Physical Methods in Biological Research and Medical Practice

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46. List the forces acting on a sedimenting molecule in a centrifuge tube!: Centrifugal force, buoyant force and frictional force.
47. Define the sedimentation constant and give its unit!: The sedimentation constant is the sedimentation velocity
of a molecule per unit acceleration, that is the
sedimentation velocity divided by the centripetal
acceleration. Unit: 1 Svedberg (S)=10^-13 sec
48. 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.
49. How does the sedimentation equilibrium depend on the form factor in the case of sedimentation equilibrium method and why?: It is independent because after reaching equilibrium
molecules stop moving.
50. What is electroforetic mobility?: Electroforetic mobility is the velocity generated by unit
electric field strength.
51. List the factors influencing the electroforetic mobility of a macromolecule!: - molecular mass
- net charge, pH of the medium
- form factor.
52. What is the principle of isoelectric focusing?: During electrophoresis in a pH gradient each compound
migrates towards the region where the pH is equal to
that of its isoelectric point (where the net charge of the
particle is zero) and does not migrate any further.
53. What is the condition of resonance absorption in NMR?: E2 - E1 = hf = g μM H , where:

E2 - E1: the energy difference between the excited and
ground state of the nuclear spin if the magnetic field
strength equals H,

h : Planck's constant
f : frequency of the applied electromagnetic radiation
g : proportionality factor (Landé factor)
μM: magnetic moment of the nucleus.
54. Why is the Bohr magneton about 2000 times greater than the nuclear magneton?: Because the magneton is inversely proportional to the
mass of the corresponding elemtentary particle, and the
rest mass of a proton is about 2000 times greater than
that of an electron.
55. What region of the electromagnetic spectrum can be used to excite nuclear and electron spins placed in a magnetic field?: Radiowaves ( âˆ¼108 Hz): NMR
Microwaves ( âˆ¼1010 Hz): ESR
56. What is the Bohr magneton?: The intrinsic magnetic moment of the electron.

μB = eh/2me
where
e - charge of the electron,
me - mass of the electron
h = h/(2Ϭ) (h is Planck's constant).
57. What kind of factors influence the resonance frequency in NMR?: Quality of the absorbing nucleus, its chemical
environment and the strength of the external magnetic
field.
58. How can the relative concentration of absorbing nuclei be determined from an NMR spectrum?: From the area under the absorption lines corresponding
to different nuclei.
59. What is chemical shift in NMR?: The local magnetic field around a given nucleus is
altered by the chemical environment resulting in a
change in its original resonance frequency.
60. Which nuclei are able to give an NMR signal?: Whose resultant nuclear spin is different from zero.
61. List at least three nuclei which can be used for NMR investigation of biological macromolecules!: 1H, 13C, 14N, 19F, 31P
62. What kind of parameters does an MRI image carry information about?: About the density of 1H nuclei in a volume unit (voxel)
and about their spin-spin and spin-lattice relaxation
rates.
63. How can we get ESR signal from a protein sample?: The protein must be coupled to a stable radical
containing an unpaired electron (spin label).
64. What kind of special characteristics does laser light have?: - monochromatic
- big coherence time and distance
- small divergence
- high light density.
65. When is electromagnetic radiation coherent?: If it consists of photons capable of forming observable
interference fringes.
66. What is the definition of electron spin?: The electron's intrinsic angular moment, which is
independent of environmental factors, is called spin.
67. What basic phenomena is a laser based on?: - population inversion
- stimulated emission.
68. What is the approximate coherence length of a laser and that of a classical light source?: 10^10 cm and a couple of cm, respectively
69. What extra information does a hologram contain compared to a normal photo?: A hologram records not only the intensity, but the phase
of light, as well, and a three-dimensional image can be
constructed from this.
70. What do we experience if we use only a part of a hologram containing an image of an object for reconstruction?: The whole object is visible, but with less spatial
resolution.
71. Give the condition of amplification for an electromagnetic wave with wavelength λ diffracted on a crystal with a grating constant of d! (angle of incidence is 90o): d cos α=kλ , where k=0,1,2,3,...n, α=angle of diffraction
72. How can the overdetermination of the Laue equations be resolved in the case of a three dimensional crystal?: Either by rotating the crystal or making powder of it.
74. In what respect does a CT image contain more information than a conventional x-ray image?: A conventional x-ray image contains only the projection
of the x-ray absorbing material, while a CT image also
reveals the third dimension (depth) of the object.
75. Write the Lambert-Beer law and interpret the variables in the formula!: lg Io/I=(ε)(c)(L)=E

