REPORT 1

MICROSCOPY

As Partial Fulfillment of the course required for Biology 22 – General

Zoology Laboratory for the Second Semester AY 2010-2011

Mark Lester V. Magabo

Researcher

Mr. Rommel Oba

Adviser

NOVEMBER 21, 2011

I. Physical Description (OVERVIEW)

Fluorescence Microscope Phase-Contrast Microscope

Scanning Electron

Microscope

Transmission Electron

Microscope

I. Principles and Information

A. Inventors

1. Transmission Electron Microscope Ernst Ruska in 1931

2. Scanning Electron Microscope Max Knoll in 1935

3. Phase-Contrast Microscope Fritz Zernicke in 1953

4. Fluorescence Microscope Marvin Minsky in 1957

B. Etymology

1. Transmission Electron Microscope transmissionem which means sending

over or across and electron which

means amber-like

2. Scanning Electron Microscope scandere which means to look closely

and electron which means amber-like

3. Phase-Contrast Microscope phainein which means to show or to

make appear and contend which

means to compare

4. Fluorescence Microscope fluorspar which means glowing with

fluorine and escentum which means

process or state of being

C. Classification 1 – Nature of Light Source

1. Transmission Electron Microscope ELECTRON Microscope

2. Scanning Electron Microscope ELECTRON Microscope

3. Phase-Contrast Microscope OPTICAL Microscope

4. Fluorescence Microscope OPTICAL Microscope

D. Classification 2 – Analysis of the Sample

1. Transmission Electron Microscope Analyze the sample all at once

2. Scanning Electron Microscope Analyze the sample via a scanning point

3. Phase-Contrast Microscope Analyze the sample via a scanning point

4. Fluorescence Microscope Analyze the sample via a scanning point

E. Basic Uses

1. Transmission Electron Microscope Used to study the internal ultrastructure

of cells

2. Scanning Electron Microscope Especially useful for the detailed study

of the surface of a specimen

3. Phase-Contrast Microscope Enhances contrast in unstained cells by

amplifying variations in density within

the specimen especially useful for

examining living, unpigmented cells

4. Fluorescence Microscope Shows the location of specific

molecules in the cell by tagging the

molecule with fluorescent dyes or

antibodies

F. What interacts with the sample to generate the image?

1. Transmission Electron Microscope Uses beam of electrons transmitted

through an ultra-thin specimen

2. Scanning Electron Microscope Uses high energy beam of electrons in a

raster scan pattern

3. Phase-Contrast Microscope Uses the phase shifting of light which

causes its amplitude and phase to

change in a way that it may give rise to

different colors and enhances contrasts

of transparent and colorless objects

4. Fluorescence Microscope Uses the principle of fluorescence (the

emission of light by a substance that

has observed light or other

electromagnetic radiation of a different

wavelength) and phosphorescence

(similar to the fluorescence but does

not immediately re-emit the radiation it

absorbs.

G. Staining of the Sample Specimen

1. Transmission Electron Microscope The specimen has been stained with

atoms of heavy metals, which attach to

certain cellular structures, thus

enhancing the electron density of some

parts of the cell more than the others

2. Scanning Electron Microscope The specimen is usually coated with a

thin film of gold

3. Phase-Contrast Microscope The specimen does not require staining

to view the slide

4. Fluorescence Microscope The sample can either be fluorescing in

its natural form like chlorophyll and

some minerals or treated with

fluorescing chemicals

H. Preparation of the Sample Specimen

1. Transmission Electron Microscope Sample preparation in TEM can be a

complex procedure. TEM specimens

are required to be at most hundreds of

nanometers thick, as

unlike neutron or X-Ray radiation the

electron beam interacts readily with the

sample, an effect that increases roughly

with atomic number squared.

Preparation includes:

1. Tissue Sectioning

2. Sample Staining

3. Mechanical Milling

4. Chemical Etching

5. Ion Etching

6. Replication

2. Scanning Electron Microscope For conventional imaging in the SEM,

specimens must be electrically

conductive, at least at the surface,

and electrically grounded to prevent the

accumulation of electrostatic charge at

the surface. Metal objects require little

special preparation for SEM except for

cleaning and mounting on a specimen

stub.

3. Phase-Contrast Microscope The cells are grown in a cell culture

container. Some cells are fixed to retain

the original morphology or structure of

cells and tissues. A microtome is use to

cut thin sections.

