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Lectures on Medical Biophysics
Department of Biophysics, Medical Faculty,
Masaryk University in Brno

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Biomolecular and Cellular Research Devices
Lectures on Medical Biophysics
Department of Biophysics, Medical Faculty,
Masaryk University in Brno

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Lecture Outline
•Biomolecular science – crucial importance for molecular medicine. We will deal with devices for
structural studies, concentration measurement (in-vitro and in-vivo), cell membrane studies
•Most common devices are based on the interactions of electromagnetic radiation with the
macromolecules
–VIS, UV and IR Spectrophotometers
–Raman spectrometers
–Circular dichroism based devices
–X-ray diffraction spectrometers
•Devices based on other properties of biomolecules (e.g., mechanical and electrical properties)
–Electrophoresis
•Cellular potentials and intra-cellular ion concentration devices

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We do not deal with….
•Devices for measurement of
–Osmolar concentration (measurement is based on cryoscopy),
–Diffusion
–Viscosity (practical exercises)
•Devices for determination of secondary and tertiary structure of proteins and nucleic acids based
in electrochemistry (interaction of macromolecules with electrodes is studied)
•Nuclear magnetic resonance (it allows to determine chemical binding of hydrogen atoms – mentioned
in lecture about MRI)
•Electron spin resonance,
•Centrifuges (other lecture) etc.

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Biophysics and Biomolecular Research
• This research is mainly oriented to structural studies which allow understanding of e.g.:
•
ØSpecificity of enzymatic and immunologic reactions
Ø
ØEffects of some pharmaceuticals (cytostatic drugs) at the molecular level.
Ø
ØMechanisms of passive and active transport processes
Ø
ØCellular motion
Ø
Ø……………..

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•Devices based on the interactions of electromagnetic radiation with the macromolecules

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Types of Spectrophotometers
ØSpectrophotometers are laboratory instruments used to study substances absorbing or emitting
infrared, visible and ultraviolet light, including studies of their chemical structure.
Ø
ØAbsorption spectrophotometers: based on the spectral dependence of light absorption.
Ø
ØEmission spectrophotometers: The light source is the analysed substance itself, which is injected
or sprayed into a colourless flame. The light emitted passes through an optical prism or grating so
that the whole emission spectrum can be obtained. The frequencies present in the spectrum enable to
identify e.g. present ions.
Ø
ØSpectrofluorimeters: light emission is evoked by light of a wavelength shorter than the wavelength
of emitted light.

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Absorption Spectrophotometers: Lambert-Beer's law
•Absorption spectrophotometry is based on the absorbance of  light after passing through a layer of
solution of a light absorbing substance. Its concentration can be found using the Lambert-Beer law:
•
•I = I0.10-ecx
•
•c solute concentration, x thickness of solution,  I0 original light intensity, I is the intensity
of light leaving the layer. The constant e (epsilon, absorption or extinction coefficient) depends
on the wavelength of light, solute and solvent. Its values for common chemical compounds can be
found in tables. These values are always given for a specified wavelength (usually the absorption
maximum). The numerical values of the coefficient depend on how the concentration of the dissolved
substance is expressed. When using mol.l-1, we speak of the molar absorption coefficient.

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•The ratio of transmitted and incident light intensities is called transmittance (transparency).
The log of reciprocal of the transmittance is called the absorbance A.
•Thus, the absorbance is directly proportional to the concentration of the solution and thickness
of the absorbing solution layer.
A = e.c.x

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Types of Absorption Spectrophotometers
ØAccording to their construction, spectrophotometers can be divided into single- and double-beam
types.
Ø
ØIn single-beam spectrophotometers one beam of light passes through the reference and then the
measured sample (the cuvettes containing the solutions must be movable). In double-beam
spectrophotometers one beam of light passes through the measured sample and the second through the
reference (or blank) sample. Double-beam instruments allow substantially faster measurements, but
they are more expensive. In simple instruments, the setting of wavelength is done manually. In more
sophisticated instruments, the setting is done automatically so that it is possible to record
directly absorption curves, i.e. plots of absorbance versus light wavelength in a given medium.

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spekol
Single-beam spectrophotometer
The light source (1) is a tungsten lamp. Its polychromatic light passes through a condensor (2)
and  reflects from a mirror (3) to the input slit (4) of the monochromator (parts 4 to 8, plus 12).
The light is collimated (5) onto a reflection optical grating (6) which forms a colour spectrum. An
almost monochromatic light is projected by an objective (7) onto the exit slit (8) of the
monochromator.

