Physical and Chemical Characterization of Biomaterials
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What is the photoelectric effect?
Electrons are emitted from matter (metals and non-metallic solids) as a consequence of their absorption of energy from electromagnetic radiation of very short wavelength, such as visible or ultraviolet light.
What are the three possibilities when an atom is impinged by a photon?
- Transverse through without interaction
- Scattering of the photon by the atomic orbital electrons resulting in partial energy loss
- Photon may interact with atomic orbital electron with total and complete transfer of photon energy to the electron, resulting in electron emission (photoelectric effect)
(T/F) A core electron can be removed from its core-level upon absorption of electromagnetic radiation (X-ray) and excited to an epty outer shell or emitted as photoelectron.
True. This is the photoelectric effect where the electron leaves from the core level rather than the valence electron.
What is emitted when an atom absorbes electromagnetic radiation (such as X-ray) in the case of the photoelectric effect?
A photoelectron, photoemitted electron, or photoionized electron from the core level of the atom (in contrast to the valence level or fermi level)
What is depth that XPS may analyze? What is one method that this may be controlled?
The depth that it may analyze is 100A (angstroms) and the depth may be controlled by tilting the specimen with respect to the detector.
What does XPS stand for?
X-ray photoelectron spectroscopy
What information may be obtained from XPS?
- All elements present except H and He.
- The amount of each element (+/- 5%)
- Molecular environment or oxidation state
- Non-destructive depth profile (100A)
- Shake-up / shake-off information
- Multiplet effect
- Surface electrical properties (charging, valence band)
What is the advantage of XPS?
- Minimal surface preparation (100A)
- Ability to work with hydrated (frozen) samples
- One experiment yields large number of information
- High surface sensitivity
- Theoretical basis well understood
What are the 5 levels of information from XPS spectra?
- Qualitative analysis - Wide scans, survey scans
- Elemental ratios - species identification, elemental ratios
- Subpeak resolution - species identification
- Shake up satellites (not with a monochrometer)
- Valence band - only do with metals
What are the secondary effects of XPS?
This is when secondary electrons leave the sample in which the deshielding of a carbon from a more electronegative atom.
What types of peaks are fit for XPS?
Narrow, detail or HR spectra in with intensities at various kinetic energies that may be fit with various peaks depending on the elements present.
What is used for charge compensation for insulators (materials that are nonconductive)?
A wire mesh may be placed about the sample and the use of a low energy flood gun to prevent positive charge building up due to the electrons leaving.
What would be observed in the case that you are looking at a non-conductive material without a charge compensator?
The material would become so "postive" due to the loss of electrons that the material will most likely not be observed. The electrons in a postive charge may be mislead or may not escape to the detector.
What is the zone of measurement for XPS?
- Composition varies with depth
- lateral variations of composition
- Inelastic mean free path (IMFP)
What is the accuracy of XPS?
What are some helpful factors that would allow for easier analysis by XPS?
- Pre-knowledge of specimen
- Ability to detect roughness, lateral and verical inhomogeneity
- Relative comparisons
How is depth profiling done with XPS?
Tilting the specimen relative to the dector and building the profile based on the angle and the gathered information.
What are some disadvantages of XPS?
- requires vacuum
- 100A depth
- charge compensation required for insulators
What is an example for the use of XPS?
Examination of protein level after SAM surface modification present after cell adhesion. (this includes basic-FGF)
Certain molecules such as nitrogen will be present in the case that a layer of proteins are placed on the surface. This would be displayed in the C1s region.
The bulk material could be silicon oxide with the SAM surface modiciation attached to silicon.
What region would show fluorine character diminishing with protein deposition?
Higher BE features would be present to (CF)x in the higher binding energy of the spectrum. In the case that (CF)x is not present, this would not show up in the spectrum.
What signals are attenuated by protein layers in XPS and what signals tend to be evident?
F and Si signals would be attenuated by protein layer and N signal would be evident. Also the C signal would be increased due to protein layer.
What difference may be observed with FGF on the surface two different surfaces that cells were trying to be grown on?
