Fluorescence Spectroscopy and Radiation Scattering Photometry Flashcards
State the difference between incandescence and luminescence
Incandescence - heated objects that emit EM radiation.
Luminescence - Light is emitted that is not due to heat. (photoluminescence is a form of luminescence)
Describe photoluminescence at the atomic level
Certain materials are able to absorb EM radiation. This is possible because electrons within that material are able to absorb specific energy levels to bring them from the ground state to an excited state. When light hits the electrons if it is the right energy level (wavelength) it will absorb the light, if it is an energy level that is too high or too low the electron will not absorb the energy.
Electrons are unstable in their excited state, so they will eventually move back to their ground state, by emitting energy in the form of visible light.
Differentiate fluorescence and phosphorescence
Both are examples of photoluminescence.
Fluorescence - Materials that are fluorescent only emit light in the presence of excitation radiation. The absorption and emission of light happen rapidly.
Phosphorescent - Materials that continue to emit light after the excitation radiation is removed (glow in the dark). Compared to fluorescent materials, phosphorescent materials will emit light for a long period of time after the excitation source is removed.
Describe the chemical structure of fluorophores
Fluorophores are substances that exhibit photoluminescence. They typically have aromatic rings, and at least one electron-donating group attached to the aromatic ring.
excitation wavelength
Excitation wavelength: Wavelengths that hit the fluorophore and allow the electrons to move to an excited state.
Emission wavelength
Wavelengths that are emitted from the fluorophore as electrons move back to their ground state. Radiation emitted is proportional to the concentration of the analyte, length of the path, and intensity of the excitation energy.
Define Stokes shift
The energy difference between an excited electron, and an electron at the ground state. Emitted photons have less energy than absorbed photons.
Define Photoluminescence
Luminescence occurs when a substance absorbs EM radiation and re-emits energy in the form of visible light.
List the main components of a fluorometer
Light source - emits the excitation light, therefore must be able to emit high-intensity radiation. Often mercury or xenon arc lamps are used. LASERs (flow cytometry) and LEDs can also be seen.
Wavelength selector - filters (fluorometers) or monochromators (spectrofluorometers) are seen. Two selectors will be used, an excitation (primary) selector before the sample, and an emission (secondary) selector after the sample.
Sample cell - cuvettes are commonly used and must be made of a material that is clear on all sides and does not fluoresce.
Detector - PMTs placed at a 90-degree angle.
List commonly used light sources found in fluorometers
Xenon Arc Lamp
Mercury Lamp
State the function of the excitation/emission filters and describe their placement with respect to the other components of a fluorometer
Primary wavelength selectors are placed in between the light and sample cell. They select the wavelength that excites the fluorophore.
Secondary wavelength selectors are placed between the sample cell and the photon detector. They select the wavelength that is detected.
Discuss the function of the sample cell (cuvette)
To hold the sample - must not interfere with the emitted light.
State the best photon detector for fluorescence instrumentation
PMT (photomultiplier tubes) are used as the light emitted off a sample may be very low, and PMTs are the most sensitive detectors.
Discuss the placement of the photon detector - what angle?
90 degrees - reduces the detection of stray light.
Discuss the following fluorescence sources of error:
Matrix effects
Background fluorescence that is caused by compounds in the sample that fluoresce that are not the analyte being tested.
Discuss the following fluorescence sources of error:
Proteins
Proteins in plasma are major contributors to background fluorescence. Most have an excitation wavelength of 260-290nm. Using a longer wavelength can minimize background fluorescence.
Discuss the following fluorescence sources of error:
Lipemia
Scatters light, but does not fluoresce. This scattered light is a problem when the analyte has a small stokes shift because the excitation light will be detected and there will be a loss of specificity. Narrow bandpass filters can minimize this effect.
Discuss the following fluorescence sources of error:
Stray light
Light other than the emission light that hits the detector. Falsely increases patient sample concentrations.
State the light source found in a fluorescence microscope
Xenon arc or mercury lamp
Discuss the purpose of the dichroic mirror in a fluorescence microscope
Reflects certain excitation wavelengths and transmits other emission wavelengths.
