Methods of Sample Preparation Preparation of Cells/Tissues for Microscopy

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

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

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

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

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

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

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

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

Fine needle aspiration cytology

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

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

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

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

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.

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

Paraffin embedded tissue block

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

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

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

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

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)

ice crystal artifacts (in cryostat)

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.

knife scratches & splitting

Shrinkage

Wrinkles

Poor fixation

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.

3-D structures in 2-D sections

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

Learning Resources Junqueira’s Basic Histology ; pp 1-3 Looking at the Structure of Cells in the Microscope http://www.ncbi.nlm.nih.gov/books/NBK28356/#A582