17 Physical methods in biology and medicine Flashcards
List the major signals being detected in a flow cytometer! Give the information provided by them.
Forward angle light scatter: size, index of refraction; Side scatter: intracellular structure/complexity; Fluorescence signal(s): the size/value of the cellular parameter for which the fluorescence probe was used for (e.g. protein expression, DNA content, membrane potential etc.).
How can a flow cytometric histogram be constructed?
Signal intensity (or the value of the respective cellular parameter) is plotted on the horizontal axis, whereas the vertical axis shows the number of cells possessing the given signal intensity
What is the dot plot and how can it be constructed?
Dot plot is a type of two-parameter data visualization, where the two parameters are plotted in a correlated fashion. Each cell is represented by a dot in the coordinate system with coordinate values corresponding to the parameters plotted on the X- and Y-axes, respectively.
What does gating mean in flow cytometry?
Gating is the restriction of flow cytometric acquisition and/or data analysis to a subset of cells having desired parameters. The cell population of interest can be defined on the basis of either one or two-dimensional plots.
How does flow cytometric cell sorting take place?
The fluid stream containing the cells is illuminated and the fluorescence/scatter signal is detected. The nozzle of the flow cytometer is shaken by a piezoelectric crystal so that the fluid stream is broken into droplets at a given distance from the place of illumination. The surface of a droplet is charged electrically depending on the light scatter and/or fluorescence signal of the cell inside the droplet.. The charged droplets pass through an electric field, where they are deflected and therefore can be collected in separate test tubes.
How does flow cytometric cell sorting take place?
The fluid stream containing the cells is illuminated and the fluorescence/scatter signal is detected. The nozzle of the flow cytometer is shaken by a piezoelectric crystal so that the fluid stream is broken into droplets at a given distance from the place of illumination. The surface of a droplet is charged electrically depending on the light scatter and/or fluorescence signal of the cell inside the droplet.. The charged droplets pass through an electric field, where they are deflected and therefore can be collected in separate test tubes.
List the advantages of flow cytometry and flow cytometric cell sorting (FACS)!
- information can be obtained for a given sample on a cell- by-cell basis;
- multiple signals can be detected simultaneously for a given cell;
- numerous cells can be studied within a short time;
- homogeneity or heterogeneity of a cell population can be
revealed; - cells can be separated on the basis of their size and
other characteristics that can be labeled fluorescently.
List the advantages of flow cytometry and flow cytometric cell sorting (FACS)!
- information can be obtained for a given sample on a cell- by-cell basis;
- multiple signals can be detected simultaneously for a given cell;
- numerous cells can be studied within a short time;
- homogeneity or heterogeneity of a cell population can be
revealed; - cells can be separated on the basis of their size and
other characteristics that can be labeled fluorescently.
What is the operating principle of a confocal laser scanning microscope?
A pinhole with a small diameter blocks the way of light beams originating from out of focal planes. This way the image of the object will be sharp. The whole process is extended to the whole plane by scanning.
What is the resolution limit of atomic force microscopy?
Angström (0.1 nm)
Explain the aim and principle of mass spectrometry.
The aim of mass spectrometry is to determine the charge-to-mass ratio of particles. The ionized form of the investigated substance should be introduced to the gaseous phase and then accelerated in an electric field. Finally, the charge-to-mass ratio is determined by different methods.
Explain the aim and principle of mass spectrometry.
The aim of mass spectrometry is to determine the charge-to-mass ratio of particles. The ionized form of the investigated substance should be introduced to the gaseous phase and then accelerated in an electric field. Finally, the charge-to-mass ratio is determined by different methods.
List forces and their directions acting on a molecule sedimenting in a centrifuge tube!
Centrifugal force pointing away from the axis of rotation, and both frictional force and buoyant force pointing towards the axis of rotation.
Define the sedimentation constant and give its unit!
The sedimentation constant is the sedimentation velocity of a molecule achieved at unit acceleration, that is the sedimentation velocity divided by the centripetal acceleration. Unit: 1 Svedberg (S)=10-13 sec
How can the density of an unknown macromolecule be determined by sedimentation experiments?
Macromolecules centrifuged in a density gradient (e.g. cesium chloride) stop sedimenting in the layer whose density is identical to their own.