The cells that had FGF believed that they had a better environment and did not try to deposit very much protein on the surface (overexcessive protein deposition may lead to cell death in an attempt to make the environment suitable).
What method does scanning electron microscopy use for analysis?
- It uses an electron beam to raster across a surface in a vacuum.
- Look at signal arising from backscattered electrons for contrast differences
- Topography data and some elemental composition
- Spot size of 10 to 150A
- Electron excitation energy of 5-30 kV
- Insulating samples can be analyzed by coating with a thin (~50) layer of Au - especially important for biological samples
What is environmental scanning electron microscopy (ESEM)?
It is a scanning electron microscope that allows for the option of collecting electron micrographs of specimens that are "wet" uncoated" or both by allowing for a gaseous environment in the speciment chamber (compared to a vacuum).
What are the conditions for envionrmental scanning electron microscopy (ESEM)?
- Natural conditions (may look at live specimens)
- Temp -200C to 1000C
- Gas environment
- Moisture content up to 100% humidity
- Resolution down to 20 - 100A (not as good as SEM)
- Operate at 0.2-1 kV to lessen beam damage
- Operation in gas or humid environment allows non-conducting samples to be examined without Au coating.
What are some examples of the use of environmental scanning electron microscopy?
- Examine dynamic physio-chemical properties such as swelling, disintegration and dissolution of drugs to understand the mechanism of drug release
- Study the effects of wetting and drying on synthetic fibers
- Observe fresh and wet tissue such as muscle tissue and fibers which connect them
- Examine live plant seedlings, pollen grains in situ, ants, fleas and dust mites
- Investigate the degradation process of environmental pollutants on materials used by painters, sculptors and architects.
What method does transmission electron microscopy use?
- Thin sections of a material are examined under high energy (100kwV electron energy beam)
- Resolution can be 1000 to 5A where lattice spacings can also be determined
- Generally samples are <300A in thickness.
Describe the confocal microscopy
- Depth of field analysis by focal plane illumination and light collection
- Problem with other optical methods - no depth resolutions due to scattering from other tissue
- epi - fluorescence can be monitored - natural fluorescence
- Can tag with fluorescently labeled antibodies to locate and image structures deep in tissues or cells down to 20-25 nm
- Resolution of <1um
- Can reassemble planes to give 3D representation of a sample
- Laser illumination in latest instrumentation.
(T?F) Confocal microscopy will have areas with infocus and out of focus areas.
- Confocal micrscopy takes several images on different focal lengths and combines them to give a crisp three dimensional image
What is the function of the pinhole in confocal microscopy?
The pinhole does not allow for light that not in focus at the point for the pinhole. This blocks out light that would be out of focus.
What is a disadvantage of confocal microscope?
- Slow (~1 sec to acquire image)
- Scans excitation spot point by point to build up image
- Low light efficiency (due to use of PMT as dtector)
- Solution: use multiple pinholes and a camera (spinning disk confocal)
What is spinning disk confocal?
A spinning disk used many pinholes at once for fast images and can scan many points at once.
What is multiphoton fluorescence microscopy?
- The use of multi (or two) photon combinations of longer wavelength to excite shorter wavelength fluorescence.
- This does not excite out of focus light so the pinhole is not necessary (the out of focus light is too dim to be registered)
- Excites area with the use of two light sources on the focused area and prevents cell death by us of IR rather than visible in some cases
- LDramatically reduces photobleaching of labeled fluorophores (good for living cells) short high powered laser pulses
- Highest concentration of photons at focus point as well as short pulses gives very low probability of excitation of other points in the specimen
- Integrated with conventional confocal optics
- Three photon excitation can give even better z-direction resolution
What is static secondary ion mass spectrometry?
- Mass spectrum of surface region (detection of charged secondary particles)
- Fragments characteristic of surface structure
- Total ion dose <10^13 ions/cm
- ~20A sampling depth
- This occurs due to collision cascades from the primary ion beam.
