Light Microscope Terms and Practices

Objectives Describe: How thin sections are prepared and stained The terms used in microscopy The limits of resolution Compare light and electron microscopy Objectives

The light microscope depends on light passing through an object in order for it to be seen. The result is the sections must be very thin, less than 5 micrometers. Light Microscope

The light microscope depends on light passing through an object in order for it to be seen. The result is the sections must be very thin, less than 5 micrometers. As a result, tissue preparation is most important. Light Microscope

Fresh tissue can not be cut this thin because it will be crushed. To avoid this the tissue is: Dehydrated with alcohol to remove water Light Microscope

Fresh tissue can not be cut this thin because it will be crushed. To avoid this the tissue is: Dehydrated with alcohol to remove water Embedded with paraffin wax to support the tissue so it can be cut. Light Microscope

Microtome

The Process

So why is this so important? Everyday, tissue samples are taken from people and they need to be evaluated for disease processes like cancer.

Stains are used to enhance the tissue slide and to allow the histologist to identify various intracellular compounds. Light Microscope

Oil Red O stains for neutral lipids Light Microscope

Haematoxylin and eosin (H&E) staining is a very common stain Haematoxylin and eosin (H&E) staining is a very common stain. Haematoxylin stains the nuclei blue and Eosin stains the cytoplasm pink. Light Microscope

PAS staining is used to identify glycogen stored in tissues. The light microscope

Compound light microscopes have two sets of lens which aid in the magnification of the specimen. The objective lens are 4x, 10 x and 40 x Magnification

Compound light microscopes have two sets of lens which aid in the magnification of the specimen. The objective lens are 4x, 10 x and 40 x The ocular lens is 10x Magnification

Compound light microscopes have two sets of lens which aid in the magnification of the specimen. The objective lens are 4x, 10 x and 40 x The ocular lens is 10x Total magnification is the product of the objective and ocular lens Magnification

Scanning 4x objective

Low Power using 10 x objective

High Power using 40 x objective

Terms used for Microscopy Resolution: The ability to distinguish 2 points as separate. The higher the resolution, the more detail is seen. Terms used for Microscopy

Terms used for Microscopy Depth of Field: The range of depth that can be focused in a specimen. A thin depth of field will need to be continuously focused. Terms used for Microscopy

Terms used for Microscopy Depth of Field: The range of depth that can be focused in a specimen. A thin depth of field will need to be continuously focused. The higher the magnification, the more shallow the depth of field Terms used for Microscopy

Terms used for Microscopy Field of View: The area of the stage that can be viewed. Terms used for Microscopy

Terms used for Microscopy Field of View: The area of the stage that can be viewed. The higher the magnification the smaller the field of view. Terms used for Microscopy

Terms used for Microscopy Working Distance: The distance between the objective lens and the microscope slide. Terms used for Microscopy

Terms used for Microscopy Working Distance: The distance between the objective lens and the microscope slide. This decreases as magnification increases. Terms used for Microscopy

Terms used for Microscopy Contrast: Difference in the intensity between the object and the background. Stains aid in the contrast between the specimen and the background. Terms used for Microscopy

Terms used for Microscopy Illumination: This is the amount of light that is transmitted through the specimen. The higher the magnification, the more light is required. Terms used for Microscopy

Terms used for Microscopy Numerical Aperture (NA): This is a complex concept. Terms used for Microscopy

Terms used for Microscopy Numerical Aperture (NA): This is a complex concept. It is the ability to gather light and resolve a fine specimen at a fixed distance. Terms used for Microscopy

Terms used for Microscopy Numerical Aperture (NA): This is a complex concept. It is the ability to gather light and resolve a fine specimen at a fixed distance. It takes in the quality of the lens, working distance, size of the lens and index of refraction. Terms used for Microscopy

Terms used for Microscopy Numerical Aperture (NA): It is represented by the formula NA= n x sin (a) where NA is the numerical aperture n is the refractive index a is ½ of the opening angle Terms used for Microscopy

Terms used for Microscopy Numerical Aperture (NA) increases with lens magnification. Terms used for Microscopy

Terms used for Microscopy The limits of resolution & magnification The greatest resolution with a light microscope is 0.2 microns. The human eye alone has a resolution of 200 microns (0.2 mm) Terms used for Microscopy

Terms used for Microscopy The limits of resolution & magnification Resolution is governed by the following relationship: MR=l/NA of the objective & condenser Terms used for Microscopy

Terms used for Microscopy The limits of resolution & magnification MR=l/NA of the objective & condenser where l is the wavelength of light Terms used for Microscopy

Terms used for Microscopy The limits of resolution & magnification MR=l/NA of the objective & condenser where l is the wavelength of light The shorter the wavelength, the greater the resolution. Terms used for Microscopy

Terms used for Microscopy The limits of resolution & magnification The wave length of light ranges from 700 nm (red) to 400 nm (violet). Using violet light the resolution of 0.2 microns is achieved. Terms used for Microscopy

An electron microscope does not use light to illuminate a specimen.

An electron microscope does not use light to illuminate a specimen. It uses the wave length of an electron which is about 0.004 nm. Electron Microscope

An electron microscope does not use light to illuminate a specimen. It uses the wave length of an electron which is about 0.004 nm. Using the previous formula, a resolution of 0.1 nm or 0.001 microns can be achieved. Electron Microscope

Electron Microscope Magnifications are typically up to 1 million times Cell ultrastructure is easily studied using this device. Electron Microscope

This require specimens to be about 0.1micrometers in thickness Electron Microscope

This require specimens to be about 0.1micrometers in thickness Specimens must be embedded in plastic and stained with heavy metals like osmium tetroxide and uranyl acetate. Electron Microscope

This require specimens to be about 0.1micrometers in thickness Specimens must be embedded in plastic and stained with heavy metals like osmium tetroxide and uranyl acetate. These will reflect electrons allowing an image to be formed. Electron Microscope

Electron Microscope

Electron Microscope

Parts of a light microscope