Methods in Cell Biology

B. Balen
 Diversity of the living world

~ 1 200 000 animal species

~ 360 000 plant species
~ 80 000 fungi species
~ 3 000 bacterial species

All diversity of the living world

can be resumed to the common
cellular basis.
 When were the first cells discovered?

Robert Hook, Micrographia, 1665.g.

-introduced term the “cell” by observing cork pieces with his light microscope

Drawing of the flee from

Micrographia

Light microscope Drawing of the cork

(magnification 30 x) from Micrographia
Antony van Leeuwenhoek, 1670. g.
- discovered the world of bacteria by watching the water droplet from the pond,
blood cells and animal sperm cells

Light microscope Drawing of the section of the

(magnification 300 x) ash tree
Matthias Schleidenn and Theodor Schwann, 1838. g.
-microscopic analyses of plant and animal tissues

 discovered that all living organisms are built

out of cells

 cell – basic unit of life

 beginning of the Cell Biology Science

 proposed cell theory or cell doctrine: all

living organisms are built out of cells or at
least one cell

Development of  Formal birth of contemporary cell biology!

the jellyfish from
the Schleiden
book Das Meer
Rudolf Virchow, 1885. g.
“Omnis Cellula ex Cellula”
 Scale of size of different biological structures
1 cm

1 mm

Eye
100 μm Plant cell

Animal cell
10 μm

1 μm bacteria

Light microscope
100 nm
virus
ribosome

10 nm

proteins

1 nm
Electron
microscope
atoms
0,1 nm
 Methods in Cell Biology

1. Microscopy

1.1. Light microscopy

1.2. Electron microscopy

1.1. Light microscopy
1.1.1. Ordinary light microscope

ocular

tubus

revolver

objective
table
clamps condenser

mirror

base

Primo Star, Zeiss

 How does the light microscope function?

Figure 9-6 Molecular Biology of the Cell (© Garland Science 2008)

 Modern light microscopes

magnification up to 1000x

disadvantage – limit of resolution (max. resolution 0.2 μm)

dmin =0.2 μm
 Microscope resolution

1. What is resolution of the microscope?

Its ability to resolve very tiny structures in the specimen

2. It is dependent on:
(a) Wavelength (λ) of the visible light (0,4 – 0,7 μm)
(b) Ability of the microscope lenses to collect light (numerical aperture)

Resolution (dmin) = 0.61 λ / NA

λ (white light) = 0,55 μm

NA = numerical aperture
0.61 = correction factor
 Numerical aperture
→ it is a function of the light-collecting ability of the microscope
NA = n x sin μ
n – refractive index of the medium which
separates the specimen from the objective and
condenser lenses (nair = 1.0)
μ – half the angular width of the cone of the rays
collected by the objective lens (max. μ=90°)

• maximal NA:
nimersion oil = 1.4
sin 90° = 1,0 → NAmax = 1,4 x 1,0 = 1,4

[Link] • max. resolution of the light microscope:

dmin = 0,61 x 0,55 μm / 1,4 = 0,22 μm

 Different objectives – different numerical apertures

[Link]

 the wider the microscope opens its eye, the more sharply it can see!
 How to increase the resolution of the light microscope?

- By applying the immersion oil

Dry objectives – air between the Immersion objectives – immersion oil

specimen and the lens between the specimen and the lens
nglass(1,4) > nair(1,0) nglass (1,4) = nimersion oil (1,4)
greater diffraction less diffraction
 Absorption and diffraction of light waves

Small details in the specimen are visible

because of:

The color – absorption of the certain

wavelengths

The difference in the refractive index

causes the change in phase of light wave
- colorless and translucent cells
(difraction and refraction)

Image - usually the combination!

