Visibility through scattering media

Lab instructions

Preparatory reading: Lectures notes on Imaging through turbid media and Light propagation and
scattering in tissues.
Personal equipment: Bring printed Lab instructions.

1 TABLE OF CONTENTS
2 Safety Considerations................................................................................................................................................... 2
3 Theory ................................................................................................................................................................................ 2
3.1 Modulation Transfer Function........................................................................................................................ 2
3.2 Extinction efficiency............................................................................................................................................ 3
4 Lab Overview ................................................................................................................................................................... 4
The first task .................................................................................................................................................................... 4
The second task .............................................................................................................................................................. 4
5 Experimental Setup....................................................................................................................................................... 4
Visibility beam path ...................................................................................................................................................... 5
Scattering beam path.................................................................................................................................................... 5
6 Experimental Exercises ............................................................................................................................................... 5
Task 1 - Familiarize with the experimental setup................................................................................................. 5
Task 2 - Visibility ................................................................................................................................................................ 6
Exercise 1) Phantom solution 1 – Intralipids solution ................................................................................... 6
Exercise 2) Modulation Transfer Function ......................................................................................................... 6
Exercise 3) Fourier filtering ...................................................................................................................................... 6
Exercise 4) Structured Illumination....................................................................................................................... 7
Exercise 5) Phantom solution 2 – Polystyrene spheres................................................................................. 7
Task 3 - Scattering phase functions ............................................................................................................................ 8
Exercise 6) Scattering phase function as function of size and wavelength ........................................... 8
Exercise 7) Blood cells ................................................................................................................................................. 8
2 SAFETY CONSIDERATIONS
This lab uses a class 3b laser system. Such lasers may cause eye damage from both direct and specular
reflections. To participate in this lab, you are required to have taken laser safety course.
To avoid any risk of injury, please adhere to these guidelines:
• Protective glasses are available but cannot be worn at all times.
• The laser system has adjustable power. Use low power whenever possible.
• Take off any wristwatches or reflective armbands before starting the lab.
• Keep your eye level significantly above table level when laser is on. Do not lean in close to the
laser and do not bend down to pick things up while laser is running.
• Turn laser off before inserting or removing new components into beam path.
• Start at low laser power and carefully increase to desired power after having inserted new
components.

3 THEORY
3.1 MODULATION TRANSFER FUNCTION
Modulation Transfer Function (MTF) can be used to quantify the quality of an imaging system. The
MTF is the degree of preservation of contrast, a value between 0 and 1, through the imaging system
as a function of spatial frequency. Contrast can be degraded due to resolution limits, poor optical
design, or challenging imaging conditions.
To calculate the MTF, an object with spatial frequencies f of known contrast Cf object is imaged using
the system. From the resulting image, the Michelson contrast at each frequency Cf is calculated as
seen in Fig. 1. The MTFf value is then given by the ratio between Cf and Cf object.

Figure 1: MTF calculation for quantifying the quality of an imaging system.

3.2 EXTINCTION EFFICIENCY
Light extinction is the gradual loss of photon flux due to scattering and absorption as light propagates
through a medium. For a medium like a dilution of particles in a liquid, the extinction cross-section
σe describes the probability that light will interact with a single particle as a virtual obstructing cross-
section. Combined with the density of particles, the extinction cross-section, then, dictates the light
extinction.
Naturally, large particles are generally more obstructing to the light flux than the same number of
smaller ones. However, the dimensionless extinction efficiency Qe describes how efficiently a particle
obstructs light in relation to its geometrical size. The extinction cross-section and the extinction
efficiency are visualized in Fig. 2.

Figure 2: Visualization of extinction cross-section and extinction efficiency. Here, σs and σa are the
scattering and absorption coefficients respectively, and G is the geometrical cross-section

Light interaction with spherical particles is described by Lorentz-Mie theory. For particles much
smaller than the wavelength of light, Mie theory can be simplified with the Rayleigh approximation
which states that the extinction efficiency is strongly wavelength dependent by a factor ~λ-4. Hence,
shorter wavelengths interact much more strongly with the particles.
For larger particles the dependence is more complex, with Mie theory predicting an oscillating
relation between extinction efficiency and particle size due to interference resonances. The
extinction efficiency of polystyrene spheres at 473 nm and 671 nm is plotted in Fig. 3.