I - intensity of light after passing through a material with
thickness L
Io- initial intensity of light when it enters the sample
E - extinction (optical density or absorption)
ε - molar extinction coefficient
c - concentration in mol/liter
L - optical pathlength.
76. What does the molar absorption coefficient depend on?: It depends on the type of the absorbing material, the
wavelength of the light, temperature, the type of the
solvent and the environment.
77. How many fold does the intensity of light decreases if the optical density (absorption, extinction) of a solution is 1?: It decreases 10-fold.
78. What is the definition of the molar absorbtion coefficient?: It is the optical density of a solution with a concentration
of 1M and an optical path length of 1 cm
79. At what wavelength are the characteristic absorption maxima of proteins and nucleic acids?: proteins 280 nm, nucleic acids 260 nm
80. Which amino acids have reasonably high absorption?: Tyr, Trp, Phe
81. What is the definition of a singlet and a triplet state?: In a singlet and a triplet state the number of unpaired
electrons is zero and two, respectively. In a singlet and a
triplet state, the value of the resultant spin multiplicity is
1 and 3, respectively.
82. What are the possible ways of relaxation of an excited electron in a molecule? (List at least 5 of them!): - vibrational relaxation
- internal conversion
- intersystem crossing
- fluorescence
- phosphorescence
- delayed fluorescence
- energy transfer to another molecule.
83. What is the definition of fluorescence lifetime?: The time during which the number of excited molecules
decreases to 1/e-times (37 %) its initial value.
84. What is a., scintillation, b., chemiluminescence, c., photoluminescence?: Processes where photon emission is elicited by
a., radioactive radiation
b., chemical reaction
c., excitation by photons.
85. How can fluorescence quantum yield be defined?: The number of emitted photons divided by the number of
absorbed photons or the rate constant of fluorescence
divided by the rate constants of all possible deexcitation
processes.
86. Why is the fluorescence quantum yield always smaller then one?: Because relaxation from the excited state can be
accomplished not only by fluorescence emission.
87. What is the lifetime range of fluorescence?: Ï„ = 10^-9 â€“ 10^-7s
88. What is the lifetime range of phosphorescence?: Ï„ = 10^-6 â€“ 10 s
89. Why is the lifetime of phosphorescence longer than the fluorescence lifetime?: Because phosphorescence is the result of a forbidden
transition, the Tâ†’S intersystem crossing.
90. What is Raman scattering?: Raman scattering is a non-elastic scattering in which the
frequency change of the scattered photon relative to the
incident one is determined by the vibrational energy
levels of the scattering molecule.
91. What are Stokes lines and anti-Stokes lines?: Lines in the spectrum of Raman scattering that have
smaller frequency (longer wavelength) and higher
frequency (shorter wavelength) than the original
frequency (wavelength) of the photon are called Stokes
and anti-Stokes lines, respectively.
92. Under what circumstances can Förster-type resonance energy transfer occur?: - The separation between the donor and the acceptor
has to be in the range of 2-10 nm
- there has to be an overlap between the emission
spectrum of the donor and the excitation spectrum of
the acceptor
- the relative orientations of the donor and the acceptor
have to be adequate.
93. Why is Förster type resonance energy transfer a sensitive method for distance measurements?: Because its probability is proportional to the inverse
sixth power of the separation between the donor and the
acceptor.
94. What can Förster-type resonance energy transfer be used for in biology?: For measuring inter- and intramolecular distances.
95. What is photoselection?: It is the selection of an oriented subpopulation from a
randomly oriented population of molecules by linearly
polarized light.
96. What is linearly polarized light?: Light in which the electric vectors of all photons point in
the same direction.
97. Describe the equation for the calculation of emission anisotropy!: A=IVV-IVH/Ivv+2IVH
A: emission anisotropy
IVV and IVH: fluorescent intensities measured in case of
parallel (vertical-vertical) and crossed (verticalhorizontal)
polarizator-analizator positions, respectively.
98. What are the advantages of flow cytometry and cell sorting over spectrofluorimetric measurements?: 1 -more information can be obtained separately about
individual cells at the same time
2 -numerous cells can be studied within a short time
3 -homogeneity or heterogeneity of a cell population can
be revealed
4 -cells can be separated on the basis of their size and
other characteristics that can be labeled fluorescently
99. Enumerate at least five parameters which can be determined measuring fluorescent intensities!: - DNA, RNA, protein and lipid content of a cell, or the
quantity of any kind of material that we tagged with a
fluorescent label.
- permeability of the cell membrane
- intracellular enzyme activities
- membrane potential
- intracellular calcium level
- intracellular pH
- presence and density of cell surface antigens and
receptors
- mitochondrial potential and the number of mitochondria
per cell.
100. What is the shortest resolvable distance in a light microscope?: approximately 200 nm
101. How can the resolving power of a microscope be increased?: - by decreasing the wavelength of light
- by increasing the index of refraction of the material
between the objective and the object
- by increasing the half angle of the objective
102. What is numerical aperture?: It is the product of the index of refraction of the material
between the object and the objective (n), and the sine of
the half angle of the objective (sin Ïˆ): n Â· sin Ïˆ.
103. Give the formula for the resolving power of a microscope!: f=1/d=2nsinÏ–/Î»

where: n = refractive index of the medium between the
coverslip and the objective, Ï‰ = half angle of the
objective, Î» = wavelength of light, d = the minimum
distance between two points at which they are
resolvable.
104. What is the purpose of the dichroic mirror in a fluorescence microscope?: It reflects the excitation light, and is transparent for the
emitted photons, therefore it separates the excitation
and emission light paths.
105. What is the purpose of the excitation filter in a fluorescence microscope?: It is transparent only in the wavelength range in which
the fluorescent dye can be excited, therefore it allows
only those photons to reach the sample which can excite
the fluorescent molecule.
106. What is the purpose of the emission filter in a fluorescence microscope?: It is transparent only in the wavelength range in which
the fluorescent dye emits photons, therefore only the
photons emitted by the fluorescent dye will reach the
detector.

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