Majmaa University Colleges of Science 1st SEMESTER 1433/1434 H

MICROSCOPY &

ELECTRON MICROSCOPE By

Dr. Alaa-Eldin Salah-Eldin Associated Professor of Cellular and Molecular Biology 1

Microscopes & Microscopy

• Basic Principles: What do Microscopes do? • Magnification: objects are made to appear larger than they are. • Resolution: the ability to see close together objects as distinct. • Microscopes are an important tool of biologists, and are used to study cells, tissues, and microorganisms in Medical Science. 2

Resolution (not magnification!) is the ability to separate two objects optically Unresolved

Partially resolved

Resolved

3

BACKGROUND THEORY AND TERMINOLOGY FOR ELECTRON MICROSCOPY What is scale all about?

Scanning Electron Microscope

4

Remember that there are 1000 micrometers (µm) in 1 mm and 1000 nanometers (nm) in 1 µm. The human eye can separate 0.2 mm at a normal viewing distance of 25 cm. The light microscope can separate 0.2 µm (0.002mm) depending on wavelength of light used. Electrons have a smaller wavelength than light therefore provide the highest resolving power – about 2 nm (0.000002mm). 5

With enough resolution we can magnify an object many millions of times and still see new detail This is why we use electron microscopes If you magnified your thumb nail just 10,000 times it would be about the size of a football pitch.

For example think of the size of Suncorp Stadium in Brisbane 6

Microscopy • Main branches: optical, electron and scanning probe microscopy. (+ less used X-ray microscopy) • Optical and Electron microscopy involves the Diffraction ‫ الحيود‬, Reflection ‫انعكاس‬, or Refraction ‫إنحراف‬ of radiation incident upon the subject of study, and the subsequent collection of this scattered radiation in order to build up an image. • Scanning probe microscopy involves the interaction of a scanning probe with the surface or object of interest. 7

Different Kinds of Microscopes:

Optical microscopy - definition • Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single or multiple lenses to allow a magnified view of the sample. • The resulting image can be detected directly by the eye, imaged on a photographic plate or captured digitally. • The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage and support, makes up the basic Light microscope. 8

Early Light Microscopes:

A Compound Light Microscope

http://micro.magnet.fsu.edu

9

Lenses of the Compound Light Microscope & their Functions: - Oculars (eyepieces): provide some magnification, focus image at eye (can be 1 or 2 of them) - Objectives: located on rotating nosepiece, provide magnification and resolving power (may be 3 to 6 of them) - Condenser: located under stage, focuses light on the specimen. It may also contain an iris diaphragm that controls the size of the cone of light entering the condenser.

10

Close-up of an Objective Lens:

http://www.bhphotovideo.com/

Total Magnification:  

  

Example: 10x / 0.25 10x: means that it magnifies the object 10 times. 0.25 is the Numerical Aperture (N.A.) of this objective. The higher the N.A., the greater the resolving power of an objective.





Easy to calculate ! Simply multiply the ocular magnification by the magnification of the objective you are using Example: Ocular magnification (15x) X Objective magnification (45x) = 675x

Hint: the magnification of each ocular and objective lens is usually written on it

11

Resolving Power:

dmin = 0.61  / N.A. 

  

dmin is the minimum distance between objects that can be seen as distinct (in μm)  is the wavelength (for light, 380 -760 nm = 0.38 - 0.76 μm) N.A. is the Numerical Aperture of the objective lens Example: green light ( = 0.52 μm): dmin = (0.61)(0.52)/1.4 = 0.23 μm (Hence, the wavelength of light limits the resolving power of light microscopes !)