- Ultra-high vacuum (10^-10 torr) is required
- Ion source such as inert gas (Ar+, Ne+, Xe+) , liquid metal (Ga+, In+), cesium (Cs+), polyatomic
- Analyzer (Quadrupole), (Electrostatic of magnetic sector), or (time of flight)
- Detector - multi-channel plate
- Neutralizer (electron flood gun)
What are the three analyzers for SIMS?
- Time of Flight
- Magnetic Sector
What are the two modes of SIMS?
- Static and Dynamic.
- Static focuses on the surface of the specimen and does not destroy as much material as dynamic which may be used to obtain information through layers.
(T/F) Static SIMS and Dynamic SIMS both are able to analyze samples <20A in depth.
False. Only static may. Dynamic is much too destructive.
(T/F) Both static and dynamic SIMS are useful for organic samples.
False. Only static is useful.
(T/F) Both static and dynamic SIMS are useful for observation of high mass fragments.
False. Only static is useful for observation of high mass fragments.
(T/F) Both static and dynamic SIMS are useful for molecular structure information.
False. Only static is useful for that information.
(T/F) Both static and dynamic SIMS are useful for depth profiling to 1 micron.
False. Only static is useful.
(T/F) Both static and dynamic SIMS are useful for element identification.
(T/F) Both static and dynamic SIMS are useful for extremely low detection limits.
(T/F) Both static and dynamic SIMS are useful for high spatial resolution (<1 micron).
(T/F) Both static and dynamic SIMS are useful for semiquantitative analysis.
(T/F) Both static and dynamic SIMS are useful for inorganics.
(T/F) Both static and dynamic SIMS are useful for powders, films, fibers etc.
What are the key capabilities of SIMS?
- Detection sensitivity (ppm or lower) (<10ng/cm^2 of protein)
- High mass resolution (exact mass determination) (isotope identification)
- Chemical identification (atomic, molecular and fragment species)
- Extreme surface sensitivity (<20A sampling depth)
- Semi-quantitative analysis
- Depth profiling
- Imaging (1 micron spatial resolution)
What is scanning tunneling microscopy? What substrate do you require?
Monitors tunneling current from a sharp tip but needs a conducting substrate.
What is atomic force microscopy? What substrates do you require?
measures deflections of a tip mounted on a cantilever. It may be done on both insulating and conducting surfaces.
What came up with the Scanning Tunneling Microscopy and Atomic Foce Microscopy?
Binnig and coworkers. Colton assisted with AFM.
What type of sensors may be used for AFM?
piezo electric sensor or laser with photodiode.
(T/F) A sharp silicon nitride tip attached to a microfabricated cantilever is used for AFM probes.
What are AFM probes typically constructed of?
A sharp silicon nitride tip attached to microfabricated cantilever.
What are the typical tip dimensions of AFM probes?
Tip dimensions 10 - 100 nm radius.
What is the normal spring constant of the cantilever?
0.01 to 100 N/m
What is the principle of operation for AFM?
- A small sharp tip is attached to the end of compliant cantilever.
- The tip is brought into close proximity with a sample.
- Forces acting between AFM tip and the sample will result in deflections of the cantilever.
- The deflection of the cantilever from its equilibrium position is proportional to the normal load applied to the tip by the cantilever
- Lateral forces result in a twisting of the cantilever from its equilibrium position.
(T/F) The normal forces experiences in AFM is a function of the normal deflection of the cantilever.
True. Small differences in the photocurrent between the upper and power pairs of diodes (A-B) will be proportional to the slope of the lever in the x-Z plane at the point of reflection X-laser.
(T/F) The lateral forces experienced in AFM is a function of friction's effect on the lateral deflection of the cantilever.
True. This is all due to friction on the surface of the sample causing twisting of the cantilever.
Differences in photocurrent between the left and right pairs of diodes (C-D) is proportional to the slope of the lever twist in the y-z plane at the point of reflection X-laser.
What are three types of image contrasts that may be obtained via AFM?