4. Fluorescence Microscope The specimen are prepared by fixation

done in three ways:

1. Cross-linking - involves treating

specimens with reagents that

penetrate into the cells and tissues

and form covalent cross-links

between intracellular components

2. Precipitation – usually done by the

immersion of sample specimen in

cool organic solvents.

3. Cryofixation – involves rapidly

freezing the cells or tissues on a

cooled back of heat-conductive

metal or plunging into a cold

medium, such as liquid nitrogen or

freon.

I. Principal Process on the use of each Microscope

1. Transmission

Electron

Microscope

A "light source" at the top of the microscope emits the electrons

that travel through vacuum in the column of the microscope.

Instead of glass lenses focusing the light in the light microscope,

the TEM uses electromagnetic lenses to focus the electrons into a

very thin beam. The electron beam then travels through the

specimen of study. Depending on the density of the material

present, some of the electrons are scattered and disappear from

the beam. At the bottom of the microscope the unscattered

electrons hit a fluorescent screen, which gives rise to a "shadow

image" of the specimen with its different parts displayed in

varied darkness according to their density. The image can be

studied directly by the operator or photographed with a camera.

2. Scanning

Electron

Microscope

The electron beam scans the surface of the sample coated with a

thin film of gold. The beam excites the electrons on the surface,

and these secondary electrons are detected by a device that

translates the patter of electrons into an electronic signal to a

video screen. The result is an image of the specimen topography.

The SEM has a great depth of field, resulting in an image that

appears three-dimensional.

3. Phase-

Contrast

Microscope

The phase contrast microscope uses the fact that the light passing

through a transparent part of the specimen travels slower and,

due to this is shifted compared to the uninfluenced light. This

difference in phase is not visible to the human eye. However, the

change in phase can be increased to half a wavelength by a

transparent phase-plate in the microscope and thereby causing a

difference in brightness. This makes the transparent object shine

out in contrast to its surroundings.

4. Fluorescence

Microscope The basic task of the fluorescence microscope is to let excitation

light radiate the specimen and then sort out the much weaker

emitted light to make up the image. First, the microscope has a

filter that only lets through radiation with the desired wavelength

that matches your fluorescing material. The radiation collides

with the atoms in your specimen and electrons are excited to a

higher energy level. When they relax to a lower level, they emit

light. To become visible, the emitted light is separated from the

much brighter excitation light in a second filter. Here, the fact

that the emitted light is of lower energy and has a longer

wavelength is used. The fluorescing areas can be observed in the

microscope and shine out against a dark background with high

contrast.

J. Direction of Light Path

1. Transmission Electron Microscope

2. Scanning Electron Microscope

3. Phase-Contrast Microscope

4. Fluorescence Microscope

K. Magnification and Resolution

1. Transmission Electron Microscope M – 50,000,000 x

R – 5 Angstroms or 50 picometers

2. Scanning Electron Microscope M – 500,000 x

R – 1 nanometer

3. Phase-Contrast Microscope M – 40x – 1600 x

R – 60 nanometers

4. Fluorescence Microscope M – 10x-120x

R – 10 nanometers

II. Advantages and Disadvantages

A. Advantages

1. Transmission Electron Microscope TEMs offer the most powerful

magnification, potentially over one

million times or more

TEMs have a wide-range of

applications and can be utilized in a

variety of different scientific,

educational and industrial fields

TEMs provide information on element

and compound structure

Images are high-quality and detailed

TEMs are able to yield information of

surface features, shape, size and

structure

They are easy to operate with proper

training

2. Scanning Electron Microscope Advantages of a Scanning Electron

Microscope include its wide-array of

applications, the detailed three-

dimensional and topographical imaging

and the versatile information garnered

from different detectors.

SEMs are also easy to operate with the

proper training and advances in

computer technology and associated

software make operation user-friendly.

This instrument works fast, often

completing SEI, BSE and EDS

analyses in less than five minutes. In

addition, the technological advances in

modern SEMs allow for the generation

of data in digital form.

Although all samples must be prepared

before placed in the vacuum chamber,

most SEM samples require minimal

preparation actions.