Phase-Contrast Microscope Light passing through thick

Differential-Interference-Contrast Microscope or dense part of the cell
Dark-Field Microscope (nucleus) is retarded
 Microphotographs of cells obtained by :

A) Regular Light Microscopy

B) Phase-Contrast Microscopy
C) Differential-Interference-Contrast Mmicroscopy

2004 Cooper and Hausmann

Four types of light microscopy. Four photographs of the same fibroblast cell

(A) Regular light microscopy, (B) Phase-contrast microscopy , (C) Nomarski differential-
interference-contrast microscopy, (D) Dark-field microscopy.
1.1.3. Fluorescence microscope
 fluorescent dies for labeling certain molecule inside fixed or living cells

 very sensitive method for analyzing the intracellular distribution of molecules

Sir George G. Stokes - Stokes shift.

[Link]
How does the fluorescence microscope work?
Figures 9-14; 9-15 Molecular Biology of the Cell (© Garland Science 2008)
DAPI → DNA visualisation (Practicum!)

A B

DNA fibers from Allium cepa labeled with DAPI: A – image

magnification 200x, B – image magnification 400x.

2006 B. Balen and P. Peharec

 GFP→ protein visualisation

Practicum!

Hairs at the surface of the Arabidopsis leaf

green fluorescent protein (GFP) incorporated in cytoskeleton proteins
(green)
chlorophyll natural fluorescence (red)
2002 Bruce Alberts, et al.
Fluorescent
microscopes at the
Division of
Molecular Biology
 Confocal microscope
 fluorescence microscope that uses laser as a light source
Figure 9-21. Conventional and confocal fluorescence microscope.

Fruit fly embryo in gastrula phase stained with fluorescent dye which binds to actin.

(A) Conventional fluorescence microscopy – blurry image due to light out of focus

(B) Confocal fluorescence microscopy – crispy image – light out of focus is excluded from
the detector

2008 Bruce Alberts, et al.

 Tridimensional reconstruction of the pollen grains by
confocal microscope

Figure 9-22.

2002 Bruce Alberts, et al.

 How to prepare the specimen?

1. Fixing – formaldehyde and glutaraldehyide Can be

(to immobilize, kill and preserve cells) replaced by
rapid freezing
2. Embedment – wax and resin – form solid block
(tissue soft and fragile)
3. Cutting – microtome (sections thickness 1 - 10 μm)
4. Staining – toluidin blue; malachite green, sudan black
(specific affinity for particular subcellular components)

-20° C

microtome
Figure 9-10 Molecular Biology of the Cell (© Garland Science 2008)
 Pl
Pl

Slika 2. Svjetlosno mikroskopska

Light microscopy of semi-
slika poludebelih prereza. Preparat
thin
obojensections.
toluidinskimDye - toluidin
modrilom,
blue, immersion
imerzijski većanja
objektiv poobjective
100:1. Prerezi stanica:
magnification A -Alista
100:1. –
hrena, B teratoma, C tumora. Pl -
J horseradish leaf, B –
plastid, J - jezgra
teratom, C – tumor.
Pl – plastid, J – nucleus
C
2006 P. Peharec
 Section of cells in urine-
collecting ducts of kidney.

Dyes - hematoxylin and

eosin

Nuclei - red
Extracellular matrix - purple

Figure 9-11a Molecular Biology of the Cell (© Garland Science 2008)

1.2. Electron microscopy
1924. De Broglie - Nobel prize → discovery of the wave nature of fast e-

λ=h/mv h – Plank constant

m – mass
v – speed

Problem:
Solution:
How to increase
Shorter wavelength 
the resolution of
better resolution!!
the microscope?

1932. Ernst Ruska and Max Knoll – prototype of the electron microscope

1939. Siemens – commercial production of EM

1940 and 1950 Claude, Porter and Palade – application of EM in analyses of

biological samples
 EM resolution

λ (electrons) = 0.004 nm in EM theoretical dmin = 0.002 nm

(λ (white light) = 0.55 μm)

but!

NA (electron lens) = around 0.01 dmin = 0.2 nm

(NA (glass lens) = 1.4)

but!

The lack of contrast in biological samples!

