1. Introduction to Histology
Al-Maarefa College

2. Objectives Understand what is Histology
Realize the size of histological tissues
Know basic steps in tissue preparation and staining
Understand the importance of different stains
Identify different techniques of imaging in histology

3. What is Histology? The name "Histology" is derived from Greek:
"Histos“: tissue
"logos" : the study of
Histology: the study of tissues

4. What is Histology?
The study of the microscopic anatomy of cells and tissues
How?
Examining a thin slice (section) of tissue under a light microscope or electron microscope
Enhanced by using different histological stains

5. Levels of body organization
Cells:
The smallest independently living highly complex structure
Tissues:
Functional unit: group of cells of similar function and origin (with matrix between cells)
Organs:
Several tissues grouped together
Systems:
Several organs working for a common function

6. Levels of body organization

7. Tissues Tissues are made of:
cells and
extracellular matrix
Intense interaction between cells and matrix
Cells and extracellular matrix form a continuum that functions together and reacts to stimuli and inhibitors together

8. Tissues Four fundamental tissues are recognized: Epithelial tissue
Connective tissue
Muscular tissue
Nervous tissue

9. The size spectrum
The small size of cells and matrix components makes histology dependent on the use of microscopes

10. equal to 10−9 m (a billionth of a meter)
The size spectrum
Electron Microscope
0.1 nm
equal to 10−9 m (a billionth of a meter)
Light Microscope
300 nm
Unaided Eye
300 um
1/1000 of a millimetre, or 0.001mm

11. The size spectrum
Unaided Eye
300 um

12. The size spectrum
Light Microscope
300 nm
Unaided Eye
300 um

13. The size spectrum Electron Microscope 0.1 nm Light Microscope 300 nm
Unaided Eye
300 um

14. Light Microscopy With a maximum magnification power of x 1000 times

15. Electron Microscopy With a maximum magnification power of x 1000000 times

16. Electron Microscopy Bronchiole, epithelial cells, X 5000
Surface of epithelial cell, X 100,000

17. Basic Techniques Preparation of histological sections Fixation
Processing (dehydration and clearing)
Embedding
Cutting
Staining
Permanent Mounting

18. Preparation of histological sections
Fixation
Fixing tissues by chemicals so they will not change their volume and shape during processing
Keeps tissue as close to their living state as possible
prevents autolysis and bacterial attack
prepares tissues for staining
Fixatives:
Acetic acid, Formaldehyde, Ethanol, Glutaraldehyde, Methanol and Picric acid.

19. Preparation of histological sections
Dehydration and clearing
removes fixative and water from the tissue and replace them with dehydrating fluid
Uses hydrophilic or alcohol solutions to extract water
Examples of solutions:
Ethanol, Methanol, Acetone

20. Preparation of histological sections
Embedding
is the process by which tissues are embedded in a medium (agar, gelatin, or wax) which when solidified provides sufficient external support during sectioning

21. Preparation of histological sections
Cutting
Small (micro) sections are cut by a Microtome
Section thickness
2 to 25 micrometers=um thick for light microscope
60 to 100 nanometers thick for electron microscopy

22. Preparation of histological sections
Staining
Various stains are used to see and highlight specific structures and molecules

23. Preparation of histological sections
Staining
Various stains are used to see and highlight specific structures and molecules

24. Hematoxylin and Eosin (H & E)
H & E stains are universally used for routine histological examination of tissue sections.

25. Staining Techniques Hematoxylin and Eosin (H&E)
Hematoxylin is a basic dye that stains acidic components of cells a blue (basophilia)
Stains the nuclei of cells, and the rER of cytoplasm

26. Staining Techniques Hematoxylin and Eosin (H&E)
Eosin is an acidic dye that stains the basic components of cells reddish-pink (acidophilia)
Most of cytoplasm of cells is stained by eosin

27. Hematoxylin and Eosin (H & E)
Nuclei - blue - with some metachromasia
Cytoplasm - various shades of pink-identifying different tissue components

28. Features of H&E Hematoxylin Eosin Blue colored
Positively charged (colors negatively charged molecules, such as DNA, rER)
The ability of such anionic groups to react with a basic dye - basophilia
Eosin
Red colored
Negatively charged (colors positively charged molecules (amino acids, cytoplasm)
The ability of such cationic groups to react with an acidic dye - acidophilia

29. Staining Techniques Periodic acid-Schiff (PAS) staining
mucus, basal lamina, glycogen (carbohydrates)
H&E stain does not preserve
Mucous in Goblet cells, which
Appear empty
PAS stain preserves Mucous in Goblet cells, which and stains it
with a magenta colour
Wheater’s Functional Histology, a text and colour atlas
Junqueira and Carneiro, Basic Histology, a text and atlas

