Microscopy

Microscopy

• Microscope is used to view objects or specimens that are too small to be seen with just the human eye.

​

• Microscopy is a technical field that involves the use of Microscopical components such as microscopes or microscope objectives to obtain greater detail of examined samples.

​

• Definition-A microscope is a high precision optical instrument that uses a lens or a combination of lenses to produce highly magnified images of small specimens or objects especially when they are too small to be seen by the naked (unaided) eye.
• A light source is used (either by mirrors or lamps) to make it easier to see the subject matter.

TERMS AND DEFINITIONS

Principle

Microscopy is to get a magnified image, in which structures may be resolved which could not be resolved with the help of an unaided eye.

Magnification

• It is the ratio of the size of an object seen under microscope to the actual size observed with unaided eye.
• The total magnification of microscope is calculated by multiplying the magnifying power of the objective lens by that of eye piece.

Resolving power

• It is the ability to differentiate two close points as separate.
• The resolving power of human eye is 0.25 mm
• The light microscope can separate dots that are 0.25µm apart.
• The electron microscope can separate dots that are 0.5nm apart.

TERMS AND DEFINITIONS

Limit of resolution

• It is the minimum distance between two points to identify them separately.
• It is calculated by Abbé equation.

Working distance

• It is the distance between the objective and the objective slide.
• The working distance decreases with increasing magnification.

TERMS AND DEFINITIONS

Numerical aperture(NA)

The numerical aperture of a lens is the ratio of the diameter of the lens to its focal length.

​

NA can be decreased by decreasing the amount of light that passes through a lens.

Diameter of the lens

Imaging Techniques

History of microscope

• In 1590 F.H Janssen & Z.Janssen constructed the first simple compound light microscope -10x to 30x .
• In 1665 Robert Hooke developed a first laboratory compound microscope.
• Later, Kepler and Galileo developed a modern class room microscope.
• In 1672 Anton Von Leeuwenhoek developed a first simple microscope with a magnification of 200x – 300x.
• In 1674, Anton was the first to see and describe bacteria, yeast, plants, and life in a drop of water- He is called as Father of microscopy.
• The term microscope was coined by Faber in 1623.
• In the early 1930’s the first electron beam microscopes were developed which were a breakthrough in technology as they increased the magnification from about 1000x or so up to 250,000x or more.

​

Compound Microscope

• Common type of microscope.
• High power microscope- The magnification (power) 40x to 1000x.
• Compound refers to the fact that in order to enlarge an image - a single light path passes through a series of lenses in a line where each lens magnifies the image over the previous one.
• In the standard form – 2 lenses
• an objective lens (closest to the object or specimen)
• an eyepiece lens (closest to the observers’ eye)
• Uses light to illuminate the specimen
• The objective lens usually consists of three or four lenses.
• The most used light method is trans-illumination.
• At 400x much detail can be seen at the cellular level of biological specimens.
• Applications: Learn about cells and microorganisms in both medical and science field.

Compound Microscope

Objective lenses

• One of the most important parts of a compound microscope, as they are the lenses closest to the specimen.
• A standard microscope has three, four, or five objective lenses that range in power from 4X to 100X.
• Objectives vary in power from 1x to 160x in compound microscopes but the most common power range is from 4x to 100x.
• Most compound microscopes have three or four (occasionally five) objectives usually of 4x, 10x, 40x, and 100x (oil immersion) which revolve on a nosepiece (turret) to give different magnifying powers.

Numerical Aperture

• NA of a microscope objective is a measure of its ability to gather light
• The more light (higher NA) the better the resolving power of the lens
• Better resolution

​

• The N.A. will be marked on the objective and the typical N.A. for the following are;
• 4x=0.10,
• 10x=0.25
• 40x=0.65
• 100x=1.25.

Ocular Lens or Eye piece

• The eyepiece consists of a series of lenses mounted in a tube (barrel) at the upper end of the microscope.
• Its basic function is to look at the focused, magnified image projected by the objective lens and magnify that image a second time before your eye looks at the image of the specimen.
• The eyepieces are usually 10x but also come in 5x, 12.5x, 15x, and 20x. The “x” refers to the amount of magnification (power) that this lens adds as a multiplier to the magnification of the objective.
• For special applications, eyepieces can have scales, pointers, crosshairs, markers, etc. on them.
• The eyepoint is the location (or position) of the eye from the eyepiece which allows for the best possible viewing of the image.

Other parts

​

• Diopter Adjustment: Useful as a means to change focus on one eyepiece so as to correct for any difference in vision between your two eyes.
• Body tube (Head): The body tube connects the eyepiece to the objective lenses.
• Arm: The arm connects the body tube to the base of the microscope.
• Coarse adjustment: Brings the specimen into general focus.
• Fine adjustment: Fine tunes the focus and increases the detail of the specimen.
• Nosepiece: A rotating turret that houses the objective lenses. The viewer spins the nosepiece to select different objective lenses.
• Specimen or slide: The specimen is the object being examined. Most specimens are mounted on slides, flat rectangles of thin glass.

​

• Stage: The flat platform where the slide is placed.
• Stage clips: Metal clips that hold the slide in place.
• Stage height adjustment (Stage Control): These knobs move the stage left and right or up and down.
• Aperture: The hole in the middle of the stage that allows light from the illuminator to reach the specimen.
• On/off switch: This switch on the base of the microscope turns the illuminator off and on.
• Illumination: The light source for a microscope. Illumination is the application of light onto an object or specimen in a microscope.
• Diaphragm: Adjusts the amount of light that reaches the specimen.
• Condenser: Gathers and focuses light from the illuminator onto the specimen being viewed.
• Base: The base supports the microscope and it’s where illuminator is located.

