1. Methods of Sample Preparation
Preparation of Cells/Tissues for Microscopy

2. Methods of Preparation
Fresh cells and tissues
Cytological techniques
Sectional methods
Sample preparation for the microtome sectioning

3. 1. Fresh cells and tissues
Direct examination
Isotonic solution (saline)
(e.g. subcutaneous connective tissue)

4. 1. Fresh cells and tissues
Direct examination
Smear
Cells suspended in fluid
(e.g. blood, urine or lymph)

5. 1. Fresh cells and tissues
Direct examination
Smear
Dissociation / teasing
Thick tissue

6. 1. Fresh cells and tissues
Direct examination
Smear
Dissociation / teasing
Natural state (^^) but lack of contrast (TT)
Specialized microscopy (e.g. Phase-contrast microscopy)
Vital staining

7. Vital Staining
stain that can be applied on living cells without killing (fixing) them
technique in which a harmless dye is used to stain living tissue for microscopical observation
neutral red, Janus green B, eosin Y, brilliant cresyl blue, nigrosin, Bismarck brown, crystal violet, methylene blue & food color

8. Vital Staining
stain that can be applied on living cells without killing (fixing) them
technique in which a harmless dye is used to stain living tissue for microscopical observation
Supra-Vital Staining
the living tissue may be removed directly and subsequently stained
Intra-Vital Staining
injection of dye into the living organism and the stained tissue removed and examined
Higher concentrations of vital stain will kill the cell
only certain cytoplasmic elements can be demonstrated

9. 2. Cytological techniques
widely applied to the diagnosis of disease
Cells obtained from all parts of body by
Fine needle aspiration (FNA) technique
Exfoliative cytology
Impression method

10. Fine needle aspiration cytology

11. Exfoliative cytology
the analysis of cells shed from the body (e.g. Pap smear)
Harvest Exfoliative Cells from the normal tissue

12. Pap smear to detect cancerous or precancerous conditions of the cervix
George N. Papanicolaou (1943)

13. Impression cytology
pressing a clean glass slide firmly against the surface of the organ or tissue and then lifting it away

14. 2. Cytological techniques
widely applied to the diagnosis of disease
Cells obtained from all parts of body by
Fine needle aspiration (FNA) technique
Exfoliative cytology
Impression method
Smear, Fix and Stain
2D-, flattened
larger than the same cells in tissue sections
Cellular details are more easily seen

15. 3. Sectional methods Why Section? Microtome Cryostat
Cutting the specimen into very thin translucent slices or sections
Microtome
Cryostat
Why Section?
Tissues and organs are usually too thick for light pass through them.

16. Sample preparation for the microtome sectioning
formaldehyde ethanol xylene paraffin
(70% → →100%)

17. Paraffin embedded tissue block

18. Microtome
1-10 μm thick
For sectioning paraffin-embedded tissues

22. Ultramicrotome For TEM sections less than 1μm thick Resin-embedding
glass or diamond knife

23. Cryostat Freezing microtome Fixed by Rapid freezing
For the frozen block of tissue
Sample preparation
Fixed by Rapid freezing
In Isopentane or liquid nitrogen
Support medium (e.g. OCT)
-20 ± 5 ℃
Control panel
Freezing chamber
(Cutting Chamber)
Handle

24. Why cryostat? Advantages of Frozen sections
Rapid preparation of sections
Better for the demonstration of histochemically sensitive molecules
enzymes
lipids
Superior for the preservation of antigenicity

25. Disadvantages Quality of sections
Inferior to paraffin sections
Less stable than paraffin- or resin-embedded sections
Inconvenient storage
Low temperature cabinet or a jar of fixative at room temperature
not ideal method for very thin section (1-3 μm)

26. ice crystal artifacts (in cryostat)

27. Problems in the study of sections
Structures seen microscopically may differ from the structures present when they were alive (=artifacts)
Shrinkage (by the fixative, ethanol or heat), Wrinkles and so on
Three-dimensional structures appear to have only two dimensions in thin sections.

28. knife scratches & splitting

29. Shrinkage

30. Wrinkles

31. Poor fixation

32. Problems in the study of sections
Structures seen microscopically may differ from the structures present when they were alive (=artifacts)
Shrinkage (by the fixative, ethanol or heat), Wrinkles and so on
Three-dimensional structures appear to have only two dimensions in thin sections.

33. 3-D structures in 2-D sections

36. Serial sections
Serial sections and reconstructing the images 3- dimensionally provides better understanding

37. Learning Resources Junqueira’s Basic Histology ; pp 1-3
Looking at the Structure of Cells in the Microscope