Human eye: about 0.2 mm



Compound Light Microscope: about 0.2 μm



Transmission Electron Microscope: about 0.2 nm 12

Greatest Useful Magnification: 

Compound Light Microscope: around 1500x



Transmission Electron Microscope: around

250,000x (biologists usually use less) 

Although you could easily make lenses for light

microscopes to magnify more, the images would be blurry due to the lower resolution 13

14

Contrast Enhancing Methods:  

What is Contrast?: The apparent difference in brightness between objects These methods are important for studying cells that lack much contrast, such as animal cells



Chemical Methods:  Stains (dyes) that color parts of cells (though many stains kill cells)



Optical Methods (can be used with living cells): − Closing down the iris diaphragm (but you lose some resolution)  Dark Field Illumination  Phase Contrast Microscopy  Differential Interference Contrast (DIC) 15

Optical microscopy - limitations OM can only image dark or strongly refracting objects effectively. Out of focus light from points outside the focal plane reduces image clarity. Compound optical microscopes are limited in their ability to resolve fine details by the properties of light and the refractive materials used to manufacture lenses. A lens magnifies by bending light.

Optical microscopes are restricted in their ability to resolve features ‫ مالمح‬by a phenomenon called diffraction.

Due to diffraction, even the best optical microscope is limited to a resolution of around 0.2 micrometres. 16

Optical microscopy - types • • • • • • • • • • •

Optical microscopy techniques Bright field optical microscopy Oblique illumination Dark field optical microscopy Phase contrast optical microscopy Differential interference contrast microscopy Fluorescence microscopy Confocal laser scanning microscopy Deconvolution microscopy Near-field Scanning OM … 17

Measurement Units in Microscopy:  

 

In light microscopy, objects are measured in micrometers (μm) 1 μm = 1/1000th of 1 mm, or 1 x 10-6 meters In electron microscopy, objects are measured in nanometers (nm) 1 nm = 1/1000th of 1 μm, or 1 x 10-9 meters 18

Cheek Cells: Bright Field Image (unstained):

Cheek Cells: Bright Field Image (stained):

Cheek Cells: a) Bright Field & b) Phase Contrast & c) DIC Compared: 19

Electron Microscopy - definition and types • Developed in the 1930s that use electron beams instead of light. • Because of the much lower wavelength of the electron beam than of light, resolution is far higher.

• Types of Electronic Microscope (EM) 1. Transmission electron microscope (TEM) 2. Scanning electron microscope (SEM) 3. Reflection electron microscope (REM) 4. Scanning transmission electron microscope (STEM) 5. Low-voltage electron microscope (LVEM) 20

Electron Microscopy - definition and types • Most Popular Types of Electronic Microscope (EM) • Transmission electron microscopy (TEM) is principally quite similar to the compound light microscope, by sending an electron beam through a very thin slice of the specimen. The resolution limit (in 2005) is around 0.05 nanometer.

• Scanning electron microscopy (SEM) visualizes details on the surfaces of cells and particles and gives a very nice 3D view. The magnification is in the lower range than that of the transmission electron microscope.

21

Transmission Electron Microscope (TEM):

Scanning Electron Microscope (SEM): is analogous to the stereo binocular light microscope because it looks at surfaces rather than through the specimen.

22

Electron beam produced here

Cross section of electromagnetic lenses

Beam passes down the microscope column Electron beam now tends to diverge But is converged by electromagnetic lenses

Sample

Diagram of Scanning Electron Microscope or SEM in cross section - the electrons are in green 23

Electromagnetic Lenses An electromagnetic lens is essentially soft iron core wrapped in wire As we increase the current in the wire we increase the strength of the magnetic field Recall the right hand rule electron will move in a helical path spiralling towards the centre of the magnetic field

24

Electron beam – Specimen Interaction. Note the two types of electrons produced.

25

Electrons from the focused beam interact with the sample to produce a spray of electrons up from the sample. These come in two types – either secondary electrons or backscattered electrons. As the beam travels across (scans across) the sample the spray of electrons is then collected little by little and forms the image of our sample on a computer screen. We can look more closely at these two types of electrons because we use them for different purposes. Inelastic scattering

-

Elastic scattering

-

+

+ Energy of electron from beam is lost to atom

A new electron is knocked out (as a secondary electron)

An incoming electron rebounds back out (as a backscattered electron)26

Example of an image using a scanning electron microscope and secondary electrons

Example of an image using a scanning electron microscope and backscattered electrons Grain containing titanium so it is whiter

Grain containing of silica so it is darker Here the contrast of these grains is all quite similar. We get a threedimensional image of the surfaces.

