Microscopy

Classification of microscope
Light Microscope:
In 1665, R. Hooke in England put forward the idea; 17 century, Lee Wenhock firstly invented

General microscope, Phase contrast microscope, Fluorescence microscope, confocal microscope, twophoton microscope Electron Microscope: In 1932, Germany scientist Ernst Ruska
firstly invented

Transmission Electron Microscope (TEM) Scanning Electron Microscope (SEM) Analytical Electron Microscope (AEM) Scanning Tunneling Microscope (STM) Scanning Force Microscope (SFM)

The Microscope
 Simple Microscope one lens  Ex Magnifying lens  Compound Microscope 2 lenses that

compound or magnify each other.

 Dissecting Microscope no special slide

preparation

Microscopes

Parts of The Compound Microscope

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 Eyepiece:  Magnifies material being viewed by 10X  The part of the microscope you look into  Sometimes contains a pointer that can be seen as you look into the eyepiece.  May also be called the ocular.

Continued
 Nose piece:  Part of microscope to which the objectives are attached  Rotates to allow for the changing of objectives to increase or decrease magnification.

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 Arm:


A secure part of the microscope to hold on to when the microscope is being carried.

Continued
 Objectives:  Low (4x)  Medium (10x)  High (40x)

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 Stage:  Platform on which

microscope slide rests  Mechanical Slide Adjuster



Used for adjusting the position of the slide for viewing

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 Coarse adjustment

knob:  Large movements of the stage  Fine adjustment knob:  Precise focusing under High power

Continued
 Diaphragm:  regulates the amount

of light passing through the slide

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 Illuminator


Light source

 Base:


provides support for microscope

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 Body tube:  Connects Ocular

to Nosepiece

TOTAL MAGNIFICATION
 Power of the eyepiece (10X) multiplied by

objective lenses determines total magnification.

Magnification
Objective Ocular Total Eyepiece Magnification Low Power 4 x 10 x 40 x

Medium Power

10 x

10 x

100 x

High Power 40 x

10 x

400 x

Field of View
 Field of View (FV) is

the illuminated circle that you see when looking through the ocular eyepiece.
 If we know the

diameter of the FV then we can estimate the size of our microorganisms.

Field of View
 With our microscopes the

diameter of the FV under low power is 4 mm

 FV is measured in

micrometers or microns.
 1 mm = 1000 microns  Therefore, our FV under

low power is 4000 microns

Using the Field of View to Estimate Microscopic Measurements

If an organism takes up of the FV under low power, it must be about 2000 microns in length

Simple Microscope
 Objective magnification  Working Distance  Eyepiece magnification

Illumination (Bright Field)

 Simple mirror (historical microscope)  Critical illumination  Koehler Illumination

Light path in a Microscope

Staining
 Staining is a biochemical technique of

adding a class-specific (DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound.  Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes.

Resolving Power of Light Microscope

Distinguished distance: d=0.61P/NA=0.61P/nsinE
Increase NA, decrease P

Numerical aperture (NA). The numerical aperture can be expressed as n sine F where n is the refractive index for the medium through which the light passes (n air =1.00; n water = 1.33; n oil = 1.4), and F is the angle of one half of the angular aperture of the lens

Light Microscope
Resolution for light microscope can be improved using oil immersion lenses, shorter wavelength and larger cone angle. Contrast can be dramatically improved using modifications such as dark field, phase contrast, and differential interference contrast. Fluorescence and confocal microscopes are specialized instruments, used for research, clinical, and industrial applications.
Phase Contrast Microscope: to view living cellular structure without pigment. The structure within the cell tend to differ in refractive indices Lights pass through specimen with different index will have different phase change, resulting in the interference Constructive interference Destructive interference

Dark Field microscopy

Poor-man`s dark field bright Dark

Dark Field

The object appears bright against a dark microscopic field. A special kind of condenser Abbe condenser. That transmits a hollow cone of light from the source of illumination. Most of the light directed through the condenser does not enter the objective. Some of the light rays will be diffracted. This diffracted light will enter the objective and reach the eye. Thus the object in this case will appear bright.

Abbe Condenser

Phase Contrast Optics

Theory & Appl. Light Microscopy

Theory & Appl. Light Microscopy

Criterion for Resolution

 Lens must capture undiffracted light plus at

least first order of diffracted rays  Combine these in image plane by interference  But most biological specimens (esp. living) are not amplitude objects  Phase Objects

Theory & Appl. Light Microscopy

Phase Objects
 Do not absorb light  Difference in index of refraction between

specimen and background

Theory & Appl. Light Microscopy

Theory & Appl. Light Microscopy

Example: Cell
 Object 1.25 Qm thick, i.r. = 1.35; i.r.

water = 1.30 (0.05 difference)  Difference in path length for light = 1.25 (0.05) = 0.0625 Qm  62.5/500 nm = 1/8 wavelength  P/8 = T/4 radians = 45  This is difference in phase of wave passing through cell against wave passing next to cell
Theory & Appl. Light Microscopy

Phase Differences
 Our eyes cannot see this  Eyes set for amplitude differences, so cell is

essentially transparent  But information is present in light beams from specimen and in image  How do we see this?

