Concept: Biologists use microscopes and the tools of biochemistry to study cells

• first compound microscope – Zacharias Jansen in 1590

• three important parameters of microscopy

– Magnification: the ratio of an object’s image size to its real size

– Resolution: the measure of the clarity of the image, or the minimum distance of two distinguishable points

• inversely related to the wavelength of the radiation a microscope uses

– Contrast: visible differences in parts of the sample

• in a light microscope (LM) - visible light is passed through a specimen and then through glass lenses

• the lenses refract (bend) the light - so that the image is magnified

• in a simple microscope - there is one lens for magnification

– e.g. magnifying lens

• in compound microscopes – more than one lens– ocular and objective lenses– improves resolution and allows for more than one

magnification

• LMs can magnify effectively to about 1,000 times the size of the actual specimen

• this allows for individual cells within a tissue to be visualized

10 m

1 m

0.1 m

1 cm

1 mm

100 m

10 m

1 m

100 nm

10 nm

1 nm

0.1 nm Atoms

Small molecules

Lipids

Proteins

Ribosomes

VirusesSmallest bacteria

Mitochondrion

Most bacteria

Nucleus

Most plant andanimal cells

Human egg

Frog egg

Chicken egg

Length of somenerve andmuscle cells

Human height

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Super-resolution

microscopy

Studying Cells: Microscopy

Brightfield(unstained specimen)

Brightfield(stained specimen)

50

m

Differential-interference-contrast (Nomarski)

Fluorescence

10 m

Light Microscopy (LM)

Phase-contrast

• various techniques enhance contrast of a LM and enable cell components to be stained or labeled

• BUT - most subcellular structures, including organelles, are too small to be resolved by an LM

• LMs cannot resolve detail finer than 0.2um - regardless of magnification

• to improve resolution and magnification – allowing for imaging of subcellular structure - development of two other microscopes in the 1950s

• called electron microscopes (EMs)- use a focused beam of electrons rather than light

• resolution increase – due to the shorter wavelength of the electron beam

• two types:– 1. Transmission Electron Microscope (TEM)– 2. Scanning Electron Microscope (SEM)

• 1. Scanning electron microscopes (SEMs) – electron beam is focused onto the surface of a subject – providing images that look 3D– SEM electron beam excites the electrons of the gold

on the subject’s surface– several kinds of electrons are produced– these electrons are detected by the scope and

projected onto a video screen as a magnified image that appears 3D

– can be colorized

Pollen grains

Blood cells

• 2. Transmission electron microscopes (TEMs) focus a beam of electrons through a specimen – subject is sliced into a very thin layer – so TEMs are used mainly to study the internal

structure of cells– subject is stained with heavy metals that adhere to

the internal structures of the cell– so some parts of the cell become more electron dense

than others– the electron beam passes through those less dense

and scattered/reflected by the more dense regions– the electrons that pass through hit a piece of film

negative or hit a detector for displaying the image