Physics 433/833, 2014 Spectroscopy and Light Scattering

Spectroscopy and Light Scattering

ABSTRACT

Light can interact with matter in different ways. In this laboratory module, you explore
how light absorption, emission and scattering provide information on the system being
probed. You then monitor the time-dependent optical density of a solution of collagen
to investigate the kinetics of collagen fibril formation.

I. OBJECTIVES

• Learn the physics principles behind absorption and fluorescence of visible light

• Learn the physics principles behind FRET and how the technique is applied to bio-
logical questions

• Learn the physics principles behind light scattering

• Determine the concentration of an unknown solution of dye molecules

• Determine the how light scattering depends on wavelength

• Determine the kinetic stages of self-assembly of collagen proteins into fibrils

II. BACKGROUND: LIGHT-MATTER INTERACTIONS

When light interacts with a sample, its intensity is attenuated due to two factors: absorp-
tion by the sample and scattering by particles within the sample. The former is described
by its absorbance density, α, and the latter by its turbidity, τ . The attenuation of intensity
as a function of distance x through a sample is given by

−dIλ (x) = (αλ + τλ ) Iλ (x) dx . (1)

Notice how each process, absorbance and turbidity, makes a separate contribution to the
loss of light. If multiple species are present in the solution, with different concentrations cj ,
the absorbance density and turbidity are a sum of the contributions of each:
X X
αλ = cj εj,λ , τλ = cj σj,λ . (2)
j j

The extinction coefficient ελ is a function of the electronic structure of the species and
relates to the strength of electronic transitions as a function of wavelength (see below). The
scattering cross-section σλ depends on particle geometry, size and refractive index, and is

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described in the following section. Integrating Eq. (1) shows that the intensity of light at a
distance x through the sample reduces exponentially with absorbance density and turbidity:

Iλ (x) = Iλ,0 (x) e−(αλ +τλ ) x . (3)

The attenuation of light intensity by a sample can be measured using an instrument known
as a spectrophotometer. Figure 1 shows a schematic of this instrument. The illuminating
light passes through the sample and the amount of light reaching the detector is recorded.
The instrument records the transmittance of light, T , through the sample, relative to a
reference sample (e.g., a blank cuvette):
I(λ)
T (λ) ≡ . (4)
I0 (λ)

FIG. 1. Light path through a spectrophotometer. Absorption or scattering of light by species in

the sample decreases the light intensity reaching the spectrometer. In the spectrometer, a prism
disperses different colours onto an array of photodetectors. The spectrum of transmitted light is
given by the intensity of light falling onto the different pixels in this array.

Here, I(λ) is the intensity of light of wavelength λ transmitted through the sample when
light of intensity I0 (λ) is incident on it.
A more common measure of the attenuation of the light by the sample is given by the
optical density (OD) of the solution, which is a function of wavelength λ:
   
I(λ) I0 (λ)
OD(λ) = − log10 = log10 . (5)
I0 (λ) I(λ)

Note that OD is occasionally defined using a natural log (ln) instead of base 10.

Pre-lab question 1: If a sample attenuates the incoming light to 10% of the incident
value, what is the observed OD of the sample?

Note that in spectroscopy, the optical density is defined as the base-10 logarithm of
the ratio of intensities, while the exponential relationship of Eq. (3) would more naturally
suggest the use of natural logarithm. Extinction coefficients and scattering cross-sections

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are likewise quantified using the spectroscopist’s convention of base-10 logarithms, which we
follow here.
Comparing with Eq. (3), we see that the optical density through a sample of thickness d
is given by

OD(λ) = (αλ + τλ ) d . (6)

The optical density is often referred to as the absorbance of a sample, though this nomen-
clature can be confusing since the attenuation of light in a sample is due to both absorbance
and scattering.

A. Scattering

Light scattering depends on the wavelength of the illuminating light, on particle size,
shape and refractive index, and on the concentration of the particles in solution. For spherical
particles much smaller than the wavelength of light,1 scattering can be described analytically
as Rayleigh scattering. In this case, the scattering cross-section for a spherical particle of
radius R is given by
2
R6 n2 − 1

σλ = f 4 , (7)
λ n2 + 2

where n ≡ nsphere /nmedium is its index of refraction relative to the surrounding medium and
f is a constant prefactor.
For a single type of scattering species in dilute solution, Eqs. (2), (5), and (7) give
2
cR6 n2 − 1

σλ ∝ 4 , (8)
λ n2 + 2

i.e., the optical density depends linearly on concentration and is inversely proportional to λ4 .
This relationship can be used to determine the relative concentration of scattering particles
in solution and can be made absolute if the relation between concentration and OD is known.