​

STM and AFM

• Both STM and atomic force microscopy (AFM) are�part of the scanning probe microscope family.
• STM uses the electronic properties between the�tip and the sample.
• AFM uses Forces between the sample and a tip on the end of a cantilever. These forces change as the tip gets closer to the sample.
• So what are these forces???

​

Atomic Forces

Force Distance Curve

• 3. The force goes to zero when the distance�between the atoms reaches a couple of angstroms, about the length of a chemical bond.
• 4. When the total van der Waals force becomes�positive (repulsive), the atoms are in contact.
• 5. The slope of the Force curve is very steep in�the repulsive or contact regime. ? As a�result, the repulsive force balances almost any�force that attempts to push the atoms closer�together. ?
• In AFM this means that when the�cantilever pushes the tip against the sample, the cantilever bends rather than forcing the tip�atoms closer to the sample atoms.

​

AFM Cantilever

.

Contact mode�

• The tip is moved over the surface by the�scanning system.
• A value of the cantilever deflection, for�example, is selected and then the feedback system�adjusts the height of the cantilever base to keep�this deflection constant as the tip moves over�the surface.
• Non-contact mode
• The cantilever oscillates close to the sample�surface, but without making contact with the�surface.
• The capillary force makes this particularly�difficult to control in ambient conditions. Very�stiff cantilevers are needed.
• �

Non- Contact Mode

• The cantilever oscillates close to the sample�surface, but without making contact with the�surface.
• The capillary force makes this particularly�difficult to control in ambient conditions. Very�stiff cantilevers are needed.

​

Tapping mode�

• Resonant frequency of the cantilever depends on its mass and spring constant stiffer cantilevers have higher resonant frequencies.�

​

​

​

​

​

​

• Question Where would you want to operate system at??? At resonance?�

Resonance frequency

Cantilever

• Used to measure long range attractive or�repulsive forces between the probe tip and the sample surface.
• Force curves( force-versus-distance curve)�typically show the deflection of the free end of the AFM cantilever as the fixed end of the�cantilever is brought vertically towards and then away from the sample surface.
• The deflection of the free end of the cantilever is measured and plotted at many points as the z-axis scanner extends the cantilever towards the surface and then retracts it again..

​

• Force measurements. The AFM can record the amount of force felt by the cantilever as the probe tip is brought close to - and even indented into – a sample surface and then pulled away

• Consider a cantilever in air approaching a hard, incompressible surface such as glass or mica.
• AB As the cantilever approaches the surface, initially the forces are too small to give a measurable deflection of the cantilever, and the cantilever remains in its undisturbed position.�BC At some point, the attractive forces�(usually Van der Waals, and capillary forces)�overcome the cantilever spring constant and the tip jumps into contact with the surface.

• CD Once the tip is in contact with the sample,�it remains on the surface as the separation�between the base and the sample decreases�further, causing a deflection of the tip and an�increase in the repulsive contact force.
• DF, FG as the cantilever is retracted from the�surface, often the tip remains in contact with�the surface due to some adhesion and the�cantilever is deflected downwards.
• GH At some point the force from the cantilever will be enough to overcome the adhesion, and the tip will break free.

Applications

• Molecular Interactions

Modern AFM imaging phase imaging

• In tapping mode, we vibrate the cantilever and we have resonance frequency. Normally we ignore any thing to do with the phase, however there is now a lot of research in using phase imaging to go beyond simple topographical mapping to detect variations in composition, adhesion, friction, viscoelasticity.

Some examples of Phase Imaging

1. Bond pad on an integrated circuit imaged by Tapping Mode (left) and phase (right). Pad contaminated with polyimide produce light�contrast with phase shifts of over 120 deg. 1.5µm scan�

2. Tapping Mode (L) and phase images (R) of a composite polymer embedded in a uniform matrix

�AFM vs STM�

• Resolution of STM is better than AFM because of the exponential dependence of the tunneling current on distance.
• STM is generally applicable only to conducting�samples while AFM is applied to both conductors and insulators.
• AFM offers the advantage that the writing�voltage and tip-to-substrate spacing can be�controlled independently, whereas with STM the two parameters are integrally linked.

​

Other Types of SPM

• Magnetic Probe Microscopy MFM
• The dots are made of permalloy and are 400 nm apart.
• The MFM image gives the field distribution for a non polarized sample, and a few different configuration are observed.�

EFM Electric Force Microscopy

• Field of view 10µm x 10µm�Different types of material are�deposited on Si wafers during processing.

�Scanning Capacitance Microscopy (SCM)

• Figure shows two sets of AFM measurements (topography and SCM) for a correctly aligned mask(left) and for a misaligned mask (right). The large pictures are a combination of both the topography (grey) and the SCM image (orange). From these�images the amount and direction of the misalignment can be observed.

AFMs as tools I Dip-pen Nanolithography

Using AFM as a tool: Nanomanipulation

�Summary�

• AFM uses force to probe the surface topology and properties of materials.
• Tapping modes vs Contact mode
• Phase imaging is a modern way to prove extra information.
• Dip- pen nanolithography is a way to perform serial writing.