The resolution power of EM is 2 nm for biological samples

(around 100 x higher than light microscope!!!).
 1.2.1. Transmission electron microscope (TEM)

Light microscope TEM

TEM  similar design with light microscope but larger and upside-down
e – scattered by collision with air molecules  air must be pumped out (vacuum!!!)
e – that have passed through the sample

2002 Bruce Alberts, et al.

Preparation of the samples:
 it is not possible to analyze living
cells! (vacuum)

1. Fixation – glutaraldehyde
osmium tetroxide
2. Dehydration – series of ethanol,
A
ethanol and acetone, acetone
3. Embedment – epoxy resin or wax
4. Cutting – ultramicrotome
(thickness 50-70 nm)
 e- have very limited penetrating B
power E

F
2006 P. Peharec
5. Constrating – contrast depends on atomic number!

Biological samples composed of atoms of very low atomic number – C, H, O, N!

- Positive contrasting
(OsO4)

- Negative contrasting
(phosphor-volfram acid)

- Metal shadowing
(gold, paladium, platinum)

Platinum shadowing
 A specimen prepared by negative contrasting and
shadowing (G. Karp pp.736)

A specimen prepared by positive contrasting and shadowing

The power of resolution of TEM.

Nucleosome in EM

Thin golden plate – distance between

the atoms is 0.2 nm

(A) Proplastid (B) Amiloplast

2002 Bruce Alberts, et al.

1.2.2. Scanning electron microscope (SEM)

 smaller, cheaper and simpler

 el – which are scattered or

emitted from the surface

 resolution – not very high

(about 10 nm)

 studying whole cells and

tissues

2002 Bruce Alberts, et al.

Cactus spines – magnification glass and SEM (2007. Balen and Peharec)

Cactus shoot with SEM - areole with SEM – a part of the

spines spines spine

An ant holding a microchip

(Science Museum)
pollen grain
 2. Cell fractionation and
centrifugation

 EM allowes a detailed analysis of cellular structures

 Cell/organell isolation - in order to analyze their

biochemical composition and physiology

Organelles have to remain intact and physiologically

active!
 2.1. Homogenization

 Ultrasound – ultrasonic vibration

 Mechanical
- mortar and pestle
- mechanical homogenizers and mixers (blenders)

 Lysis in hypotonic solution (osmotic shock)

 Enzymes
• Celullase and pectinase – plant cell walls
• Lysosyme – bacterial cell walls
1. Mortar and pestle
2. Mixer (Omni-mixer Sorval)
3. Homogenizer (Retsch MM 200)

2
Things in common:

 disruption of cell and ER membrane

 mild, quick and cold

 Keep the conditions like in the cell:

• pH (buffers)
• isotonicity (sucrose, sorbitol, mannitol 0,2-0,6 mol/L)
• ion composition and concentration
• antioxidants (mercaptoethanol, dithiotreithol, serum albumin,
ascorbic acid) → oxidative protection
• stabilization chemicals (PVP)
 2.2. Centrifugation

→ separation of compounds:

2.2.1. Differential centrifugation

2.2.2. Centrifugation in concentration gradient

A) velocity
B) equilibrium
 2.2.1. Differential centrifugation

→ sedimentation of compounds at increasing speed in a certain

time
→ important:
• rotation speed → (g)
• time (min or h)
• temperature (oC)

Figure 8-9. Ultracentrifuge

2002 Alberts, et al.
 800 x g (10 min)

homogenate

supernatant 15.000 x g supernatant

(10 min)

Sedimentation
pellet
of nuclei supernatant 100.000 x g
(60 min)

Sedimentation
of plastids,
mitochondria,
peroxisomes,
lysosomes supernatant 200.000 x g
Sedimentation coefficient:
(3 h)
Sedimentation of – unit Svedberg (S)
cell membrane
and ER
membrane – 1S = 10-13 s
cytosol