Pre-lab question 2: Would you expect to detect more transmitted light for 300 nm
or 500 nm illumination? What ratio of attenuated light intensity would you predict for
these two conditions, assuming Rayleigh scattering?

1
Rayleigh scattering is conventionally assumed to hold if the particle radius R < λ/20.

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Pre-lab question 3: If you wanted to determine the power-law relationship between

optical density and wavelength (i.e., determine or verify the exponent of λ in Eq. (8),
what measurements would you perform and how would you plot and analyse your data?

B. Absorption

Molecules that absorb visible light absorb a photon and promote an electron to a higher-
lying molecular orbital. These electronic excitations occurring in the visible range of the
spectrum (750–400 nm) correspond to electronic states separated by energies of 1.6–3.1
eV. Electronic transitions can also occur to states of higher energy; some molecules are
colourless and their lowest accessible electronically excited states lie in the ultraviolet range
of the spectrum. By contrast, absorption of infrared light is associated with much lower
energy vibrational transitions, and rotational energy transitions are yet lower in energy, in
the microwave region of the spectrum. Selection rules of quantum mechanics determine
which transitions are allowed based on the symmetries and spins of the participating states.
Figure 2 shows an example of a Jablonski diagram, illustrating the types of transitions
that can occur between different electronic states of a molecule. Here, the notations S0 ,
S1 , and S2 refer to electronic states of singlet spin, where S0 is the ground state of the
molecule. Transitions from a singlet state to a triplet state, such as the lowest-energy T1
triplet state, are not quantum mechanically allowed, and so these transitions happen rarely.
If a triplet state is populated, it is generally long lived, since transitions out of this to the
ground electronic state, which is usually a singlet state, are also disallowed.
Each molecule possesses a unique spectroscopic fingerprint, by which it can be identified
under specified experimental conditions. For example, DNA absorbs strongly at 260 nm,
where an electron in a π-bonding orbital can be excited to a π ∗ -antibonding orbital. (The
notation π refers to electronic wavefunctions possessing a nodal plane.) By contrast, proteins
absorb most strongly around 280 nm, which is due mostly to electronic excitations from π-
bonding to π ∗ -antibonding orbitals on the aromatic amino acids tryptophan and tyrosine.
(π to π ∗ excitation of electrons in the peptide bonds gives rise to a large absorbance peak
further into the UV, at 200 nm.) Thus, the purity of a DNA sample relative to contaminating
proteins can be determined from the ratio of its absorbance at 260 nm to 280 nm.
For a solution containing a single type of absorbing species, Eqs. (2) and (6) give

Abs(λ) = OD(λ) = ελ cd . (9)

This is the well-known Beer-Lambert law of spectroscopy, which describes the linear relation-
ship between the solution concentration and the measured absorbance. Here, ελ represents
the wavelength-dependent extinction coefficient of the absorbing molecule, c its concentra-
tion and d the optical pathlength through the sample cuvette (generally 1 cm).
The quantity ελ is specified in units of M−1 cm−1 and is usually given for the wavelength
corresponding to the maximum absorbance. The extinction coefficient can vary with solution
conditions, so may depend on the solution’s ionic strength, pH, and temperature, among

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FIG. 2. Jablonski diagram showing possible relaxation pathways and their timescales following
absorption of a photon and excitation of an electron. Lines represent energy levels of specific
quantum mechanical states, where for illustration purposes a few vibrational states are included
within each electronic manifold. Figure courtesy of Marcia Levitus, Arizona State University.

other possibilities. The linear relationship of Eq. (9) holds over a range of concentrations
that is spectrometer dependent; a good rule of thumb is that it holds from 0.1 < OD < 1.0,
and often to an order of magnitude lower OD.
By measuring the absorbance spectrum of a solution containing a molecule of known
extinction coefficient under the solution conditions used, one can determine its concentration
in solution. Alternatively, by making a dilution series of a molecule in solution, one can
determine its extinction coefficient from a plot of A vs. c. Spectrophotometers generally
have built-in software that reports absorbances A(λ) as output, though it is important to
keep in mind, as stated above, that this measured attenuation of light can result not only
from absorption of light by a sample but also from scattering.