3. Phase-Contrast Microscope The capacity to observe living cells

and, as such, the ability to examine

cells in a natural state

Observing a living organism in its

natural state and/or environment can

provide far more information than

specimens that need to be killed, fixed

or stain to view under a microscope

High-contrast, high-resolution images

Ideal for studying and interpreting thin

specimens

Ability to combine with other means of

observation, such as fluorescence

Modern phase contrast microscopes,

with CCD or CMOS computer devices,

can capture photo and/or video images

4. Fluorescence Microscope The fluorescence microscope objective

serves first as a well-corrected

condenser and secondly as the image-

forming light gatherer. Being a single

component, the objective/condenser is

always in perfect alignment.

B. Disadvantages

1. Transmission Electron Microscope TEMs are large and very expensive

Laborious sample preparation

Potential artifacts from sample

preparation

Operation and analysis requires special

training

Samples are limited to those that are

electron transparent, able to tolerate the

vacuum chamber and small enough to

fit in the chamber

TEMs require special housing and

maintenance

Images are black and white

2. Scanning Electron Microscope SEMs are expensive, large and must be

housed in an area free of any possible

electric, magnetic or vibration

interference.

Maintenance involves keeping a steady

voltage, currents to electromagnetic

coils and circulation of cool water.

Special training is required to operate

an SEM as well as prepare samples.

The preparation of samples can result in

artifacts. The negative impact can be

minimized with knowledgeable

experience researchers being able to

identify artifacts from actual data as

well as preparation skill. There is no

absolute way to eliminate or identify all

potential artifacts.

In addition, SEMs are limited to solid,

inorganic samples small enough to fit

inside the vacuum chamber that can

handle moderate vacuum pressure.

Finally, SEMs carry a small risk of

radiation exposure associated with the

electrons that scatter from beneath the

sample surface.

3. Phase-Contrast Microscope Annuli or rings limit the aperture to

some extent, which decreases

resolution

This method of observation is not ideal

for thick organisms or particles

Thick specimens can appear distorted

Images may appear grey or green, if

white or green lights are used,

respectively, resulting in poor

photomicrography

Shade-off and halo effect, referred to a

phase artifacts

Shade-off occurs with larger particles,

results in a steady reduction of contrast

moving from the center of the object

toward its edges

Halo effect, where images are often

surrounded by bright areas, which

obscure details along the perimeter of

the specimen

4. Fluorescence Microscope Fluorophores lose their ability to

fluoresce as they are illuminated in a

process called photobleaching.

Photobleaching occurs as the

fluorescent molecules accumulate

chemical damage from the electrons

excited during fluorescence.

Photobleaching can severely limit the

time over which a sample can be

observed by fluorescent microscopy

Fluorescence microscopy with

fluorescent reporter proteins has

enabled analysis of live cells by

fluorescence microscopy, however cells

are susceptible to phototoxicity,

particularly with short wavelength

light.

Sources

A. Books

Bacallao, R., and Steltzer, E. (1989) Meth. Cell Biol. 31, 437-452

Campbell, N.A., Reece, J. B. et al. Biology Eight Edition. Pearson Education Incorporated-Benjamin

Cummnigs.2008. pp 95-97

Danilatos, G,D. "Foundations of environmental scanning electron microscopy". Advances in

Electronics and Electron Physics 71: 109–250.

Manuel Gunkel, Fabian Erdel, Karsten Rippe, Paul Lemmer, Rainer Kaufmann, Christoph Hörmann,

Roman Amberger and Christoph Cremer. Dual color localization microscopy of cellular

nanostructures. In: Biotechnology Journal, 2009, 4, 927-938. ISSN 1860-6768

Porter, K and Blum, J (1953). "A study in Microtomy for Electron Microscopy".The anatomical

record 117 (4): 685–710.

B. Internet

http://en.wikipedia.org/wiki/Fluorescence_microscope

http://en.wikipedia.org/wiki/Microscope#The_rise_of_modern_light_microscopy

http://en.wikipedia.org/wiki/Phase_contrast_microscopy

http://en.wikipedia.org/wiki/Scanning_electron_microscope#Sample_preparation

http://en.wikipedia.org/wiki/Transmission_electron_microscopy#Electrons

http://serc.carleton.edu/research_education/geochemsheets/browse.html

http://typesofmicroscopes.blogspot.com/2009/07/scanning-electron-microscope.html

http://www.biologymad.com/cells/microscopy.htm

http://www.etymonline.com/

http://www.microscopemaster.com/scanning-electron-microscope.html

http://www.microscopemaster.com/transmission-electron-microscope.html

http://www.nobelprize.org/