Chapter 9 – Emerging Biophysics Techniques  433

9.8 A synthetic DNA molecule was designed to be used for optical data trans­

mission by turning the DNA into a molecular photonics wire comprising five

different neighboring dye molecules, with each having an increasing peak

wavelength of excitation from blue, green, yellow, orange through to red. Each

dye molecule was conjugated in sequence to accessible sites of the DNA, which

was tethered from one end of a glass coverslip. Each dye molecule was spaced

apart by a single-​DNA helix pitch. The mean Förster radius between adjacent

FRET pairs was known to be ~6 nm, all with similar absorption cross-​sectional

areas of ~10⁻¹⁶ cm².

a

When a stoichiometrically equal mix of the five dyes was placed in bulk solution,

the ratio of the measured FRET changes between the blue dye and the red dye

was ~15%. How does that compare with what you might expect?

b

Similar measurements on the single DNA-​dye molecule suggested a blue-​red

FRET efficiency of ~90%. Why is there such a difference compared to the bulk

measurements?

9.9 For the example data transmission synthetic biomolecule of Question 9.8, a blue

excitation light of wavelength 473 nm was shone on the sample in a square wave

of intensity 3.5 kW cm⁻², oscillating between on and off states to act as a clock

pulse signature.

a

If the thermal fluctuation noise of the last dye molecule in the sequence is

roughly ~kBT, estimate the maximum frequency of this clock pulse that can

be successfully transmitted through the DNA-​dye molecule, assuming that

the emission from the donor dye at the other end of the DNA molecule was

captured by an objective lens of NA 1.49 of transmission efficiency 80%, split

by a dichroic mirror to remove low-​wavelength components that captured

60% of the total fluorescence, filtered by an emission filter of 90% transmis­

sion efficiency, and finally imaged using a variety of mirrors and lenses of very

low photon loss (<0.1%) onto an electron-​multiplying charge-​coupled device

(CCD) detector of 95% efficiency.

b

How would your answer be different if a light-​harvesting complex could couple to

the blue dye end of the photonic wire? (Hint: see Heilemann et al., 2004.)

9.10 The integrated intensity for a single molecule of the yellow fluorescent protein

mVenus was first estimated to be 6100 ± 1200 counts (±standard deviation) on a

camera detector in a single-​molecule fluorescence microscope. Each protein sub­

unit in the shell of a carboxysome was labeled with a single molecule of mVenus

with biochemical experiments suggesting that the distribution of stoichiometry

values of these specific carboxysomes was Gaussian with a mean of 16 and standard

deviation sigma width of 10 subunit molecules. The initial integrated fluores­

cence intensity of a carboxysome population numbering 1000 carboxysomes was

measured in the same fluorescence microscope under the same conditions as was

used to estimate the single-​molecule brightness of mVenus, with the stoichiom­

etry of each carboxysome estimated by dividing the initial fluorescence intensity

obtained from a single exponential fit to the photobleach trace (see Chapter 8) by

the single-​molecule fluorescence intensity for mVenus. Estimate with reasoning

how many carboxysomes have a calculated stoichiometry, which is precise to a

single molecule.

9.11 Discuss the key general technical scientific challenges to developing a lab-​on-​chip

biosensor for use in detecting early-​stage bacterial infections that are resistant to

antibiotics and strategies to overcome them.

9.12 If there are no “preferred” length and time scales in biology, with complex and multi­

directional feedback occurring across multiple scales, then what can the isolated

study of any small part of this milieu of scales, for example, at a single-​molecule level,

a cellular level, a tissue level, a whole organism level, or even at a whole ecosystem

level, actually tell us?