C. Fluorescence

A fluorescent molecule, or fluorophore, absorbs light of energy corresponding to allowed

transitions of electrons in the molecule (see above), and then emits light at longer wave-
lengths when the electron that was excited by the absorbed light relaxes to a low-lying state.
The possible types of transitions and associated timescales are shown in Fig. 2, the Jablon-
ski diagram familiar to spectroscopists. Because of rapid relaxation to low-lying vibrational
states within each electronic state, the downward transitions (e.g. fluorescence) are of lower
energy than the excitations (absorbance). Thus, a molecules fluorescence/emission spectrum
is said to be red shifted (shifted to longer wavelengths / lower energies—i.e., towards the
red region of the spectrum) compared to its absorbance spectrum.
Figure 3 shows the absorbance and fluorescence emission spectra for the dye molecule

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Rhodamine 6G, which will be used in this laboratory module. Illumination with the com-
mon laser wavelength 532 nm excites rhodamine near the peak of its absorbance spectrum.
Its fluorescence emission can be detected at wavelengths longer than this excitation wave-
length. For this reason, Rhodamine 6G is also used in the module on fluorescence correlation
spectroscopy (FCS).

(a) (b)
FIG. 3. Rhodamine dye molecule: (a) Absorbance (green) and emission (red) spectra. (b) Chemical
structure.

Generally, we measure fluorescent light at 90◦ with respect to the excitation source.
Since, on the slow timescale of these measurements, emission of photons by the fluorophore
is isotropic, any detection angle should give rise to an equal fluorescence signal. On the
other hand, the forward direction contains the highest intensity of the excitation source.
The signal-to-background ratio is thus better if we measure fluorescence by detecting at 90◦ ,
rather than in the forward direction.

D. Förster Resonance Energy Transfer (FRET)

Förster Resonance Energy Transfer (FRET)2 is an extremely popular technique in current

biophysics research, employed to provide information about the spatial separation between
two parts of a molecule or different molecules. It relies on the spectral overlap between the
emission of a donor fluorophore and the absorbance of an acceptor fluorophore. If these
coincide, the two species are spatially close, and other conditions are met, as described
below, one fluorophore (donor) can absorb light and a second (acceptor) emit fluorescence.
Measuring the emitted light intensity at wavelengths corresponding to donor fluorescence
and comparing with the emitted light intensity at wavelengths corresponding to acceptor
fluorescence provides information about whether the two fluorophores are separated by short
distances (acceptor emission observed) or larger distances (donor fluorescence observed).
FRET occurs due to coupling of the transition dipoles for the two transitions involved
(de-excitation of the donor and excitation of the acceptor); see Fig. 4. This dipole-dipole
interaction depends on the spatial separation between the two dipoles, r, as well as the
2
The technique is commonly but incorrectly called fluorescence resonance energy transfer. It is not
fluorescence that is transferred; the form of energy transfer was first described by Förster as dipole-dipole
coupling.

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(a) (b)
FIG. 4. FRET. (a) Schematic illustrating the spatial dependence to emission of light intensity by a
donor or acceptor species, in response to excitation of the donor. Figure from Marcia Levitus. (b)
Absorption and emission spectra of a possible FRET pair, cyan fluorescent protein (CFP) and red
fluorescent protein (DsRFP). The emission spectrum of CFP shows extensive spectral overlap with
the absorption spectrum of DsRFP, meaning CFP could act as a donor and DsRFP could act as
an acceptor. A common small-molecule FRET pair is Cy3-Cy5, two cyanine-based dye molecules.
Figure from Olympus.

relative orientation of the dipoles. The separation is usually defined relative to the critical
Förster distance R0 , the separation at which 50% of absorbed intensity is transferred to the
acceptor. The efficiency of energy transfer is given by

1
E(r) =  6 . (10)
r
1+ R0

where the Förster distance is given by

R06 ∼ QD κ2 n4 J , (11)

Here, QD is the fluorescence quantum yield of the isolated donor, κ2 is a geometric factor
that depends on the relative angular orientation of the two dipoles, n is the refractive index
of the medium, and J is the integrated spectral overlap of the donor and the acceptor, as
illustrated schematically in Fig. 4b.
For most FRET pairs, R0 lies in the range of 2–6 nm, which is conveniently similar to the
size of proteins and on the scale of many biological processes. Examples of studies employing
FRET include protein and nucleic acid folding, protein-protein binding interactions (in vitro
and in vivo), molecular motor stepping, and vesicle fusion. Many of these studies can be
done in “bulk” (using a cuvette and sample in solution); however, much richer information
emerges when FRET is studied at the single-molecule level and the kinetics of transitions,
e.g., between folded and unfolded states of a system, can be monitored in real time. You will
not study FRET in this module, but please keep it in mind for your independent project.