Theory & Appl. Light Microscopy

Theory & Appl. Light Microscopy

Theory & Appl. Light Microscopy

It is used to observe unstained living cells. It uses a conventional light microscope fitted with a phase contrast objective & a phase contrast condenser. It is based on the fact that, light passing through one material in to another of slightly different refractive index or thickness will undergo a change in phase. These differences in phase are translated in to variations in brightness of the structures. hence they are detectable by the eye.

Theory & Appl. Light Microscopy

Theory & Appl. Light Microscopy

Problems with Designs

 Image deteriorates with higher magnification

objectives (40x max)  Optical path differences in different scopes  Contrast is lost with open aperture  Condenser and Objective must be specially modified and are not useable for other optics

Theory & Appl. Light Microscopy

Theory & Appl. Light Microscopy

Theory & Appl. Light Microscopy

APPLICATIONS
Very useful in the examination of intracellular components of living cells at relatively high resolution. It is used to observe motility of the structures such as mitochondria, mitotic chromosomes, vacuoles .

LIMITATIONS
It is only suitable for observing single layers or thin cell layers. Since the separation of deviated & undeviated rays is incomplete halo formation results.

Fluorescence Microscope
What is fluorescence?

The molecules absorbs high energy light (blue, for example). This increases the energy of the molecules, represented as the top black line in the diagram (an "excited" molecules). Some of the energy from the blue photon is lost internally (represented by the red squiggly arrow in the picture). The molecules then emits a photon with less energy, green in this example.

Fluorescence Microscope
How does a fluorescent microscope work?

Dichroic mirror reflects light shorter than a certain wavelength, and passes light longer than that wavelength.

The advantage of fluorescence for microscopy is that you can often attach fluorescent dye molecules to specific parts of your sample

FLUORESENCE MICROSCOPY
Light source - High intensity mercury lamp- emits white light The specimen is stained with a fluorescent dye. Stained portion absorb blue light & emits green light. Two types of filters:Barrier filter- Transmits only blue light to the specimen & blocks out all other colors. Exciter filter- Allows the green light to pass to the eye. Thus the image can be seen.

APPLICATIONS
Detection of macromolecules such as antigens, DNA & RNA within the cell. To study the banding patterns of chromosomes. Detection of chromosomal anomalies.

Electron Microscope (TEM)

Magnetic focus F=qvBsinE=mv2/R R=mv/qB T=2 m/qB

Magnetic condenser The acceleration voltage determine the velocity of electron, thus the penetrating depth <100KV for biological sample

The Transmission Electron Microscope (TEM)

The Transmission Electron Microscope (TEM)

TEM has three essential systems: (1) an electron gun, which produces the electron beam, and the condenser system, which focuses the beam onto the object, (2) the image-producing system, consisting of the objective lens, movable specimen stage, and intermediate and projector lenses, which focus the electrons passing through the specimen (3) recording system Electron gun The TEM is an evacuated metal cylinder (the column) about 2 meters high with the source of illumination, a tungsten filament (the cathode), at the top. If the filament is heated and a high voltage (the accelerating voltage) of between 40,000 to 100,000 volts is passed between it and the anode, the filament will emit electrons. These negatively charged electrons are accelerated to an anode (positive charge) placed just below the filament, some of which pass through a tiny hole in the anode, to form an electron beam which passes down the column. The speed at which they are accelerated to the anode depends on the amount of accelerating voltage present.

The Transmission Electron Microscope (TEM)

Image producing system: As the electron beam passes through the specimen, some electrons are scattered whilst the remainder are focused by the objective lens either onto a phosphorescent screen or photographic film to form an image. Unfocussed electrons are blocked out by the objective aperture, resulting in an enhancement of the image contrast. The contrast of the image can be increased by reducing the size of this aperture. The remaining lenses on the TEM are the intermediate lens and the projector lens. The intermediate lens is used to control magnification. The projector lens corresponds to the ocular lens of the light microscope and forms a real image on the fluorescent screen at the base of the microscope column.

Electro-magnets, placed at intervals down the column, focus the electrons, mimicking the glass lenses on the light microscope. The double condenser lenses focus the electron beam onto the specimen which is clamped into the removable specimen stage, usually on a specimen grid.

Resolution in the TEM

The resolving power of a microscope is a function of the wavelength, P, of the source of illumination. For an electron, the wavelength is given by de Broglies equation: p=mv=h/ P, P=h/mv
Where, h is Plancks constant, m is the mass of the electron, and v is the velocity. Better resolution is achieved by using lower wavelengths.

Velocity v increases, wavelength decreases, resolving power increases mv2/2=eV,

where V is the accelerating voltage, e is the charge of the electron, m is the mass.