- Lateral foces
(T/F) AFM may be used to measure single cell adhesion and mechanisms for cell elasticity.
True. It may also be used to measure things such as breaking force between protein's secondary structure.
(T/F) AFM may be used to record time series of mechanical beating of a single cell.
True. Pulse mapping on active cells is possible.
What is the "tappingmode" of AFM suppose to prevent?
Dragging/damaging of the cell speciment by periodically lifting the cantilever
(T/F) AFM may be used to measure forces between complementary strands of DNA, forces between single molecules, and forces in the secondary structure of proteins.
What is ellipsometry?
- The use of optical properties of thin films to measure the thickness of the film and is extremely surface sensitive.
- Can measure from angstrom thickness to several wavelengths of thickness
- Any transparent media is useable
- No labeling is necessary and requires little sample preparation
Describe what happens to the light in ellipsometry
- Light are vibrations in all planes along the the propagation axis
- Polarized light vibrates only in the plane of the polarization
- Polarization parallel (p) to the plane of incidence is differentiated from polarization perpendicular (senkrecht S) to plane of incidence.
What are the states of polarization in ellipsometry?
- Figures traced as a function of time
- Elliptical is traced in a clockwise or counterclockwise sense, as viewed in the direction of propagation
- Circular: ellipse axis are equal
- Linear: Minor axis of the ellipse is zero
What is the plane of incidence in ellipsometry?
Plane formed by the impinging and reflected beam, perpendicular to a surface
WWhat is atomic emission?
- Emission of light from excited atoms in a flame.
- Normally Na+, K+, Ca++
- Li+ is internal standard
- Only 1% efficient
What is atomic adsorption?
- Adsorption of light by atoms in a flame.
- Hollow cathode lamp as source
- Can monitor Ca, Pb, Cu, Zn, Fe, and Mg
What is fluorometry?
- Detection of fluorescent emissions
- Need separate emission and exictation lines
- Hg lamp source (and others)
- Higher sensitivity than Atomic emission/adsorption.
- Generally for molecular species but pH sensitive.
What is chromatology?
- Separation of species using a solid or liquid stationary phase and liquid or gas mobile phase.
- Detection - flame ionization, thermastors etc
- Gas Chromatography (GC)
- HPLC - High pressure liquid chromatography
What is electrophoresis?
- Movement of ionic species under influence of an applied potential.
- Normally in a gel (agar or polyacrylamide usually)
- Measures components in protein in plasmas, urine, CSF as well as antibodies, isoenzymes, metabolites and other biological materials
What is automated differential counts?
- Pattern recognition.
- Nuclear morphology, cytoplasmic morphology, nuclear/cytoplasmic ratio, chromatic pattern and cytoplasm characteristics.
What is hematology? What does it include?
- Ratio of different cells in blood as well as absolute cell count.
- Includes Hematocrit (HCT) - ratio of all blood components to sample volume
- Hemoglobin - cyanmthemoglobin detection
- WBC and RBC
- -Coulter counter size separation
- -Flow cytometry - single stream of cells and measured by light scatter or other light technique (fluorescent labeling)
Who received the Nobel Prize for Polmerase Chain Reaction (PCR)?
KKary Mullis - 1993 Chemistry
What is the basic principle of PCR?
- Elevated temperature to melt doublestranded DNA.
- DNA polymerase - enzyme for polymerizing bases
- Enzyme will recognize certain "ques" or primers"
- Specific DNA strand or "primer" to recognize certain sequences at both ends of the DNA of interest
- Comes at it from different ends, repeats 25x and eventually have pure amplified DNA strand
Where does the polymerase that is used in PCR orignate from?
- Thermophile bacteria - Taq for elevated temperatures.
- Don't have to replace enzyme but errors occur
- Pwo or Pfu from archea bacteria - has internal proofreading mechanism to reduce errors.
What are some applications of PCR?
- Simple amplification as tool
- DNA fingerprinting
- Site- directed mutagenisis
- DNA sequencing
- Paternity testing
- SNPs for detection for hereditary diseases or predisposition to disease
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