Microscopy - light microscopy, light microscope, specifimen preparation, staining

What are microscopes used for

studying structures that are too small to see with the naked eye

Light microscopy

• most commonly used microscopy technique 

• often requires staining of the specimen 

When is light microscopy limited to

• a minimal resolution of approximately 200 nm

minimal resolution

distance between two points that are still distinguishable as two separate entities

what is resolution limited by

• physical properties of light and the lense of the microscope

• wavelength of light

• aperture value of objective lense 

what length of wavelength has the better resolution

shorter wavelength 

what helps to tune the aperture value to its limits

• placing a drop of immersion oil between the coverslip slide and the objective lens

• without immersion oil the light is refracted, immersion oil diminishes the effect and minimal resolution can be achieved

light microscope

• illuminates specimen by focusing light through a lense onto the microscope slide 

• light passes through the specimen and into the objective lense 

Total magnification

objective magnification x eyepiece magnification

what does it mean that the objectives are parfocal

they stay in focus upon switching to the next magnification

steps of sample preparation

• fixation

• dehydration 

• embedding 

• sectioning 

• mounting 

• staining 

staining methods 

• needed in order to better visualize cells

• Dehydration 

• permeabilization 

• fixation 

• mordant application

dehydration

replacing water with another chemical

permeabilization

dissolving cell membranes to allow larger dye molecules to penetrate the cell

fixation

involves several techniques, such a creating chemical bonds between proteins to increase their rigidity 

mordant application

a substance that forms a strong complex with the dye and tissue