Microscopy Flashcards
Microscope
An instrument that allows you to magnify an object that may not be visible with the naked eye.
Types of Microscopes (4)
Light
SEM
TEM
LSCM
SEM
TEM
Scanning Electron Microscope
Transmission Electron Microscope
Eyepiece Lens
x10 Magnification
Eyepiece graticule can be inserted
Coarse-focus knob
Allows for quick focus
Fine-focus knob
Allows for intricacy in focusing on an image
Turret
Allows to switch between different objective lenses
Objective Lens
x4,x10,x40
Specimen Stage
Where slide is kept
How a microscope is held
Arm and Base
How an SEM works
A beam of electrons sent across the surface of a specimen.
Reflected electrons collected
How a TEM works
A beam of electrons is transmitted through a specimen and focused to produce an image.
How an LSCM works
A single spot of focused light is moved across a specimen, causing fluorescence from components labelled with a dye.
Emitted light from specimen filtered through a pinhole aperture.
Only light radiated from near to the focal plane is detected.
Properties of a Light Microscope
Wavelength is longer
Resolution of 200nm
Max Magnification of x2000
Chemical Stain
Properties of a TEM
shorter wavelength
Resolution of 0.2nm
Max Magnification of x2,000,000
Heavy Metal Stain
Properties of an SEM
the same wavelength of TEM
Resolution of 3nm
Max Magnification of x500,000
Heavy Metal Stain
Properties of LSCM
Uses Light
Allows for 3D imaging whilst object can be alive
Light Microscope Image
2D
Organelles
Transparent until Stained
The specimen can be alive
TEM image
2D
Organelles
Black and White unless stained
The specimen is not alive
SEM image
3D
No Organelles: only Surface
Black and White unless stained
The specimen is not alive
LSCM
3D
produces z sections
Colored with ‘dye’
The specimen can be alive
Properties of Staining
Increases contrast within cells
Allows components to become visible
Differential Staining
Used to distinguish between two types of organisms that would otherwise be hard to identify.
Gram stain
Crystal violet and then iodine is applied to the slide.
Gram-positive retain the stain- purple
Gram negative do not- too thin of a cell wall- Sarafin (stain) turns pink
Acid Fast acts on
Differentiates Species of Mycobacterium from other bacteria
Acid Fast Process
Lipid solvent is used to carry carbolfuchsin dye.
Cells washed with a dilute acid-alcohol solution.
Mycobacterium not affected by wash and retain stain.
Magnification=
Size of image/Actual size
Actual Size=
Size of Image/Magnification
Size of Image=
Magnification x actual size