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spekol
The grating is rotated by means of a wavelength selection control (12) to choose wavelength
directed into the exit slit. The light beam passes through a cuvette (9) with the sample. Intensity
of the transmitted light is measured by a photodetector (10, 11). Signal from the detector is
amplified by an amplifier (13). The value of absorbance is displayed (14). Intensity of the light
transmitted through the reference solution is always compared with the intensity of the same beam
passed through the measured sample.
Single-beam spectrophotometer

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•Modern UV/VIS/NIR Spectrophotometer
uv spec_fig2%20diode%20array spec_fig1%20scanning
Light of one selected wavelength or also whole transmitted spectrum can be measured
NIR = near infrared

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UV Absorption spectrophotometry
ØThe ultraviolet (UV) light is absorbed by various compounds, namely by those having conjugate
double bonds. Both proteins and nucleic acids absorb strongly UV light, which can be used for their
investigation.
•
–The amino acids tryptophan and tyrosine have absorption maximum at about 280 nm. Phenylalanine at
255 nm.
–
–Nucleotides (nitrogen bases) have absorption maximum in the range of 260 - 270 nm.
–
–Chromophores – their absorption properties vary according to chemical composition of the medium.

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Absorption spectra of amino acids
•
According: http://www.gwdg.de/~pdittri/bilder/absorption.jpg
Wavelength [nm]

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Hypochromic Effect (HE)
ØAbsorption of light is influenced by dipole moments of chemical bonds which interact with photons.
Stochastically (randomly) oriented dipole moments (denatured protein) absorb light better than in
the state with ordered structure (helices). In proteins, the HE is derived from peptide bonds,
which have UV absorption maximum at about 190 nm.
ØThe double helix of DNA absorbs UV light less than when the molecule is denatured.
•
ØHelicity – percentage of ordered parts of the macromolecule

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Hypochromic effect in polyglutamic acid. At pH 7 this polypeptide forms random coil (1), at pH 4 it
adopts helical structure (2).  Absorption maximum of peptide bonds is lowered due to their spatial
arrangement. e is the molar absorption coefficient and l is wavelength of UV light. [according
Kalous and Pavlíček, 1980]

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IR Spectrophotometry
ØIR interacts with rotational and vibration states of molecules. Complex molecules can vibrate or
rotate in many different ways (modes). Various chemical groups (-CH3, -OH, -COOH, -NH2 etc.) have
specific vibration and rotation frequencies and thus absorb IR light of specific wavelength.
•
ØTherefore, infrared absorption spectra have many maxima. A change in chemical structure is
manifested as changes of the position of these maxima.

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IRspektrum CH2AsymStr CH2SymStr CH2ScissorBend CH3UmbrellaBend CH2Rock
Infrared transmittance spectrum of hexane http://www.columbia.edu/cu/chemistry/edison/IRTutor.html

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Raman spectrometry
ØRayleigh scattering of light. Interaction of photons with molecules can take place with no or very
little change of wavelength. The intensity of the scattered light depends on molecular weight and
also scattering angle which can be used for estimation of the macromolecule shape.
•
ØRaman spectrometry. In scattering of photons a small change of wavelength occurs (wavelength
shift), which is caused by a small decrease or increase of scattered photon energy during
transitions from original to changed vibration or rotational states of interacting molecules. These
states can change due to structural changes of molecules.
•
ØThus, changes in the Raman spectra (signal intensity vs. wavelength shift or wave number values)
reflect conformational changes of molecules.

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Raman spectrometry
Raman spectrum of giant chromosomes of a midge (Chironomus).
At selected wave number values it is possible to run Raman microscopy. Excited by 647.1 nm laser
light.
According to: http://www.ijvs.com/volume2/edition3/section4.htm
Wave number [cm-1]

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Otto6.tif (7235218 bytes)
Micrograph in normal white light
(chromosome Chironomus Thummi Thummi)
•
Confocal Raman micrograph showing DNA backbone (vibration at 1094 cm-1)
Confocal Raman micrograph showing the presence of aliphatic chains in proteins at 1449 cm-1
according: http://www.ijvs.com/ volume2/edition3/section4.htm
aaaHPA

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Optical rotation dispersion - optional
•In optical rotation dispersion method (ORD) we measure dependence of optical activity on the light
wavelength. This method was replaced by more sensitive method of circular dichroism (CD), which
gives similar information.

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Circular Dichroism (CD) - optional
Ø Measurements of optical activity (ability to rotate plane of polarised light). Conformation
changes of molecules can be followed as changes of optical activity using a special polarimeter.
Ø We compare absorbances of laevorotatory and dextrorotatory circularly polarised light, the
wavelength of which is near the absorption maximum of the protein.
Ø CD can be used also for studying the structure of nucleic acids.
t1
The figure shows changes of elipticity of a synthetic  polypeptide containing long poly-glu
sequences after addition of the trifluoroethanol (TFE), which increases percentage of the a-helix.
http://www-structure.llnl.gov/cd/polyq.htm
Wavelength [nm]

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X-ray diffraction Spectrometers
•The crystal lattice acts on X-rays as an optical grating on visible light. Diffraction phenomena
occur and diffraction patterns appear. These patterns can be mathematically analysed to obtain
information about distribution of electrons in molecules forming the crystal.
http://cwx.prenhall.com/horton/medialib/media_portfolio/text_images/FG04_02aC.JPG
•
•
•
X-ray source
Protein crystal
Diffracted X-rays

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fig4b
Electron density map of an organic substance calculated from an X-ray crystallogram

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The crystallogram of B-DNA obtained in 1952 by Rosalind E. Franklin, on the basis of which Watson
and Crick proposed the double-helix model of DNA structure.
watson crick xrayhelix franklin watson
F
W
C