Here the differing contrast of the grains tells us about composition

27

So how does this work – telling composition from backscattered electrons? The higher the atomic number of the atoms the more backscattered electrons are „bounced back‟ out This makes the image brighter for the larger atoms

Titanium – Atomic Number 22

Silica – Atomic Number 14

28

Understanding compositional analysis using X-rays and the scanning electron microscope If the yellow electron falls back again to the inner ring, that is to a lower energy state or valence, then a burst of + X-ray energy is given off that equals this loss.

Inelastic scattering

This is a characteristic packet of energy and can tell us what element we are dealing with 29

EDS output from X-rays

Amount of packets

1050 900

Characteristic carbon peak

006

CKa

1200

Characteristic oxygen peak Characteristic chlorine peak ClKa

600

OKa

Counts

750

450 300 150 0 0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

keV

Energy of packets in thousands of electron volts

30

Using X-rays to investigate composition in this way is called Energy Dispersive Spectroscopy (EDS) since it produces a spectrum graph We can get quite detailed information about mass and atomic percentages in materials from EDS phi-rho-z Method Standardless Quantitative Analysis Fitting Coefficient : 0.4050 Element (keV) mass% Error% At% Compound C K 0.277 65.88 0.08 74.01 O K 0.525 28.12 0.72 23.71 Cl K 2.621 6.00 0.20 2.28 Total 100.00 100.00

mass%

Cation

K 75.5733 34.1444 13.7857

31

The electromagnetic spectrum

32

Light v Electron

33

Magnifying using the Light Microscope

34

Magnifying using the Electron Microscope

35

THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE FEATURE

Electromagnetic spectrum used Maximum resolving power

Maximum magnification Radiation source

Lenses Interior Focussing screen

LIGHT MICROSCOPE

ELECTRON MICROSCOPE

Visible light

Electrons

760nm (red) – 390nm Colours visible

app. 4nm Monochrome

app . 0.2 mm (200nm)

0.2nm Fine detail

x1000 – x1500

x500 000

Tungsten or quartz halogen lamp

High voltage (50kV) tungsten lamp

Glass Air-filled

Magnets Vacuum

Human eye (retina), photographic film

fluorescent (TV) screen, photographic film 36

THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE

FEATURE Preparation of specimens

Fixation Embedding

LIGHT MICROSCOPE Temporary mounts living or dead

ELECTRON MICROSCOPE Tissues must be dehydrated = dead

Alcohol

OsO4 or KMnO4 Resin

Sectioning

Wax Hand or microtome slices  20 000nm Whole cells visible

Microtome only. Slices  50nm Parts of cells visible

Stains

Water soluble dyes

Heavy metals

Glass slide

Copper grid

Support

37

Different Kinds of Microscopes: In General

• Compound Light Microscope (transmitted light) • Dissecting Light Microscope (reflected light) • Transmission Electron Microscope (transmitted electron beam) • Scanning Electron Microscope (reflected electron beam)

38

Comparison of Light Microscope & TEM: http://www.lab.anhb.uwa.edu.au

39

40

Summary of Electron Microscope Components 1. Electron optical column consists of: – electron source to produce electrons – magnetic lenses to de-magnify the beam – magnetic coils to control and modify the beam – apertures to define the beam, prevent electron spray, etc. 2. Vacuum systems consists of: – chamber which “holds” vacuum, pumps to produce vacuum – valves to control vacuum, gauges to monitor vacuum 3. Signal Detection & Display consists of: – detectors which collect the signal – electronics which produce an image from the signal 41

