University:
Texas A&M University
Course:
BIOL 213 | Molecular Cell Biology
Academic year:

2024
Views:

374

Pages:

38
Author:

diamundgalx45p

Introduction to Cells, Tissues, and Microscopy Part II OBJECTIVES 1. Preview cellular ultrastructure 2. Preview cells, tissues, and organs 3. Overview of light and electron microscopy 4. Preparation of specimens – types of visions 5. Section orientation Magnification vs. Resolution 1. magnification - increase in image size 2. resolution - smallest distance between two points that can be seen (distinguished) Calculated by: 0.61 (wavelength) / numerical aperture 0.25 um for light microscope 0.1 nm for electron microscope Sample Preparation 1. Fixation 2. Embedding A. Paraffin B. Plastic 3. Sectioning A. 0.5 um for Light Microscopy B. 60-80 nm for Electron Microscopy Staining 1. Light Microscopy A. Hematoxylin and Eosin (H&E) B. Periodic Acid/Shiff (PAS) C. Toluidine Blue 2. Electron Microscopy (TEM) A. Osmium B. Lead Citrate Staining 1. Light Microscopy A. Hematoxylin and eosin (H&E) B. Periodic Acid/Shiff (PAS) Color provides clues C. Toluidine Blue Shape Size Intensity of staining Staining 1. Light Microscopy A. Hematoxylin and Eosin (H&E) B. Periodic Acid/Shiff (PAS) C. Toluidine Blue 2. Electron Microscopy (TEM) A. Osmium B. Lead Citrate Fundic stomach (H&E) Fundic stomach, monkey (PAS) Surface mucus cells of Fundic stomach, rabbit (toluidine blue) Toluidine Blue Eosin (H&E) of retina 19709 Transparency of unstained tissue Phase Contrast Dead stained cells Bright Field Live unstained cells Phase Contrast Nomarksi “differential interference contrast” Dark Field Other Light and Electron Microscope Procedures • • • • Immunofluorescence Autoradiography In Situ Hybridization (ISH) Freeze Fracture – (Membrane Analysis) AUTORADIOGRAPHY self radioactive Photomicrograph of a section of human epithelial tumor (condyloma) in which in situ hybridization with the DNA of the human papilloma virus type II (HPVII) was performed. Observe dark staining of several nuclei, indicating the presence of the genome of this virus in the tumor, suggesting its possible participation in the genesis of the tumor. Carbon Replica 1. Heavy metal evaporated from a filament “shadows” the specimen. 2. A strengthening film of carbon evaporated from above. 3. The replica is floated onto the surface of a powerful solvent to dissolve away the specimen. 4. The replica is washed and picked up on a copper grid for examination. Typical TEM Carbon replica TEM Section orientation Nerve axons Section Orientation Section Orientation