Sedimentation of
ribosomes

2004. Cooper and Hausmann (Fig. 1-38)

 2.2.2. Centrifugation in concentration gradient

→ organelles are separated by sedimentation through density

gradient

A) Velocity centrifugation
 shallow sucrose gradient (5 - 20%)

 separation according to size and shape

 molecules of the different size sediment by different speed →

zones of the molecules of the same size
B) Equilibrium centrifugation

 steep sucrose gradient → linear or gradient

 separation according to density

 molecules travel through the gradient and stop where their

density is equal to the gradient density

 very sensitive method→ it is possible to separate

macromolecules which have incorporated heavy isotopes 13C
or 15N from macromolecules with normal isotopes (12C or 14N)
 Centrifugation in concentration gradient

according to
size and according to
shape density

2008 Alberts, et al. (sl. 8-11)

3. Cell culture

great amount of material and large number of cells required

for biochemical analyses
 sample – tissue part  heterogeneous cell population
 it is necessary to dissociate cells from tissues and to
separate different cell types
 the aim: homogenous cell population which can be cultured
in vitro
 animal as well as plant cells
3.1. Disruption of extracellular matrix
 Proteases (trypsin, collagenase) – degradation of extracellular matrix proteins
 Reagents (EDTA) bind Ca2+ - important for cell-cell adhesion

3.2. Separation of different cell types

 Centrifugation (large from small cells, dense from light ones etc.)
 Growing them on the plastic or glass surface (different adhesion strength)
 Antibodies labeled with fluorescent dies  fluorescence-activated cell sorter
 From thin tissues section laser capture microdissection
3.3. Cells can be grown in a culture dish

 majority of the animal and plant cells can live, multiply and even
express differentiated properties in culture cell vessel
 different analyses can be performed:
• microscopy
• biochemical analyses
• monitoring of influence of different compounds (hormones,
growth regulators, biotic or abiotic stressors)
• mixing of two different cell types

 experiments on cell in culture “in vitro”

 experiments on living organisms  “in vivo”
3.3.1. Animal cell culture

 First culture of animal cells - 1907. Harrison

Controversy in neurobiology:
Is nerve fiber an outgrowth of a single
nerve cell or a product of the fusion of
many cells?

 answer  one cell

 established foundation for cell culture
development

2000 Geoffrey M. Cooper

 Requirements for cell culture

 1955. Harry Eagle → described the first medium for animal cell culture

 Requirements:
• growth vessel (Petri dish) - most cells are not adapted to live in suspension
• ingredients of extracellular matrix (collagen, laminin)
• nutrient medium (salts, glucose, vitamins, growth regulators, amino acids)

 Subcultivation every few weeks

 Maintain the characteristics of differentiated cells :

• fibroblasts→ secrete collagen
• neural cells → form axons and make synapses
• embryonic muscle cells → produce muscle fibers; spontaneous contractions
 Cell culture types

Animal cell cultures can be divided in two groups:

 Embryonic and tumor cells  immortal cell lines

 Differentiated cells  restricted time of division in culture

→ limited proliferation capacity
→ fibroblasts 25-40 divisions before they stop
→ reason: progressive shortening of telomeres due to turning off the
telomerase (enzyme that maintains telomeres)
→ it is possible to incorporate the gen encoding the telomerase
catalytical subunit – immortalized cell line
 Animal cells in culture

A – phase-contrast micrograph of fibroblasts in culture

B – myoblasts in culture – cells fusing to form multinucleate muscle cell
C – oligodendrocyte precursor cells

Figure 8-4 Molecular Biology of the Cell (© Garland Science 2008)

3.3.2. Plant cell culture

 unlimited cell growth on appropriate

nutrient medium
 regeneration, cloning
 plant cell transformation
 production of genetically altered plants
 synthesis of secondary metabolites
Sugar beet cell lines. A) normal callus N; B) habituated
line HNO; C) tumor line Tz; D) tumor line Tc; E) tumor
line Tb. (Prepared by D. Pavoković). White line = 1cm.

Mammillaria gracilis Coleus blumei Armoracia lapathifolia

Production of genetically altered
plants