42

Transmission Electron Microscope (TEM)   

 

Is capable of much higher resolution than the light microscope An electron beam is transmitted through a very thin section Instead of glass lenses, magnetic lenses are used, which bend and focus the electron beam much like a glass lens bends and focuses light Specimen preparation is time-consuming Only fixed and stained (dead) specimens can be examined 43

Transmission Electron Microscopy (TEM) • Beam of electrons is transmitted through a specimen, then an image is formed, magnified and directed to appear either on a fluorescent screen or layer of photographic film or to be detected by a sensor (e.g. charge-coupled device, CCD camera. • Involves a high voltage electron beam emitted by a cathode, usually a tungsten filament and focused by electrostatic and electromagnetic lenses. • Electron beam that has been transmitted through a specimen that is in part transparent to electrons carries information about the inner structure of the specimen in the electron beam that reaches the imaging system of the microscope. • Spatial variation in this information (the "image") is then magnified by a series of electromagnetic lenses until it is recorded by hitting a fluorescent screen, photographic plate, or CCD camera. The image detected by the CCD may be 44 displayed in real time on a monitor or computer.

Transmission Electron Microscopy (TEM) Neuron growing on astroglia

Black Ant

House Fly House Fly

Human stem cells

Human red blood cells

Neurons CNS 45

TEM Image: rubra)

(thin section of Myrionecta

(Hansen & Fenchel, Mar. Biol. Res., 2: 169-177, 2006)

46

Scanning Electron Microscope (SEM)  



Is capable of higher resolution than the light microscope An electron beam is “bounced off” the specimen to a detector, instead of being passed through it It produces a detailed image of the surface of the specimen, but not its internal structure

47

Scanning Electron Microscopy (SEM) • type of electron microscope capable of producing highresolution images of a sample surface. • due to the manner in which the image is created, SEM images have a characteristic 3D appearance and are useful for judging the surface structure of the sample.

Resolution • Depends on the size of the electron spot, which in turn depends on the magnetic electron-optical system which produces the scanning beam. • is not high enough to image individual atoms, as is possible in the TEM … so that, it is 1-20 nm 48

Scanning Electron Microscope (SEM): http://www.engr.uky.edu/emc/facilities/sem.html

49

SEM Image:

(Emiliania huxleyi, a haptophyte alga) http://starcentral.mbl.edu/

50

Feeding tube from a moth under the scanning electron microscope

51

X-ray microscopy • less common, • developed since the late 1940s, • resolution of X-ray microscopy lies between that of light microscopy and the electron microscopy. • X-rays are a form of electromagnetic radiation with a wavelength in the range of 10 to 0.01 nanometers, corresponding to frequencies in the range 30 PHz to 30 EHz. 52

Fluorescence & Microscopy: Fluorescence: when a molecule is excited by light at a shorter wavelength, and emits light at a longer wavelength  Autofluorescence: some substances, such as chlorophyll, are naturally fluorescent  Fluorescence Microscope: a microscope that excites and detects fluorescent materials  Fluorescent Dyes: are widely used in biology to label cell organelles, molecules, and genes in cells 

53

Chloroplast Autofluorescence: (Paramecium bursaria) http://starcentral.mbl.edu/

54

Confocal Laser Scanning Microscopy (CLSM) 

   

The higher the magnification the less “depth of field” - only a narrow slice is in focus The CSLM gets around this problem Lasers scan the specimen at different depths A computer reconstructs a clear 3D image Usually used with fluorescent specimens

55

Confocal Scanning Laser Microscope (CLSM): http://pict-ibisa.curie.fr

56

Myrionecta rubra & Geminigera cryophila: a & b): Fluorescence images, c) TEM image, d) Confocal FISH image (Johnson et al., Nature, 445:426-428, 2007)

57

Phase Contrast Image:

(Flagellate Protozoa from Termite Intestines) http://visualsunlimited.photoshelter.com/

58