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FIG. 5. Jablonski diagram illustrating the resonant energy transfer between a donor and an
acceptor, where this transfer can occur if (a) both transitions are the same energy; (b) symmetries
of the states; and (c) spatial orientation and separation of the two species make the transfer allowed.
Figure from Olympus.

III. BACKGROUND: COLLAGEN FIBRIL FORMATION

Collagens are a class of proteins with a unique triple helical structure. They are the pre-
dominant proteins in vertebrates, comprising greater than 25% of our total protein content.
They form the basis of the majority of our connective tissues (including tendons, cartilage,
bone and skin) and of the extracellular matrix, the network of supports within which our
cells grow. The mechanics of collagen are critical to physiological function and can exert
profound influences over cell fate, as seen in fetal development, in cancer progression, and
in stem cell differentiation in engineered devices that utilize collagen as a biomaterial.
Most collagens are fibrillar, meaning that they assemble into well-ordered, higher-order
structures called “fibrils.” Fibrillar collagens are triple helical proteins of about 300 nm (1000
amino acid residues, 100 kDa per chain) in length and about 1.5 nm in diameter. The fibrils
formed by these collagens are extremely well ordered structures, as seen by the distinct
dark-light “D-banding” pattern observed in electron microscopy, resulting from staggered
overlap and gap regions of collagen proteins in the fibrils (Fig. 6). Fibrils are typically a
few hundred nanometres in diameter and can be tens of micrometers in length. These long,
rope-like structures in turn can undergo further assembly (sometimes alone and often with
other proteins or minerals) into fibres, networks or other structures that form our connective
tissues. The process of assembly from collagen proteins into fibrils is so well specified by
the chemical sequence of collagen that this process can occur in vitro, i.e., in a tube in the

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lab, with no chaperones or other assisting proteins normally found in the cellular milieu
necessary.

(a)

(b) (c)
FIG. 6. The structure of a collagen fibril. (a) Collagen triple helices pack laterally, offset from one
another, giving rise to bands of protein overlap and gaps (fewer overlapping regions) transverse to
the fibril axis. (b) A collagen fibril can extend for tens of micrometres, exhibiting high degrees of
order along its axis. The gap regions are preferentially bound by heavy-atom stains used in electron
microscopy, giving rise to dark bands when negatively stained and imaged using transmission
electron microscopy (TEM). Scale bar = 500 nm. (c) A close-up view of the collagen fibril in part
b reveals substructure in the overlap and gap regions. Scale bar = 100 nm. TEM images of type
II collagen fibrils were recorded at SFU’s Nanoimaging Facility. Images courtesy of Clara Chan,
former SFU Biological Physics honours undergraduate student.

Collagen proteins are generally stored in an acidic solution, as they are highly positively
charged at low pH, leading to electrostatic repulsion between proteins. To initiate fibril
formation, the solution is neutralized, usually by diluting the collagen into a solution buffered
around pH 7, and salts are added. Under these conditions, collagens can associate and start
to pack into fibrils.

Pre-lab question 4: Why might increasing the pH to 7 allow collagen molecules to

associate? Why might adding salt to the solution allow collagen molecules to associate?

Collagen fibril formation, during which collagen changes from being isolated as a triple
helical protein in solution into being tightly packed in a fibril, is entropically driven. At first