Discuss how an automated blood culture system detects bacterial growth
A baseline fluorescence measurement is taken.
Fluorescence is then measured at regular intervals.
A microprocessor monitors the rate of change in fluorescence - determines if there is bacterial growth in the bottle.
A tech is signalled that there is bacterial growth.
Name the gas that is produced via bacterial metabolism
CO2
Describe the relationship between fluorescence and pH
As pH decreases, fluorescence increases.
Define the acronym FRET
Forster Resonance Energy Transfer
Explain the term “radiationless energy transfer”
Excitation energy is absorbed by the first fluorophore and transferred to a second fluorophore without the emission of light. The second fluorophore will emit light.
Describe the FRET process
Two fluorophores (donor and acceptor) must be in close proximity and have overlapping emission spectrums.
Excitation energy is absorbed by the first fluorophore and transferred to a second fluorophore without the emission of light.
The second fluorophore will emit light.
Detection is based on recording the fluorescence signal of the acceptor or quenching (loss) of the donor signal.
List the conditions required for FRET
Donor and acceptor fluorophores must have overlapping emission spectrums.
Discuss applications of FRET
Studying biological phenomenon that produces changes in molecular proximity (protein folding, the structure of nucleic acids, protein interactions.
Detection of protease activity
Detection of DNA hybridization
Describe how light scatter acts as a source of error in absorbance-based methods
Light scatter in absorbance-based methods increases %T, decreasing absorbance, and therefore decreasing patient results.
Discuss the relationship between wavelength of incident light, particle size, and the type of scatter produced
Rayleigh scatter is produced when the diameter of a particle is smaller than the wavelength of light hitting it. Light is mostly scattered towards the front and back of the particle symmetrically.
The intensity of scattered light is proportional to the particle size, and inversely proportional to the wavelength of light - shorter wavelengths are scattered more.
Mie scatter is produced when the diameter of a particle is larger than the wavelength of the light hitting it. Mostly forward scatter is produced. Bacteria and blood cells produce Mie scatter.
All wavelengths are scattered equally.
Define turbidance
The measurement of decreased incident radiation as it passes through a solution containing particles. %T is measured.
Increased turbidity = increased scatter = decrease %T
Measurements are usually made on particles that are large enough to produce Mie scatter.
Discuss the components of light scattering instrumentation
Light source (visible)
Collimating lens
Wavelength selector
Cuvette
Photodetector
Describe the main difference between a turbidimeter and a nephelometer
Detection of scattered light is at a fixed angle in nephelometry - Transmitted light is measured in turbidimetry.
Turbidimetry - Increased particle concentration = increased scatter = decreased %T = Increased sample concentration
Nephelometry - Increased particle concentration = increased scatter = increased light striking photon detector = increased sample concentration.
Discuss how the following sources of error will affect turbidimetric and nephelometric measurements: Matrix effects
Turbidimetry - False decrease in % T, False increase in the patient sample.
Nephelometry - Flase increase in a patient sample
Discuss how the following sources of error will affect turbidimetric and nephelometric measurements: Stray Light
Turbidimetry - False increase in %T, false decrease in the patient sample.
Nepelometry - Flase increase in a patient sample
List some applications which use the measurement of light scatter
Water testing
Antimicrobial susceptibility testing
Immunoassays
Antibody-antigen complexes (nephelometry)
Define flow cytometry
The analysis of cells based on light scatter. A flow cytometer is able to characterize a heterogeneous population of cells and identify specific subpopulations.
Cells are pushed through a tube single file, once in the tube, a LASER is shined at the cell and the amount of light scattered is measured.
Forward light scatter determines the cell’s size.
Side scatter determines the cell’s complexity.
Describe hydrodynamic focussing and discuss its importance
Forces the cell’s single file in the sheath fluid, when the LASER beam is shined onto it. This helps reduce cells being examined together.
List the two angles of light scatter that are typically measured by a flow cytometer and discuss what information is determined by each of these measurements
Forward light scatter determines the cell’s size.
Side scatter determines the cell’s complexity.
