CE70009 - Product Characterisation Flashcards
How do monochromatic and polychromatic and polarized light differ?
Mono - one wavelength
Poly - multiple wavelengths
Polarised - waves all in one direction
What’s collimated light?
When light rays are parallel to each other
What’s coherent light?
When there is no interference between waves
List key parts of transmission microscope:
Ocular / eyepiece
Objective lens
Stage
Condenser lens
Condenser diaphragm
Focusing knobs
How is numerical aperture, NA, calculated?
NA = nsinØ
n = refractive index of medium in which lens is working, = 1.0 for air, 1.33 for water, and up to 1.56 for oils
How does resolution, light transmission, working distance, and depth of field vary with numerical aperture, NA?
Resolution increases with NA
Light transmission decreases with NA
Working distance decreases with NA
Depth of field decreases with NA
What is bright and dark field illumination?
Bright field microscopes usually have halogen lamp or LED light sources. This type of microscope tends to have low contrast owning to the biological samples transmitting most of the light. Staining if often required to combat this problem, which comes with the disadvantage that live imaging is difficult due to staining killing the cells.
Dark field microscopy is generally preferred therefore over light field. With a dark field microscope a special aperture is used to focus incident light meaning the background stays dark. The light does not pass directly through the sample being studied. Instead light is reflected off the specimen, making it appear to be emitting light. Brightfield microscopy shows clear magnification while the dark field image shows minute details.
Bright field – sample is dark while background is bright
Dark field: sample is bright while background is dark. Light is shone from top and we look at the light that is reflected back. Hollow cone of light with great obliquity. no transmission: reflection or scattering
What’s confocal microscopy?
An optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation.
Confocal microscopy offers several advantages over conventional optical microscopy, including shallow depth of field, elimination of out-of-focus glare, and the ability to collect serial optical sections from thick specimens. In the biomedical sciences, a major application of confocal microscopy involves imaging either fixed or living cells and tissues that have usually been labelled with one or more fluorescent probes.
When fluorescent specimens are imaged using a conventional widefield optical microscope, secondary fluorescence emitted by the specimen that appears away from the region of interest often interferes with the resolution of those features that are in focus. This situation is especially problematic for specimens having a thickness greater than about 2 micrometers. The confocal imaging approach provides a marginal improvement in both axial and lateral resolution, but it is the ability of the instrument to exclude from the image the “out-of focus” flare that occurs in thick fluorescently labelled specimens, which has caused the recent explosion in popularity of the technique.
In confocal microscopy a pinhole is used in the focal plane both at illumination and at detection.
In this way out of focus emitted light is effectively rejected by the detection pinhole and an increased resolution is obtained .
How does a TEM (transmission electron microscope) work?
• Gun emits electrons
• Electric field accelerates
• Magnetic (and electric) field control path of electrons
• Electron wavelength @ 200KeV = 2x10-12 m
• Resolution normally achievable @ 200KeV = 2 x 10-10 m = 2Å
• High Vacuum
The sample will appear dark. Shadowing technique.
Very thin sample required
How is the wavelength of electrons determined?
By accelerating voltage (V) on the filament from which they were emitted.
Wavelength = 0.1*(150/V)^0.5
Very high voltages (~100kV) required.
Why is high vacuum needed in TEM?
For transmission electron microscopy, vacuum needed as the mean free path of electrons is very short in air.
High vacuum of 10^-5 mbar aimed for.
Also
- tungsten filaments burn out in air
- columns must be kept dust free
The vacuum is achieved via pumps
What is SEM (scanning electron microscopy) used for?
• Topography and morphology
• Chemistry
• Crystallography
• Orientation of grains
• In-situ experiments:
– Reactions with atmosphere
– Effects of temperature
In brief: we shoot high-energy electrons and analyze the outcoming electrons/x-rays
• A SEM typically has orders of magnitude better depth of focus than a optical microscope making SEM suitable for studying rough surfaces
• The higher magnification, the lower depth of focus
What are issues with SEM?
Needs to be conducted in vacuum.
Samples need to be dry.
If electrons get absorbed by the sample, the surface becomes negative and repels further electrons. So, the sample should be coated with a conductor e.g. gold.
What is STM?
How does it work?
Scanning tunnelling microscopy
A scanning tunnelling microscope (STM) is an instrument for imaging surfaces at the atomic level.
The STM is based on the concept of quantum tunnelling. When a conducting tip is brought very near to the surface to be examined,
a voltage difference applied between the two can allow electrons to tunnel through the vacuum between them. The resulting tunnelling current is a function of tip position, applied voltage, Information is acquired by monitoring the current as the tip’s position scans across the surface, and is usually displayed in image form.
STM can be a challenging technique, as it requires extremely clean and stable surfaces, sharp tips, excellent vibration control, and sophisticated electronics.
STM requires a conducting material to tunnel.
What is AFM?
How does it work?
Atomic force microscopy
AFM consists of scanning a sample with a probe mounted on a cantilever and determining the surface topography from the deflection of laser light on the probe with a position sensitive detector.
The tip (or the sample) is positioned using an extremely precise piezo-electric unit, reaching Å resolution (in x,y,z).
The probe-sample interactions, which flex the cantilever, are repulsive at short distances. The tip can either be in permanent contact with the sample – contact mode –, or oscillating at its resonance frequency (~100 kHz), tapping briefly the sample – tapping mode.
Further, a feedback mechanism keeps the oscillating amplitude constant.
What are issues with AFM (atomic force microscopy)?
As the sharp point moves along the surface of the sample, if the sample is soft, the point may cause the sample surface to deform.
AFM may then detect what is under the surface instead of the surface itself.
What are 3 common types of scattering techniques to identify particle size?
Light scattering
X-ray scattering
Neutron scattering
What are the issues with scattering?
You can’t identify the structure inherently from the scatter / crystal pattern. Estimates and guesses must be made.
What are the 4 outcomes of shining a light on a substance?
Reflection
Transmission (passing through)
Absorption
Scattering
What is the key law of reflection?
Angle of incidence = angle of reflectiom
What is snells law?
Snells law calculates n, the refractive index
Sin (i) / Sin (r) = n
Where I and r are angle if incidence and refraction
What does the Beer-Lambert law for absorption show?
The Beer-Lambert Law (also called Beer’s Law) is a relationship between the attenuation of light through a substance and the properties of that substance.
A = ƐlC
A - absorbance
Ɛ - molar absorptivity
l - length of light path
c - concentration
What is constructive and destructive interference?
Constructive - waves in phase amplify each each other
Destructive - waves out of phase cancel each other out
What is the condition for constructive interference?
n * λ= 2 d sin (Ø /2)
Bragg equation
Where:
λ - wavelength
d - spacing between crystal lattice planes