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glance, this entropic mechanism might seem impossible: as collagen proteins are bound into
fibrils, they lose their configurational entropy and are constrained to lie relatively straight
the well-ordered structure of a fibril. However, the necessity of packing three amino acid
chains into a triple helix means that the side-chains (so-called R-groups) must protrude into
solution, rather than being shielded inside the folded protein core, as is the case for globular
proteins. To keep these side-chains solvated requires structured water molecules, which form
water bridges and contribute to salt bridges. As the collagen proteins associate laterally in
their preferred staggered configuration, the structured water molecules are able to return to
the bulk water, where they gain significant entropy, more than offsetting the configurational
entropy loss of the collagen proteins.
The kinetics of fibril formation are easily monitored via light scattering measurements.
Initially, the collagen proteins are isolated in solution and are too small to scatter significant
amounts of visible light. As they start to encounter each other in solution, small aggregates
can nucleate (nucleation phase of fibril formation). These are proposed to have a well-defined
structure consisting laterally of five or six collagen molecules in a so-called microfibril. These
structures are also too small to scatter significant amounts of visible light, but as they
assemble into fibrils, the optical density of the solution rapidly increases as the fibrils grow
sufficiently large to scatter light. This is the growth phase of fibril formation, and the
slope of scattered light intensity versus time in this region gives the rate at which fibrils
grow. Finally, the solution becomes depleted of free collagen molecules, as the majority has
been incorporated into fibrils, and so the amount of scattered light no longer continues to
increase (the plateau region). You will observe and characterize the kinetics of collagen fibril
formation in this lab.

IV. INVESTIGATIONS

1. Measure the absorbance spectrum of Rhodamine 6G dye at a range of known concen-

trations in solution, to determine its extinction coefficient at 532 nm. Can you find a
literature value to compare to?
2. Measure the fluorescence spectrum of Rhodamine 6G dye in response to excitation at
532 nm, and determine the fraction of the emitted intensity that falls above 550 nm,
the cutoff wavelength of the filter used in the Fluorescence Correlation Spectroscopy
module.
3. Determine whether 30 nm diameter polystyrene spheres scatter light as expected for
Rayleigh scattering, choosing an appropriate range of wavelengths for your analysis.
4. Measure the absorbance and fluorescence spectra of a solution of 36 nm diameter
“fluorospheres” over the wavelength range 250–900 nm. (Decide what part of the
spectrum is useful.) Determine the scattering, absorption, and fluorescence properties
of the spheres.
5. Measure the kinetics of collagen fibril formation by taking advantage of the enhanced
light scattering of fibrils compared with isolated proteins in solution.

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V. MATERIALS AND EQUIPMENT

• Spectrophotometer equipped for absorbance and fluorescence measurements.

• Disposable cuvettes.
• Stock solution of Rhodamine 6G (10 µM in water) IMPORTANT: Rhodamine is a
health hazard. Dispose in chemical waste; wear gloves and eye protection.
• Concentrated solution of 31-nm-diameter polystyrene spheres (8% w/v).
• Concentrated solution of 36-nm-diameter fluorescently labelled polystyrene spheres
(1% w/v).
• Concentrated solution of type I collagen, extracted from rat tail tendons, in acetic acid
(2.5 mg/ml).
• Concentrated PBS buffer solution, pH 6.9 with excess phosphates, for fibril formation
(5X working concentration).3

VI. SUGGESTED TIMELINE

Day 1: Measure the absorbance and fluorescence spectra for each of the Rhodamine 6G
and nanosphere solutions. To determine the extinction coefficient of Rhodamine 6G, you
will need to measure its absorbance spectrum at a range of concentrations. To measure its
fluorescence spectrum, you will need to determine the appropriate concentration, as well as
integration time on the spectrometer, in order to see a curve of good signal-to-noise ratio.
To measure the spectra of the microspheres, dilute them so the solution is not too optically
dense. Try a 100-fold dilution.

Day 2: Measure the kinetics of collagen fibril formation. Come to lab having analyzed the
spectra from Day 1 experiments and answered the following pre-lab questions.

Pre-lab question 5: What features in the spectra from Day 1 arise from light scatter-
ing?

Pre-lab question 6: What type of measurement (e.g., absorbance or fluorescence

mode) are you going to perform to determine the amount of light scattering during
collagen fibril formation? Justify your choice.

3
Working (1X) stock concentration of the buffer is 273.8 mM NaCl, 5.4 mM KCl, 42.2 mM dibasic sodium
phosphate (Na2 HPO4 ) and 8.8 mM monobasic potassium phosphate (KH2 PO4 ).

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You will need to measure the intensity of scattered light as a function of time. Protocol—
Using the Spectrophotometer explains how to set up the instrument to record a time series
of measurements. Start your measurements rapidly after initiating fibril formation, so that
you have enough time to observe the plateau phase of fibril assembly and growth. To ini-
tiate fibril formation, mix—on ice—the 5X buffer solution, pure water, and concentrated
collagen solution to obtain working concentrations of

1X buffer and ≈ 0.3 mg/ml collagen.

