Microscopy

INTRODUCTION TO MICROSCOPY

Components of Microscope Light source: light or electron Condenser lens Objective lens Projector lens Suitably prepared specimen

Image formation in reflected illumination

Image formation in transmission illumination

Condenser Lens  Collects light to direct it at the small area of the object which is to be examined. It makes the object brighter (better contrast) and enables to control the angle at which the illumination reaches the object.  The condenser lens can converge the light beam on object or can illuminate it with parallel rays.  Condenser aperture: controls the area of specimen to be illuminated

Magnification Objective lens provides an inverted image of object at B with magnification, M1 = (v1–f1)/f1 Projector lens: the lens gives a final upright image at a further magnification, M2 = (v2–f2)/f2 image distance (v) and focal length (f) The total magnification: M = M1x M2

Comparison of OM, TEM and SEM

Transmission Electron Microscope Similar to an optical instrument in that lenses are used to form images Scanning Electron Microscope Not similar to an optical instrument (no image forming lens) but uses electron optics to form a fine probe on the specimen and the signals emitted are detected and characterized.

Resolution Limit Abbe (Diffraction) limit: Defines the minimum resolvable distance between the image of two point objects using a perfect lens In any magnifying system, a point object (i.e. zero dia) can not be imaged a point but as a distribution of intensity having a finite width;

0.61λ ρ= η sin(α ) λ η α

wavelength of the imaging radiations refractive index of the lens illumination semi angle

α

Resolution The ability to discern fine details. It is represented by the minimum distance between two points such that the two points are perceived as separated image ρ = 0.61λ/N.A. N.A. = η sin α, Numerical aperture λWavelength of light α - the half acceptance angle of the lens.

Minimum distance between two diffraction maxima still projected separately

Resolution limit of a light microscope λ can decrease to 400 nm (green light) µ sin α is limited to ~ 1.6 Thus R. P. = λ 0.61/N.A. = 0.61x 400/1.6 = 152 nm The resolution is about 150 nm (0.15 µm)

Resolution limit of an electron microscope λcan be as small as 0.001 nm; µ sin α is very small, because µ is unity and α is about a degree. 0.61λ R.P. = α

For wavelength of 0.0037 nm and α = 0.1 radians, the resolution is about 0.02 nm

Important Features for Imaging Wave length λ of the electrons accelerated by a potential of V volts is given by the expression,

h λ= 2meV h is Planck’s constant (6.626x10-34 Js), m is electronic mass (9.11x10-31kg) and e is electronic charge (1.602x10-19C).

Important Features for Imaging (contd.) For large values of V, electrons can attain velocities comparable with the speed of light and relativistic increase in mass should be taken into account. This is done by replacing V by Vc (relativistic accelerating voltage) Vc = V[1 + eV/2m0c2] where c is the velocity of light (2.998x108 m/s). For 100 kV electrons, wavelength is 0.04Å (without relativistic correction) and 0.037Å with correction.

Resolution of the Microscope Resolution is given by the expression, Δrdiff = (0.61λ/μsinβ) = (0.61λ/β), since μ is 1 and β is small. Compromise aperture size taking diffraction and spherical aberration into account is β ~ 0.88 (λ/Cs)1/4. Corresponding value for resolution Δr, is given by Δr = 0.69λ3/4Cs1/4.

Light VS Electrons Light Microscope

Electron Microscope

λ = 0.5µm

λ~

η = 1.5 (glass) α = 70° ρ = 0.2µm=2000 Α

η= 1.0 (Vacuum) α = 1° ρ = 0.00016µm=1.6

150 = 0.055 A (@ 50kV ) V 0

Depth of Field The distance parallel to the optical axis of the microscope that a feature on the specimen can be displaced without loss of resolution Optical Microscope

Electron Microscope

λ wavelength η refractive index α semi angle M total magnification NA numerical aperture of the lens

Depth of Field Magnification 10 100 1000 10000

Depth of field Optical SEM 60µm 8µm 0.2µm --

1000µm 100µm 10µm 1µm

http://www.matter.org.uk/tem/depth_of_field.htm

Scanning Electron Microscope

Scanning electron microscope  A scanning electron microscope is similar to a light microscope being used in reflection.  The major difference is that instead of imaging the entire specimen at once, the electron beam is scanned back and forth over the specimen, imaging only one point at a time (much like how a television works--there is only one electron beam, but it scans every spot on the screen).  The interactions of the electrons with the surface are registered, and from this data an image can be constructed.

