Visualizing Cells

Molecular Biology of the Cell - Chapter 9

Resolution, Detection Magnification

Interaction of Light with matter:

Absorbtion, Refraction, Reflection, Fluorescence

Light Microscopy

Absorbtion of Light (dyes, stains)

Refraction methods

Fluorescence Microscopy

Green Fluorescent Protein

Confocal Microscopy

Total internal reflection fluorescence

Electron microscopy

Staining methods

Scanning Electron Microscopy

> 2 meters (2x100

meters)

1.5 centimeter (1.5x10-2

meters)

Average human hair (diameter) .1

mm = 100 micrometers (um) =

100x10-6 meters

Typical cell is 20

micrometers (um) = 20x10-

6 meters

Large organelles, 2

micrometers (um) = 2x10-6

meters

Macromolecular

complexes, 0.2

micrometers (um) = 2x10-7

meters

Figure 9-1

Size

Resolution vs Magnification

Resolution

Resolution is determined by the wavelength of

light and the numerical aperture of the objective

lens. Resolution IMPROVES if D gets SMALLER

Resolution is directly proportional to the lens

numerical aperture

Resolution is inversely proportional to the

wavelength used for imaging

Visible light wavelength are between about 360

and 780 nm

The resolution limit of the light microscope (using

near UV illumination) is about .2µm (200 nm). (A

red blood cell is 7µm across)

Long wavelength light

is less damaging and

less easily scattered

than short wavelength

light and can

illuminate deep

structures

Cy5 dyes emit near-

infrared light

Long Wavelength Light

Objects smaller than the resolution limit of a

microscope can be detected

Microtubules24 nm diameter

(DIC image)Actin (8nm) on myosin lawn

(Fluorescent actin)

MBoC supplemental video

Resolution vs Detection

Objects smaller than the resolution

limit can be detected but are not

resolved

Imaging devices have discrete detector elements

Rods and Cones in Eye

Pixels in Digital Camera

What is magnification good for?

The minimal adequate magnification is one that

allows the smallest object you want to resolve to fall

on 3 discrete elements of the imaging device.

Magnification

Four properties of the interaction between light and

matter influence the design of microscopes used to

produce contrast.

Properties of Interaction

Kidney Collecting

Duct Fig 9-11

Adipose Tissue Purkinje neuron

Stains are compounds that absorb light or electrons.

Black stain absorbs all colors of light

Colored stains absorb some colors of light, others to pass

Absorption methods: Stained tissue

sections

Hematoxylin (blue) stains nucleic

acids

Hematoxylin & eosin (proteins stain

pink)

Periodic acid Shiff’s stains

carbohydratesOsmium stains lipids in neuron sheath

Chemical compounds of some stains bind

preferentially to specific cellular components

Different components of a cell can be

selectively stained

Antibodies are used to detect specific

cell components

Delta I gene expression in developing

somites (beta-gal staining)

Craniofacial neurons in E10.5 mouse

(horseradish peroxidase labeling), Sahay et

al., J. Neurosci 2003. 23:6671-80

Antibodies can be linked to enzymes

that produce colored products

Very little incident light is absorbed, reflected or

refracted by a living biological specimen

Brightfield Imaging: refracted light from the specimen is poorly detected

Refraction Techniques in Light

Microscopy

Methods for imaging refracted light I.

Darkfield Imaging

Phase Contrast

Imaging

Living Cells

Living cells are seen clearly using phase contrast microscopy to

image the difference between refracted and non-refracted

light.

Shelden

Features of living cells can be seen clearly using phase contrast

microscopy.

Fluorescent molecules absorb high energy light and then emit less energetic,

longer wavelength light. The shift in wavelength between absorbed and

emitted light is called the Stokes shift.

Excitation

spectrumEmission spectrum

Rhodamine anti-MAP in cultured neurons

maxima

Fluorescence microscopy:

Fluorescence microscopes use excitation, emission and dichroic

filters to take advantage of the Stokes shift

Fig 9-11

Fluorescence Microscopes

Chemical compounds of some fluorescent stains bind

preferentially to specific cellular components

Fluorescent

phalloidin (red)

stains actin

DAPI (blue)

stains nucleic

acids

Shelden

Fluorescent Stains

Antibody linked fluorophores detect specific cellular

antigens

Anti-tubulin (green)

Anti-neurofilaments

(green)

Fluorophores

Fluorescence in situ hybridization (FISH) uses synthetic fluorescent RNA

probes to detect compatible mRNA in cells and tissue

Figure 9-12

mRNA

Fluorescent RNA

probe

FISH

Light emitting dyes reveal changes in ion concentrations

Calcium changes during fertilization

visualized with aequorin

Figure 9.32

Calcium signaling in renal epithelial cells

(fluo-5 indicator) Shelden

Green fluorescent proteins can be expressed in

living organisms

Aequorea victoria

GFP

Fluorescent Protiens

Cell-type specific gene promoters can be used to express GFP

in specific cells or tissues

GFP Coding

Sequence

Neuron specific

promoter

Figure 9-25 (transgenic fruit fly larva)

Gene Promoters

Green fluorescent fusion proteins can be used to label proteins

in living cells

Relocation of GFP-tagged proteins in

muscle cells

Recombinant DNA

Fluorescent Fusion Protiens

Dynamic studies of fluorescent probes can be conducted using

Fluorescence Recovery After Photobleaching (FRAP)

Fig 9-31

Fluorescent Probes

Fig 9-30

Fluorescent Probes

Dynamic studies of fluorescent probes can be

conducted using photo-activatable probes

The electron microscope uses electrons to resolve fine structure of the cell

The wavelength of an electron can be .004 nanometers, so the theoretical

limit of resolution of an electron microscope is 1/20 angstoms, or 1/20 the

diameter of a hydrogen atom.

Fig 9-42

Electrons pass

through the

specimen in TEM

Electron Microscope

Stains used for electron microscopy (EM) are very dense (metals) so they absorb electrons

EM stains are generally soluble, reactive forms of metals such as lead, uranium, gold, silver

and tungsten

Water

Osmium

tetroxide

Uranyl

acetate

Shelden

EM Stains

Gaseous metals can be

directionally applied (shadowing)

Sputter coatter

Fig 9-52

Metal shadowing can be applies to surface structures or

interior structures using freeze fracture and freeze etching

methods

Cryoelectron microscopy of freeze-

etched skeletal muscle fibers

Cryoelectron microscopy of freeze-

fractured intestinal microvilli

Specimens are frozen, then split using a microtome and exposed surfaces prepared and imaged.

Metal Shadowing

EM Negative staining allows isolated macromolecules

to be seen Actin filaments

Bacteriophage virus

Gene Meyer

University of S. Carolina

Macromolecules on

substrate

Stain in

excess

Excess stain removed and

dried

Fig 9-54

Shelden

EM Negative Staining

Computational methods can produce three

dimensional structural inormation from multiple

views of an object

Figure 9-55 reconstruction of the Hepatitis B virus

Computational Methods

Antibodies can be attached to metal particles to detect

specific cellular components

Immuno-gold labeling of microtubules in an

interphase cells

Immuno-gold labeling of centromere proteins.

Fig 9-46

Antibodies

Dynamic studies of molecules can be conduced at the EM level

by pulse chase radio-labeling.

Fig 9-38

Pulse Chase Radio-Labeling

Reflection techniques in microscopy:

Scanning EM:

Fig 9-50Fig 9-49