30. Staining Techniques Periodic acid-Schiff (PAS) staining
mucus, basal lamina, glycogen (carbohydrates)
Glycogen

31. Staining Techniques Periodic acid-Schiff (PAS) staining
mucus, basal lamina, glycogen (carbohydrates)
Carbohydrate content in liver tissue stained magenta with PAS stain
Clarke F. Millette, Univ. of South Carolina, USA

32. Staining Techniques Toluidine blue (Metachromatic stain)
Blue stain that stains specific components of tissues a purple color
Metachromasia is seen in the matrix of hyaline cartilage, or in the granules of mast cell
IJDVL, Omar El Safoury
Science Photo Library
Blue stain of Hyaline cartilage
Metachromasia of Mast cells, skin

33. Staining Techniques Oil Red O Sudan black
Stain lipids red-orange in unfixed frozen sections
Sudan black
Stains lipids black in unfixed frozen sections

34. Staining Techniques Impregnation
Silver impregnation techniques are also widely used to demonstrate reticular fibers
Lymph node, Silver Impregnation
Seoul National University

35. Staining Techniques Reticulin
Reticular fibers are stained dark. Used esp. in liver
Smooth Muscle, Reticulin stain
Clarke F. Millette, Univ. of South Carolina, USA

37. A liver biopsy stained using the reticulin demonstrating the normal hepatic plate thickness.

38. Plasmodiun Falciparum (Malaria)
Staining Techniques
Giemsa stain
Blood smears, for disease
Wuchereria bancrofti
Causing filariasis
Plasmodiun Falciparum (Malaria)

39. Plasmodiun Falciparum (Malaria)
Staining Techniques
Giemsa stain
Blood smears, for disease
Trypanosoma sp.
Plasmodiun Falciparum (Malaria)
CDC /Dr. Myron G. Schultz 1970

40. Preparation of histological sections
Mounting
preserves and supports a stained section
mounted on a clear glass slide, and covered with a thin glass cover-slip

41. Basic Techniques Total preparation time: 16 hours
Immediate Frozen section time: 5 minutes
For immediate results – (in tumor surgery)
Results not as accurate as formalin fixed, wax embedded specimens

42. Basic Techniques
In some cases the tissue to be examined is a very thin membrane (Cell Smears)
blood or bone marrow

43. Basic Techniques
In some cases the tissue to be examined is a very thin membrane (Cell Smears)
blood or bone marrow

44. Basic Techniques
In some cases the tissue to be examined is a very thin membrane (Cell Smears)
blood or bone marrow
epithelial cells
(e.g. from the oral cavity, cervix uteri)
Crvical carcinoma, PAP stain
Univ of Uklahoma Health Science Center
NATIONAL CANCER INSTITUTE / SCIENCE PHOTO LIBRARY

45. Some definitions Histology: Biopsy: Autopsy:
microscopic study of tissues
Biopsy:
removal of tissues for diagnostic purposes
Autopsy:
examination of organs of a dead body (to find clues to determine cause of death)

46. Phase-Contrast Microscopy

47. Phase-Contrast Microscopy
An optical microscopy illumination technique
A small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image.
Does not require staining to view the slide
Epithelial cell from cheek

48. Polarizing Light Microscopy
Polarization is a property of Electromagnetic waves, such as light that describes the orientation of their oscillations
Polarizer is rotated to transmit the reflections as well as possible
By rotating the polarizer by 90° , almost all reflected sunlight is blocked 

49. Polarizing Light Microscopy
Polarization is a property of Electromagnetic waves, such as light that describes the orientation of their oscillations

50. Polarizing Light Microscopy
Collagen fibers
Yellow birefringence

51. Fluorescence Microscopy
A sample is illuminated with light of a one wavelength which causes fluorescence in the sample. The light emitted by fluorescence, which is at a different, longer, wavelength than the illumination, is then detected through a microscope objective

52. Fluorescence Microscopy
Epifluorescent imaging of the three components in a dividing human cancer cell.
DNA is stained blue,
a protein called INCENP (Inner centromere protein) is green, and
the microtubules are red.
Each fluorophore is imaged separately using different filters, and images are captured sequentially using a digital CCD camera( charge coupled device), then overlaid to give a complete image.

53. Fluorescence Microscopy

54. Scanning Electron Microscopy
Clarke F. Millette, Univ. of South Carolina, USA

55. Scanning Electron Microscopy
Bruce Wetzel/Harry Schaefer, courtesy National Cancer Institute

56. Scanning Electron Microscopy

57. Scanning Electron Microscopy

58. Scanning Electron Microscopy
Cut end of human hair
Catchrandom.blogspot

59. Scanning Electron Microscopy
Human skin

60. Summary What is histology
The size spectrum and organizational structure
Light microscope / Electron microscope

61. Summary Preparation of tissue Different stains and special techniques
fixation
processing
embedding
cutting
staining
mounting
Different stains and special techniques

62. Thank you