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Methods based on measurements of mechanical and electrical properties of macromolecules
• Size and shape of macromolecules can be studied by measurement of:
 Osmotic pressure (size, see lecture ″Thermodynamics and life″)
 Diffusion coefficient (size, see lecture ″Thermodynamics and life″)
 Viscosity (shape, see practical exercises)
 Sedimentation (size, see lecture ″Devices for electrochemical analysis. Auxiliary laboratory
devices″
–
–We can also use:
 Electron microscopy (size and shape, see lecture ″Microscopy″)
 Chromatography - molecular sieve effect in gel permeation chromatography (see chemistry)
 Electrophoresis (end of this part of lecture)

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Electrophoretic Device
Electrophoresis
http://library.thinkquest.org/C0122628/showpicture.php?ID=0064

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Electrochemical properties of colloids
•Colloids are solutions containing particles 10 – 1000 nm in size. Some molecular and micellar
colloids are polyelectrolytes with amphoteric properties. These ampholytes behave like both bases
and acids depending on pH of the medium.
0
In proteins changes the number of –NH3+ and –COO- groups.
Resulting charge
Isoelectric point (Ip)

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Origin of electric double layer on the surface of colloid particle
•Two mechanisms:
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ØIon adsorption (also in hydrophobic colloids)
ØElectrolytic dissociation (prevails in hydrophilic colloids)
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•The double layer on the particle surface differs in concentrated and rarefied electrolytes.
•
•In rarefied electrolytes we can distinguish between stable, diffusive and electroneutral region in
the whole ion cloud around the particle.
•Electrokinetic potential – z (zeta)-potential(také zeta-potenciál nebo ζ-potenciál) Rozdíl
potenciálů na pohybovém rozhraní, který se ustavuje při relativním pohybu tuhé fáze s  elektrickou
dvojvrstvou vůči roztoku (viz modely elektrické dvojvrstvy, obr. 1). Znaménko ζ-potenciálu je
opačné než znaménko iontů vnější vrstvy elektrické dvojvrstvy (podle konvence je potenciál
v objemové fázi roven nule). Elektrokinetický potenciál nebývá vyšší než 0,1 V; je značně
ovlivňován přídavkem elektrolytů, a to i v malých koncentracích. Je příčinou
vzniku elektrokinetických jevů.

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zeta potential

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Electrophoresis
ØElectrophoresis – movement of charged molecules in an electric field. In uniform rectilinear
motion of spherical particles with radius r, the electrostatic force acting on the particle is in
equilibrium with the frictional force arising from the viscosity. The frictional force is given by
Stokes formula:
•F = 6.p.r.h.v
•     where v is particle velocity and h the dynamic viscosity of medium.
ØThe electric field acts on the particle by force:
•F = z.e.E
•     where z is number of elementary charges of the particle, e is the elementary charge
(1,602.10-19 C) and E is intensity of electric field in given place.
ØSince both forces are equal, velocity of the particle equals:

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Electrophoretic mobility
ØThe electrophoretic mobility u does not depend on intensity of the electric field. It is defined
as a ratio of particle velocity and the electric field intensity. It holds:
Note. Electrophoresis with sodium dodecylsulphate. This compound carrying one negative elementary
charge binds in defined way to proteins and eliminates their own electric charge. Protein molecules
then move with different velocities only due to their different radii.

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Measurement of membrane potentials
ØMembrane potentials are measured by means of glass microelectrodes, i.e. glass capillaries with
very fine narrow tips. The diameter of the opening in the end of the tip must be below 1 mm to
avoid substantial damage to the cell. The inner space of the capillary tip is filled by KCl
solution with concentration of 3 mol.l-1. A silver chloride electrode placed in the extracellular
space is used as reference electrode.
•
ØGlass microelectrodes are characterised by high internal resistance (about 10 MW), so we need high
quality amplifiers for the measurement to avoid distortion of the voltage to be measured.

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Experimental setup for measurement of membrane potential by capillary microelectrodes.
When using glass microelectrodes, it is possible to measure also other electrochemical parameters
of the cells and membranes, e.g., concentrations of some ions. They can be prepared as ion
selective electrodes for Na+, K+, Ca2+, H+ etc.
amplifier
oscilloscope
cell

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Patch-clamp Method
Some ion channels may be closed or opened in advance, the microelectrode filling may even contain
ligands capable of interaction with ion channels, and in general any substances that can affect the
function of the membrane. This discovery enabled to examine the activity of the individual or small
groups of ion channels.
membrane-mesurement
A blunt glass microelectrode clings to the surface of the cell or an isolated part of the
biological or artificial membrane. The opening at the end of the microelectrode is completely
sealed by the membrane “patch” and the measured electric voltages or currents thus relate to only a
small part of the membrane, in which a small number of ion channels are found.

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Author:
Vojtěch Mornstein
Content collaboration and language revision:
Viktor Brabec, Carmel J. Caruana
Presentation design:
Lucie Mornsteinová
Last revision: September 2015