106 Questions

3.7  SUMMARY POINTS

◾Elastic scattering light spectroscopy—​using VIS light, UV, and IR—​is a robust tool

to determine the concentration of biological scattering particles in solution.

◾Fluorescence spectroscopy and FACS can characterize and help isolate different

cell types.

◾Image contrast can be improved in bright-​field microscopy using a range of tools,

especially those involving optical interference, which includes phase contrast and

DIC microscopy.

◾Single-​photon excitation fluorescence microscopy is one of the most widely used

and valuable biophysical tools to investigate functional biological material, espe­

cially when combined with multiple color dye tags.

◾Of the many different types of dye tags used in fluorescence microscopy, FPs offer

the greatest physiological insight but have suboptimal photophysical features and

often cause steric hindrance of native biological functions.

◾There are several different modes of illumination for fluorescence microscopy,

from which TIRF offers huge enhancements in contrast for monitoring processes

in cell membranes in particular.

QUESTIONS

3.1

Give an example of how a biological process spans multiple length and time scales

and crosses over and feedbacks at several different levels of length and time scale.

Describe an experimental biophysical technique that can be used to generate infor­

mation potentially at the whole organism, single-​cell, and single-​molecule levels sim­

ultaneously. Should we try to study even broader length and time scales regimes, for

example, at the level of ecosystems at one end of the spectrum or quantum biology at

the other? Where should we stop and why?

3.2

Transmission electron microscopy (see Chapter 5) on a layer of cells in a tissue

suggested their nuclei had mean diameters of 10.2 ± 0.6 μm (± standard deviation).

Negative-​phase contrast microscopy images from this tissue suggested that the nuclei

were the brightest features in the image when the nuclei were most in focus.

a

Derive a relation between the length through a cell over which the phase of propa­

gating light is retarded by one quarter of a wavelength, stating any assumptions.

b

Estimate the range of refractive index for the nuclei.

3.3

What do we mean by an isotropic emitter in the context of a fluorescent dye mol­

ecule? Derive an expression relating the geometrical efficiency of photon capture of

an objective lens of numerical aperture (NA) (i.e., what is the maximum proportion

of light emitted from an isotropic emitter, neglecting any transmission losses through

the lens). What factors may result in fluorescence emission not being isotropic?

3.4

Fluorescence anisotropy experiments were performed on a GFP-​tagged protein in

aqueous solution, whose effective Stokes radius was roughly twice that of a single GFP

molecule. Estimate what the minimum sampling frequency in GHz on the photon

detector needs to be to detect anisotropic emission effects. (Assume that the viscosity

of water is ~0.001 Pa·s.)

3.5

The same protein of Question 3.3 under certain conditions binds to another protein

in the cell membrane with the same diameter, whose length spans the full width of the

membrane. If this GFP-​tagged protein is imaged using a rapid sampling fluorescence

microscope, which can acquire at a maximum sampling time of 1000 image frames

per second, comment on whether it will be possible to use fluorescence polariza­

tion images to determine the state of the membrane protein’s angular rotation in the

membrane.

3.6

A yellow FP called “YPet,” at peak emission wavelength ~530 nm, was used to tag a

low copy number cytoplasmic protein in a spherical bacterial cell of radius ~1 μm.

A slimfield microscope, using an objective lens of NA 1.45 with EMCCD camera