Comparison of Optical and Scanning Electron Microscopes Characteristic Illumination Wavelength Lens

SEM Electron beam ~ 0.05A Electrostatic lens for demag crossover and electromag lens for magnification

Optical Light beam 2000-7000A Optical lenses for magnification

Comparison of Optical and Scanning Electron Microscopes (contd.) Characteris- SEM tic Resolution ~ 50 Å

Depth field Magnification

Optical Visible region 2000Å UV region 1000 Å ~ 0.1µ m

of 30 µ m at 1000 X Continuously 10X to 2000X variable from 10X to 200,000X

Comparison of Optical and Scanning Electron Microscopes (contd.) Focusing Obtainable images Contrast

Electrical Several Shape chemical property controlled

Mechanical Transmitted and reflected and Colour and brightness controlled

The basic premise of an SEM is that signal produced from a scanned area of the specimen is displayed as an image with the exact same scan pattern on a CRT

The scan pattern on the specimen is created by a set of deflection coils in the column that move the beam in a coordinated X/Y pattern. This is referred to as a scan or “raster” pattern

Cathode Ray Tube accelerates electrons towards the phosphor coated screen where they produce flashes of light upon hitting the phosphor. Deflection coils create a scan pattern forming an image in a point by point manner

Color CRTs usually have three separate e-guns, one each for red, green, and blue (RGB)

Rastering

The scan generator coordinates the movement of the primary beam with the movement of the e-gun in the back of the CRT

Magnification is accomplished by scanning a progressively smaller portion of the specimen and displaying the image on the CRT. Thus total magnification is square area of CRT divided by area scanned.

In contrast focus is accomplished by bringing the beam to its crossover point on the surface of the specimen. In this way focus and magnification are completely separate from one another in the SEM.

In the TEM the specimen lies very close to the objective lens resulting in a relatively large half angle of illumination. In SEM since the image is not formed by an objective lens the half angle can be very small resulting in a large depth of field.

10X An SEM focused at high magnification will still be in focus at low magnification

110X

200X

400X

4K

16K

45K

Strong Lens: Small probe size, high resolution, short working distance and shallow depth of field

Weak Lens: Larger probe size, low resolution, long working distance, and larger depth of field

A smaller final lens aperture can reduce the half angle and therefore increase the depth of field. This is true on a relatively strong lens which has a fairly short working distance and therefore high resolution.

The SEM forms an image by generating a number of signals as a result of the beam interacting with the specimen.

Micrographs of blood corpuscles (a) optical, (b) scanning

SE and reflected light micrographs of Fe-1.0%C steel

BSE

SE

BSE and SE Images of Fractured 7075 Al Alloy

Variation of image contrast with variation in accelerating potential

X-ray Mapping

TEM

Basic Electron Optics

 Electrons and ions are charged particles; they can be accelerated in an electric field  The trajectory of an accelerated charged particle can be changed (deflected) by E and/ or B field.  The accelerated particles also behave like waves (de Broglie)

Schematic of TEM

Electron optical elements &attachments  Electron source  Lenses  Deflection coils  Stigmators  Electron detectors  Photon/X-ray detectors

Source

TEM TOM 100-400 kV electron gun Light source high current densities: 5 x 104 Am-2 for tungsten filament 1 x 106 Am-2 for field-emission source

Condenser lens

Electromagnetic lens, focus adjusted by controlling the lens current

Glass lens, focus adjusted by lens position

Specimen stage

Allows for specimen tilt as well as some z-adjustment

Allows for specimen tilt as well as some z-adjustment

Objective lens Fine focusing of the image by adjusting the lens current

Fine focusing by adjusting the position of the specimen and the objective lens

Final imaging system

Employs electromagnetic lenses to produce image on a fluorescent screen

Eye piece forming image for direct viewing

Recording system

Computer monitor or TV

Normal viewing or photographic films

Experimental set-up in

Vacuum, better than 10-6 torr

Air, at atmospheric pressure

Electron Source Generation of electrons that can be accelerated by high tension to obtain the illuminating electron beam. Thermionic gun:  triode or self-biasing gun, W, LaB6, CeB6 Field Emission Gun:  Single crystal W

Electron Sources Thermionic Emitters

Field Emitters

Thermionic Electron Gun • Electrons are emitted from a heated tungsten filament and then accelerated towards an anode; a divergent beam of electrons emerges from the anode hole. • Commonly used electron source • Robust, cheap and does not require relatively high vacuum

Schematic of thermionic electron gun

W

LaB6

Common Modes of Operation of TEM • Bright Field (BF) Microscopy • Selected Area Diffraction • Dark Field (DF) • Weak Beam (Special case of DF)

If a monochromatic ebeam of known λ strikes a crystal at the appropriate Bragg’s angle a number of the diffracted electrons will be forward scattered. Like the transmitted electrons these diffracted electrons will have nearly their same energy but will have been significantly altered from their trajectory.