Start recording your spectra immediately after mixing and putting in the cuvette (i.e.,
ensure the spectrophotometer is properly zeroed and ready to go before mixing your sample).
The measured kinetics of collagen fibril formation depend very strongly on initial conditions
(see Wood and Keech,1960). For one experimental run, as you will perform here, these are
less critical, but it is important to keep materials cold so that fibril nucleation does not
initiate prior to the start of your measurements.

Pre-lab question 7: What amounts of buffer, collagen and water will you mix to create
a final sample 1 ml in volume?

• 5X buffer: µl

• ddH2 O: µl
• collagen (2.5 mg/ml stock): µl

VII. ITEMS TO INCLUDE IN LAB WRITE-UP

• Answer all pre-lab questions.

• Plot the Rhodamine 6G absorbance spectrum at a given concentration (and state the
concentration).
• Determine the extinction coefficient of Rhodamine 6G at 532 nm from a plot of ab-
sorbance versus concentration.
• Plot Rhodamine 6G-fluorescence spectrum at a given concentration (and state the
concentration).
• Determine the percentage of emitted light intensity at wavelengths longer than 550
nm from Rhodamine 6G excited by a laser at 532 nm.
• Plot the relationship between OD and wavelength of 31 nm diameter polystyrene
spheres, and determine whether this follows Rayleigh scattering.

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• Plot absorbance and fluorescence spectra of 36 nm diameter polystyrene “fluoro-

spheres,” labelling contributions from scattering, absorption and fluorescence

• Plot light scattering versus time throughout the course of collagen fibril formation,
labelling nucleation, growth and plateau phases.

• Determine growth rate of fibrils from the kinetics measurements.

• Discuss the above results, as well as errors and experimental difficulties encountered,
if any.
• Discuss at least two (Phys 433) or all (Phys 833) of the points from the following
section.

VIII. POINTS TO PONDER

• How would the fluorescence spectrum of Rhodamine 6G change if the solution were
excited at 500 nm? At 550 nm?
• Describe and sketch the expected OD(λ) curve for a solution containing (a) 30 nm
diameter polystyrene spheres; (b) 100 nm diameter polystyrene spheres; and (c) equal
numbers of the two sizes of spheres.

• Find an example in the literature of using FRET to study some aspect of a biological
system. Citing the reference, summarize in one or two paragraphs a key example of
how the experiment was conducted and what was learned.
• How would the kinetics of fibril formation change if the collagen concentration were
decreased? Describe any changes expected in each of the three kinetic phases.
• How would the kinetics of fibril formation change if the temperature were increased?
Describe any changes expected in each of the three kinetic phases.

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IX. ADDITIONAL READING

• R.N Zare, B.H. Spencer, D.S. Springer and M.P. Jacobson, “Light Scattering from
Disordered Systems” in LASER Experiments for Beginners. University Science Books
(1995).
• C. Garland, J. Nibler and D. Shoemaker, “Spectroscopy,” in Experiments in Physical
Chemistry. McGraw-Hill (2008).
• C. Joo, H. Balci, Y. Ishitsuka, C. Buranachai and T.J. Ha, “Advances in Single-
Molecule Fluorescence Methods for Molecular Biology.” Annual Review of Biochem-
istry 77, 51–76 (2008).
• An introduction to FRET from Olympus (the microscope company).
• C.G. Wood and M.K. Keech, “Formation of fibrils from collagen solutions 1. Effect of
experimental conditions: kinetic and electron-microscope studies,” Biochemical Jour-
nal 75, 588–598 (1960).
• C.G. Wood, “Formation of fibrils from collagen solutions 2. Mechanism of collagen-
fibril formation,” Biochemical Journal 75, 598–605 (1960).
• B.R. Williams, R.A. Gelman, D.C. Poppke and K.A. Piez, “Collagen fibril formation.
Optimal in vitro conditions and preliminary kinetic results,” Journal of Biological
Chemistry 253, 6578–6585 (1978).

Some more detailed articles about FRET:

• “Recent advances in FRET: distance determination in protein-DNA complexes.” A.

Hillisch, M. Lorenz and S. Diekmann. Current Opinion in Structural Biology 11,
201–207 (2001).

• “Fanciful FRET.” S.S. Vogel, C. Thaler and S.V. Koushik. Science STKE 2006 (331).

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