UNIVERSITY OF GLASGOW

Degrees of MEng, BEng, MSc and BSc in Engineering

MICROSCOPY AND OPTICS M (ENG5289)

Wednesday 9th December 2020
Release time: 14:00 (GMT) for 4 hours

This exam should take you: 2 hours to complete

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Attempt all PARTS

The numbers in square brackets in the right-hand margin indicate the marks allotted to the
part of the question against which the mark is shown. These marks are for guidance only.

A calculator may be used. Show intermediate steps in calculations.

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Q1 (a) You are viewing a specimen of white blood cells using brightfield microscopy
however the contrast is low. Describe two methods you could use to increase
the contrast of the image. [5]

(b) For an experiment you need to count the number of red blood cells within a 1
mm square section of a microscope slide. The cells are eight microns in
diameter and have sedimented down into a single layer. You have two
objectives you can use a 5x with Numerical Aperture (N.A.) 0.1 and a 100x
objective with N.A. 0.95. Your light source peak wavelength is 530 nm.
Determine the optical resolution limit of the two lenses and using this describe
which would be more appropriate for the cell counting task.
[5]

(c) You are required to design a microscope to produce a 200x magnified image of
a sample on a camera that is fixed 300 mm from the sample. You may use a 20x
objective that produces an intermediate image which is 20x larger than the
sample and is 160 mm from the objective (e.g. S1'=160 mm) and you may use
one further lens in a configuration as shown in Figure 1. Calculate the distance
the second lens will have to be placed from the camera (S2') and the focal length
it will need to be. [10]

Figure 1, 100x microscope.

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Q2 (a) Discuss the relative advantages and disadvantages of using optical microscopy
or a Scanning Electron Microscope for imaging blood platelets which are 2 to
3 microns in diameter. [5]

(b) A microscope objective contains four glass lenses with refractive index of glass
= 1.5. Assuming the light is incident perpendicular to the lenses and that each
interface causes Fresnel reflections calculate the percentage of light that can be
transmitted through the objective. [5]

(c) A brightfield reflective microscope is to be used to observe a cell which has an

average refractive index of 1.36 for its cytosol and contains a vacuole filled with
fluid of refractive index 1.34. The cell sits within an aqueous medium of
refractive index 1.33. Calculate the percentage of the light we could expect to
be reflected back from the medium/cell interface and from the cytosol/vacuole
interface and hence deduce which interface would give greater contrast on the
image. [5]

(d) You are to design a microscope to image two organelles within a cell which are
600 nm apart. Assume the microscope has an infinity corrected objective that
can be thought of as a single lens and a standard distance between the tube lens
and a camera capturing the intermediate image of 200 mm. Your camera has a
pixel spacing of 14 microns. Decide how many pixels you wish to use to image
the two organelles and using this decision calculate the maximum working
distance (distance between the objective and the sample) we will be able to use.
[5]

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Q3 (a) Describe why light from a LASER is different from light from a conventional
incandescent bulb and why it is useful. [5]

(b) Discuss the effect the laser cavity has on the output spectrum of the laser. How
would the spectrum differ between two lasers; one with a low optical gain but
a high quality optical cavity and the other with a higher gain and a lower quality
cavity. [5]

(c) For a laser dissection experiment you need to deliver 5 nJ of energy to a small
location on a cell which absorbs strongly in the visible using a standard
brightfield upright microscope with an additional optical path for the laser light.
The Numerical Aperture of the objective is 0.8.

(i) Choose a laser wavelength and power (continuous not pulsed) to deliver
this energy. Assuming all the energy from the laser light is absorbed by
the cell how long will it take to deliver the 5 nJ. [5]

(ii) How many photons will this involve? [3]

(iii) If you assume all the optical power is delivered to an optical spot that has
a radius equivalent to the optical resolution limit what is the average
optical intensity in this spot? [2]

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Q4 (a) You work in a lab that has been asked to evaluate a new drug using living tissue
samples of XXX m thickness, where XXX are the last three digits of your
matriculation number + 50 (e.g. if your matriculation number is 123456, XXX
is 506 m).

You should detect the morphological and volumetric changes of individual cells
within the tissue. You have access to the following techniques: 1)
epifluorescence microscopy, 2) confocal laser scanning microscopy, 3) atomic
force microscopy, 4) scanning electronic microscopy, and 5) two-photon
microscopy.

(i) Select one technique that will detect morphological and volumetric
changes of individual cells and justify your choice. Describe any sample
treatment required. [5]

(b) Focal adhesions are dynamic protein complexes through which a cell interacts
with the surrounding extracellular matrix. The size of focal adhesions can range
from ~200 nm at the early stage of formation up to 10 m after maturation.

(i) Design an assay for real-time imaging of the early state formation of
focal adhesions in living cells that migrate on a glass coverslip. Describe
the procedure of the technique you would use and any sample treatment
required. [5]

(ii) If you are given patient samples and are not allowed to treat the cells,
propose a method to image the contact areas of cells on a substrate and
describe the principle. Comment on whether this method can be used to
measure focal adhesions smaller than 200 nm size. [5]

(c) You are asked to detect a virus gene (ATTTGGCATTGC) in clinical samples.
Design a method for this that makes use of the quenching phenomena. Describe
the procedure and sample treatment required. [5]

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Q5 (a) Figure Q5 shows the Resonance Raman spectra of a photosynthetic cell that
was cultured in water saturated with either 12CO2 or 13CO2. The laser
wavelength for Raman acquisition is 532 nm. The three Raman peaks are
characteristics of an intracellular metabolite called carotenoids.

(i) Explain what causes the curve to tilt upward? Suggest a method to
overcome this problem. [2]

(ii) The position of the three peaks move towards the lower wavelength for
cells cultured in 13CO2 water. The position of the v1 peak in the blue
spectrum is 1520 cm-1. Assuming the shift between the two spectra is
XX cm-1 for the v1 peak, calculate this shift in units of “nm”. XX is the
last two digits of your matriculation number +5 (e.g. if your
matriculation number is 123456, XX is 61).
[3]

(iii) If we use a 785 nm laser instead to acquire Raman spectra of these

samples, explain how these three peaks would change and how the shifts
between the two spectra would change.
[3]

Figure Q5. Raman spectra of a cyanobacteria cell cultured in water with

12
CO2 or 13CO2.

Part (b) & (c) can be found overleaf…

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(b) You are asked to measure the stiffness of the following samples using Atomic
force microscopy (AFM): 1) living cancer cells; 2) PDMS elastomer substrate;
and 3) a bone specimen.

(i) You have access to four AFM cantilevers with the following spring
constants: 1) 0.03 N/m; 2) 0.3 N/m; 3) 3 N/m; 4) 30N/m. Comment on
which cantilever could be suitable for each sample and justify your
choice. [4]

(ii) Describe the procedure for the stiffness measurement and illustrate your
answers with a diagram. Propose the environments under which the
measurement can be made and justify your choice.
[4]

(c) You are asked to design an assay to measure dissolved oxygen levels within
living cells. You can access the following methods: 1) bright-field and phase
contract microscopy; 2) all fluorescence technologies; 3) all Raman associated
technologies; 4) Atomic Force Microscopy; 5) Scanning Electron microscopy.
Select one appropriate method and justify your choice. Explain how the
measurement is done and any sample treatment as required.
[4]

End of question paper

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