The transmitted electrons will be brought to convergence in the back focal plane of the objective lens (Y). Likewise the diffracted electrons will also be brought to convergence in the back focal plane of the lens but at a different spot (X).

Normally an aperture is placed in the back focal plane of the objective lens to stop widely scattered electrons from reaching the viewing screen but in the case of diffraction it is these same scattered electrons that contain the information about the diffraction event.

To operate the TEM in diffraction mode the objective aperture is removed from the beam path and the scope is adjusted to focus an image of the back focal plane of the objective lens, not the image plane.

This is most easily accomplished by adjusting the strength of the objective lens so that an image of the back focal plane is projected onto the viewing screen.

The result is an electron diffraction (ED) pattern. The pattern one obtains is completely dependent on the d-spacing and composition of the crystal that is being analyzed.

An ED from a single crystal will result in a series of diffraction spots arranged in concentric rings around the central bright spot which is comprised of transmitted electrons.

Changes in diffraction pattern – spots to rings

Diffraction Pattern from Amorphous Material – diffused rings reflecting short range order; often seen on contamination layer or C support

ELECTRON DIFFRACTION PATTERNS

MOSAIC SINGLE CRYSTAL

PLATELIKE TEXTURE

POLYCRYSTA L

Selected Area Electron Diffraction (SAED):

V irtual A perture Specim en Low er O bjective Lens B ack FocalPlan

b* a* D

SA A perture

An SAED pattern of a crystal. Lattice plane have spacing of d θ

Camera length

K

L

Ewald Sphere D

K+g

SAED use parallel illumination and limits the sample volume by an aperture in the image plane of the low object lens.

D = tan ( θ ) = tan ( 2θ B ) ; L 2dSinθ B = λ

1 D ≈ d Lλ

SAD patterns for beam directions of [001] and [111], BCC Crystal

(a)

(b)

Formation of (a) Bright Field (BF) and (b) Dark Field (DF) Images

Bright Field Imaging • If the main portion of the near-forward scattered beam is used to form the image

Dark Field Imaging • If the transmitted beam is excluded from the image formation process – off-axis imaging – tilted beam imaging

http://www.matter.org.uk/tem/stem_images.htm

~ ATOMIC FORCE MICROSCOPE ~ HOW DOES IT WORK? Feedback Loop

Laser V Piezo Crystal

Photodiode Mirror

Tip

ThermoMicroscopes Explorer AFM

Substrate

The atomic force microscope (AFM), uses a sharp tip attached to the end of a cantilever rasters across an area while a laser and photodiode are used to monitor the tip force on the surface. A feedback loop between the photodiode and the piezo crystal maintains a constant force during contact mode imaging and constant amplitude during intermittent contact mode imaging.

As with the STM the probe tip of an AFM must be very small but because there is no need to establish a tunneling current one can use a variety of materials, not just those with a low workfunction.

Similar to a phonograph needle the probe is actually in contact with the specimen and is physically moved up and down due to the repulsion of van der Waals forces

The AFM records the position of the probe by bouncing a laser off the back surface of the probe and recording how the light is deflected

By using a four quadrant detector the relative amount of laser light hitting each quadrant can be used to determine how the tip has been deflected as it moves over the surface of the specimen

AFM of Chromosome Since an AFM relies on AFM derived models of contact rather than nuclear pore complex current many nonconductive materials can be

Since the contact of the tip with the specimen can cause physical damage to the specimen many AFMs employ a “tapping” mode in which the probe vibrates up and down as the sample is moved.

~ ATOMIC FORCE MICROSCOPE ~ WHAT CAN WE LEARN?

Imaging direction Elasticity TIP

Height

Rc

Friction Width

AFM Image and manipulation of an Adenovirus.

Binding