Microscopes (Ch. 6)

PART 1

Dead White Men Who Discovered (and were made of) Cells:

Anton Van Leeuwenhoek Robert Hooke

History of Cells

Robert Hooke - Observed

cells in cork.

Coined the term "cells” in

1665.

History of Cells

1833 - Robert Brown, discovered the nucleus.

1838 - M.J. Schleiden, all plants are made of cells.

1839 - T. Schwann, all animals are made of cells.

1840 - J.E. Purkinje, coined the term “protoplasm”.

Cell Theory

All living matter is composed

of one or more cells.

The cell is the structural and

functional unit of life.

All cells come from pre-

existing cells

Light Microscopy

TECHNIQUE RESULTS

Brightfield (unstained specimen).

Passes light directly through specimen.

Unless cell is naturally pigmented or

artificially stained, image has little

contrast. [Parts (a)–(d) show a

human cheek epithelial cell.]

(a)

Brightfield (stained specimen). Staining

with various dyes enhances contrast, but

most staining procedures require that cells

be fixed (preserved).

(b)

Phase-contrast. Enhances contrast

in unstained cells by amplifying

variations in density within specimen;

especially useful for examining living,

unpigmented cells.

(c)

50 µm

Differential-interference-contrast (Nomarski).

Like phase-contrast microscopy, it uses optical

modifications to exaggerate differences in

density, making the image appear almost 3D.

Fluorescence. Shows the locations of specific

molecules in the cell by tagging the molecules

with fluorescent dyes or antibodies. These

fluorescent substances absorb ultraviolet

radiation and emit visible light, as shown

here in a cell from an artery.

Confocal. Uses lasers and special optics for

“optical sectioning” of fluorescently-stained

specimens. Only a single plane of focus is

illuminated; out-of-focus fluorescence above

and below the plane is subtracted by a computer.

A sharp image results, as seen in stained nervous

tissue (top), where nerve cells are green, support

cells are red, and regions of overlap are yellow. A

standard fluorescence micrograph (bottom) of this

relatively thick tissue is blurry.

50 µm

50 µm

(d)

(e)

(f)

Electron Microscopy

TECHNIQUE RESULTS

Scanning electron micro-

scopy (SEM). Micrographs taken

with a scanning electron micro-

scope show a 3D image of the

surface of a specimen. This SEM

shows the surface of a cell from a

rabbit trachea (windpipe) covered

with motile organelles called cilia.

Beating of the cilia helps move

inhaled debris upward toward

the throat.

(a)

Transmission electron micro-

scopy (TEM). A transmission electron

microscope profiles a thin section of a

specimen. Here we see a section through

a tracheal cell, revealing its ultrastructure.

In preparing the TEM, some cilia were cut

along their lengths, creating longitudinal

sections, while other cilia were cut straight

across, creating cross sections.

(b)

Cilia 1 µm

Longitudinal section of

cilium

Cross section of cilium

1 µm

Cell Fractionation APPLICATION

Cell fractionation is used to isolate

(fractionate) cell components, based on

size and density.

TECHNIQUE

First, cells are homogenized in a blender to

break them up. The resulting mixture (cell

homogenate) is then centrifuged at various

speeds and durations to fractionate the cell

components, forming a series of pellets.

Tissue

cells

Homogenization

Homogenate 1000 g

(1000 times the

force of gravity)

10 min Differential centrifugation

Supernatant poured

into next tube

20,000 g

20 min

Pellet rich in

nuclei and

cellular debris

Pellet rich in

mitochondria

(and chloro-

plasts if cells

are from a

plant)

Pellet rich in

“microsomes”

(pieces of

plasma mem-

branes and

cells’ internal

membranes)

Pellet rich in

ribosomes

150,000 g

3 hr

80,000 g

60 min RESULTS

In the original experiments, the researchers

used microscopy to identify the organelles in

each pellet, establishing a baseline for further

experiments. In the next series of

experiments, researchers used biochemical

methods to determine the metabolic

functions associated with each type of

organelle.

Researchers currently use cell fractionation

to isolate particular organelles in order to

study further details of their function.

Why Cells Are So Small: The SA:V Ratio Surface area increases while

total volume remains constant

5

1 1

Total surface area

(height width

number of sides

number of boxes)

Total volume

(height width length

number of boxes)

Surface-to-volume

ratio

(surface area volume)

6

1

6

150

125

12

750

125

6

Prokaryote

bacteria cells Types of cells

Eukaryote

animal cells

- no organelles

- organelles

Eukaryote

plant cells

Why organelles? • Specialized structures

– specialized functions

• Containers

– Compartments = different local environments

• pH, concentration differences

– distinct & incompatible functions

• lysosome & its digestive enzymes

• Membranes as sites for chemical reactions

– Unique lipids & proteins

– embedded enzymes & reaction centers

• chloroplasts & mitochondria

Proteins do all the work!

cells DNA

proteins

organism Repeat after me…

Proteins do all the work!

Cell functions

• Building proteins

– copy DNA

– DNA -> RNA

– build proteins

– process proteins

• Folding, modifying

–Remove amino acids

–Add molecules (e.g. glycoproteins)

– address & transport proteins

Protein Synthesis • Organelles involved

– nucleus

– ribosomes

– endoplasmic reticulum (ER)

– Golgi apparatus

– vesicles

nucleus ribosome ER Golgi

apparatus vesicles

The Protein Assembly Line

The Endomembrane System

Cell Wall

Nonliving jacket that

surrounds some cells.

Found in:

Plants

Prokaryotes

Fungi

Some Protists

Plant Cell Walls

All plant cells have a Primary

Cell Wall.

Some cells will develop a

Secondary Cell Wall.