Micro Tools of the Laboratory: Microscopy

ocular lens 

Eye piece you look through, provides minimal magnification

diopter adjustment

alters focus for people with different vision correction

mechanical stage

Holds sample and moves sample round. X and Y axis controlled by Stage

Control

condenser 

Focuses light through the sample

course adjustment

Moves the stage up and down to change focus rapidly

fine adjustment

Moves the stage up and down to change focus slowly (more precise)

objective lens

Major step in magnification. Different lenses have different magnification and

N.A

magnification

The ability to enlarge the apparent size of an object

Why is magnification important

We need a way to magnify the organism (make it appear bigger) to study certain aspects.

resolution

the ability to distinguish two separate objects

what is resolution based on

the wavelength of light and the property of the microscope objective

what is the problem with light microscopy

resolution

What happens because the wavelength of visible light is not able to change

there is a maximum resolution of light microscopy

what happens when there is a maximum resolution of light microscopy

you will never be able to distinguish two different objects

light microscopy Illumination Source

visible light

light microscopy maximum resolution

200nm

pro of Light Microscopy

Cheap and easy to use; can use live or fixed samples

con of light microscopy 

not high resolution or magnification. Hard to see fine structure. Samples are basically colorless

Fluorescence Microscopy illumination source

Visible Light (laser or halogen)

Fluorescence Microscopy maximum resolution

200 nm

Fluorescence Microscopy pros

Can identify specific structures with labeled molecules. Some samples can be live or fixed, depending on the detection method

Fluorescence Microscopy con

not high resolution or magnification. Hard to see fine structure.

Confocal Microscopy illumination source

Visible Light (laser or halogen)

cofocal microscopy maximum resolution

200nm but apparent resolution decreases that

confocal microscopy pros

Can identify specific structures with labeled molecules. Can isolate focal planes to eliminate noise from other focal planes. Can build 3D images

confocal microscopy cons 

Can be slow (needs to scan image). More expensive that regular light microscopy and needs special training

what is resolution intrinscially linked to

wavelength of illumination source

Transmission Electron Microscopy illumination source

electron beam

Transmission Electron Microscopy maximum resolution

0.2 nm

Transmission Electron Microscopy pros

high resolution and high magnification. Can see very fine detail

transmission electron microscopy cons

Slow. Requires very expensive equipment and extensive training. Cells have to be fixed with toxic ”electron dense” materials

Scanning Electron Microscopy illumination source

electron beam

Scanning Electron Microscopy maximum resolution

10 nm

Scanning Electron Microscopy pros

High resolution and high magnification. Can see very fine external detail. Scans across the outside of a cell to reveal structures that can be destroyed by other microscopy methods

Scanning Electron Microscopy cons

Slow. Requires very expensive equipment and extensive training. Cells have to be fixed with toxic ” electron-dense” materials.

what is fluorescence

A molecule that absorbs light at one wavelength and emits light at a slightly

larger wavelength

what can fluorescence molecules be used to do

detect specific molecules within a system

gram stain

A stain used to classify bacteria based on their cell wall structure

gram postive 

large cell wall 

• Stains a deep purple color.

gram negative

outer membrane and cell wall

• little stain- pink color 

what do some bacteria have

waxy, or lipid rich cell walls that do not easily Gram stain

example of a bacteria that doesnt easily stain because of their cell walls 

Mycobacterium tuberculosis – causative agent of TB

how to do a acid fast stain

Use Carbol Fuchsin which is lipid soluble and can stain the cell wall and become trapped by the waxy cell covering