By substitution, we obtain P=1.22/V1/2 The above equation is not useful for calculating the resolution because lens aberration( ), refractive index of the medium, and aperture angles limit the resolution. However, it is possible to demonstrate that the highest practical resolution of TEM is 0.4 to 1 nm, with a practical magnification of 100,000 to 200,000

a Leica AFS Freeze-Subsitution System: for low temperature freeze-substitution and resin-embedding of samples

Gatan CT3500 Cryo-Transfer System: for cryotransfer of particulate samples on TEM grids for examination by cryo-TEM a Leica Ultracut Ultramicrotome( ) with cryo-sectioning capability: for cutting of routine and cryo thin sections for examination in the TEM

an axially-mounted Gatan Ultrascan 1000 CCD camera: for the acquisition of 2K X 2K pixel, 16 bit digital image output from the TEM

a Leica EM CPC Cryo-Preparation System: for the cryo-fixation of cells, tissues and particulate samples on TEM grids

Scanning Electron Microscope (SEM)

SEM is based on the interaction of an electron beam with a specimen. The incident beam (Primary electrons) displaces orbital electrons from the sample, giving rise to secondary electron (Reflect Electron Microscope, REM)

Scanning Electron Microscope (SEM)

Scanning Electron Microscope (SEM)

While all these signals are present in the SEM, not all of them are detected and used for information. The signals most commonly used are the Secondary Electrons, the Backscattered Electrons and X-rays

structure of SEM: Electron gun; Detecting system; Imaging system; Recording system

Deflector coils bend the primary beam for scanning across the specimen. Deflector coil in SEM connect to the CRT deflector plates

CRT: cathode ray tube

SEMs and TEMs have many features in common, primarily they both use electron beams to visualize a sample. However, they differ in the way the beam interacts with the specimen.
TEM accelerating voltages are 100KV SEM accelerating voltages are limited to around 40 KV

Advantages of SEM
3D image: the positioning of the primary scanning beam and the location of the detector affect the image contrast, therefore giving a sense of three dimension. Wide range of magnification: 10~100000 Easy sample preparation: For TEM, thickness is less than 100nm. Convenient keeping imaging ( see page 172) The SEM has a large depth of field, which allows a large amount of the sample to be in focus at one time. The SEM also produces images of high resolution, which means that closely spaced features can be examined at a high magnification. Preparation of the samples is relatively easy since most SEMs ony require the sample to be conductive. The combination of higher magnification, larger depth of focus, greater resolution, and ease of sample observation makes the SEM one of the most heavily used instruments in research areas today

This is the SEM in the Iowa State MSE Department

Analytical Electron Microscope

X ray micro-analyzer Attachment-AEM

Electron beam microanalysis is based on the excitation of characteristic x-rays which identify the elements present (limited to atomic numbers >3) and whose intensity is proportional to the relative quantity of the element present (accurate quantitative analysis is limited to atomic numbers >10). Electron beam microanalytical instruments include the scanning electron microscope/electron probe microanalyzer and the analytical electron microscope/scanning transmission electron microscope. The electron probe microanalyzer utilizes both wavelength dispersive (WDS) and energy dispersive (EDS) x-ray spectrometers for measurement of the x-ray energies and intensities from samples in the form of solid targets or particles. An important feature of the instrument is the capability to form scanning electron microscope (SEM) images of the sample with spatial resolution of morphological details as fine as 10 nm. The spatial resolution of x-ray microanalysis of thick specimens is limited to a volume with dimensions of approximately 1 micrometer due to electron scattering effects. The analytical electron microscope (AEM) is based on a conventional 200 keV transmission electron microscope (TEM) which has been augmented to include the functions of a scanning transmission electron microscope (STEM) and the scanning electron microscope (SEM). Since the electrons must penetrate through the specimen, the specimen dimensions must be no greater than 1 micrometer thick, and preferably in the range of 100 nm. Bulk specimens may be thinned to this range by a combination of mechanical, chemical, and ion sputtering techniques, all of which are available in the microanalysis laboratory. As an imaging tool, the AEM is capable of 0.3 nm spatial resolution in the TEM mode, 1 nm resolution in the STEM mode, and 3 nm resolution in the SEM mode

Atomic force microscope (AFM)

Atomic force microscope (AFM)

Scanning Tunneling Microscope (STM)

STM, along with the scanning force microscope (SFM), belong to the more general group of scanning probe microscope (SPM or SXM)

Scanning Tunneling Microscope (STM)

Specimen Preparation-SEM
There are two basic types of SEM's. The regular SEM, which we have in the Iowa State Materials Science Department, requires a conductive sample. An environmental SEM can be used to examine a nonconductive sample without coating it with a conductive material. Three requirements for preparing samples for a regular SEM such as in the Iowa State Materials Science Department are: 1) Remove all water, solvents, or other materials that could vaporize while in the vacuum. 2) Firmly mount all the samples. 3) Non-metallic samples, such as bugs, plants, fingernails, and ceramics, should be coated so they are electrically conductive. Metallic samples can be placed directly into the SEM.

Projects: Describe the theory and application of the following microscope:

Fluorescence microscope (including the labeling of fluorescence probe) Confocal microscope Two photon microscope Transmission Electron Microscope(TEM) Scanning Electron microscope(SEM) Atomic Force microscope (AFM)