to plasma membrane seen in EM difficult to visualize; special stains are used to observe many times thicker than plasma membrane Polysaccharide in nature part of intrinsic proteins of plasmalemma found in tubular organs; extends to lumen negatively charged

19. the glycocalyx

20. Cell Junctions

21. Tight or occluding junctions ( zonula occludens ) linear fusions of membrane between adjoining epithelial cells usually extend around the entire circumference appears as a five layered or pentalaminar at junction site

22. Adherens junctions 2 types Intermediate junction or zonula adherens forms a continuous "belt" around the cell the intercellular space remains constant at junctional site a vague intercellular line parallel to junctional membranes cytoplasmic surface has a density receives actin filaments

23. Zonula adherens

24. Desmosome or macula adherens plaque like structures contains a definite intermediate dense line in intercellular space parallel to cell surface Intermediate filaments of keratin ( tonofilaments ) make hairpin turns at the junctional density Freeze fracture reveals them as aggregations of intramembranous particles of unequal size

25. Hemidesmosomes or half desmosomes cell surface specializations found at interface b/w epithelium and the underlying basal lamina appearance at the cell surface is one whereby the junction resembles a desmosome joined to a basal lamina.

26. Gap junctions or nexus demonstrate a seven­layered ( septalaminar ) appearance the opposing membranes of the cells come close to each other, but do not fuse intercellular space 2 to 4 nm Gap junction particles ( connexins ) are seen in thin sections work in intercellular communication Connexins contain pores that transport molecules of 1,200 kd molecular weight

27. Junctional complexes join all epithelial cells at their lateral surface near the apical aspect of the cell present where the lateral surface of the epithelial cell meets a lumen; e.g. in the gastrointestinal tract The tight junction is the component closest to the lumen followed by the intermediate junction , and then the hemi- desmosome , which is most basally positioned desmosomes are only periodic structures

28. Scanning electron microscopy

29. Vacuum system Electron beam generation system Electron beam manipulation system Beam specimen interaction system Detection system Signal processing and display system

30. SEM working principle Vacuum system: Prevents air electron collisions Electron beam generation system: Electron gun Electron beam manipulation system: electromagnetic lenses and coils- control the size, shape, and position of the electron beam on the specimen surface Beam specimen interaction system: Electron beam and specimen interaction 5. Detection system: A secondary electron detector Signal processing and display system: visualization of an electronic signal

31. How scanning is achieved: The Scanning Coils As the beam passes through the final condenser lens, two sets of magnetic scanning coils move the beam: These radially opposing, magnetic coils allow scanning in both the X and Y directions The scan pattern is called a raster (rectangular) pattern The scan begins in the upper left corner and proceeds to the right, then dropping down one line with each line scanned these coils give the scanning electron microscope its name

32. electron Detection; Signal processing and display system: Each point on the specimen that is struck by the accelerated electrons emits signal in the form of electromagnetic radiation Selected portions of this radiation, usually secondary (SE) and/or backscattered electrons (BSE), are collected by a detector the resulting signal is amplified and displayed on a TV screen or computer monitor

33. Sem specimen stubs

34. Glial Cells (Red) & Neurons (Blue)

35. SEM V/S TEM SEM TEM Resolving power 400 pm based on scattered electrons Reports morphology and relation large area of sample analyzed at a time Projects picture via CRT to monitor 3D Resolving power 50 pm based on transmitted electrons Reports internal composition only small area of sample analyzed at a time fluorescent screens / monitor 2D

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