Figure 3: Extinction efficiency of polystyrene spheres as function of sphere diameter, at 473 nm and
671 nm illumination. Commonly available spheres diameters are indicated.
4 LAB OVERVIEW
The first task of the exercise is to investigate light propagation and visibility when imaging in two
scattering solutions: a solution of intralipids (lipid emulsions used as a component of intravenous
nutrition for people who are unable to get nutrition via an oral diet) and a solution of single size
polystyrene spheres of unknown diameter.
You will use different illumination wavelengths and deploy optical filtering techniques to image an
object that is hidden inside the scattering solution. Specifically, you will investigate how spatial
Fourier filtering and structured illumination can improve visibility by suppressing signal
contribution from multiple light scattering. You will quantify the visibility by obtaining the
modulation transfer function of the optical system.

The second task is to investigate the scattering phase functions in dilute dispersions with varying
particle sizes and how they vary with illumination wavelength. Additionally, we compare the phase
functions to that of a dilute blood sample.

5 EXPERIMENTAL SETUP
The experimental setup (Fig. 4) used for this lab has an RGB laser diode system consisting of three
CW diode lasers at 473, 532 and 671 nm. The beam is coupled into one of two possible optical fibers:
One is used for the visibility experiment (task 1) and the other is used for the scattering phase
function experiment (task 2). The fibers rest on a vibrating fiber disruptor for suppressing speckles.

Figure 4: The optical setup used in the lab. The two beam paths, Visibility and Scattering phase
function, will be used for task 1 and 2 respectively. The fiber selector is used to switch between the
beam paths.
Visibility beam path: Light exiting the optical fiber is collimated and incident on one of two cuvettes
containing scattering solutions. Inside the first scattering solution is a hidden object which will be
imaged using a 5Mpixel sCMOS camera. At the Fourier plane of the camera objective is an aperture,
covered by a black cloth. In front of the cuvettes is a sinusoidal grating that can be flipped in or out
of the beam path.
Scattering beam path: Light exiting the optical fiber is collimated and incident on one a holder for
10 mm cuvettes, which will contain dilute scattering solutions. Scattered light from the cuvettes is
collected by a large lens, with focal on a white screen. The center of the screen has a small hole to
allow the strong ballistic light component to pass.

6 EXPERIMENTAL EXERCISES
TASK 1 - FAMILIARIZE WITH THE EXPERIMENTAL SETUP
• Make sure that the laser system is coupled to the correct fiber (Visibility/camera beam path)
and that the large cuvette (intralipids solution) is in the beam path.

• Turn on the three lasers while having the keys on OFF. Activate one laser after another by
turning the key to ON. Illuminate the sample first separately and then all together to create a
white illumination.

• Check the illumination profile for one color by flipping the paper before the cuvette. Is the
pattern homogeneous or is it “grainy”? How can you homogenize the beam profile?

Q1: Why does vibrating the fiber reduce the laser speckles pattern?

Q2: Which of the following is the dominant process in the solution (justify your answer):
(1) Scattering (σs ≫ σa), (2) Absorption (σa ≫ σs), or (3) Both are equally prominent (σs≈σa)?

• Start the imaging system: Make sure all lasers are off.

• Turn on the power switch on the back of the camera.

• Start the MATLAB script controlling the camera named: “MTF_v2” located on
the desktop.

• Press the Camera switch to “On” in the MATLAB interface.

• Wait until the camera has reached operation temperature (box turn blue)

• Start the Live stream of the camera.

TASK 2 - VISIBILITY
Exercise 1) Phantom solution 1 – Intralipids solution
An object is hidden within a cubic volume containing a scattering solution of intralipids, the first goal
will be to try to visualize the object:

• Start with the 473 nm laser and illuminate the sample. Gently increase the laser power to
~50% saturation. What do you see?

• Try with the other lasers of different wavelengths.

Q3: Which wavelength provides the best visibility in solution 1, and why?

Exercise 2) Modulation Transfer Function

The goal is to quantify the contrast at all spatial frequencies for the 671 nm illumination, at varying
depths in the medium. This corresponds to extracting the modulation transfer function.
• Move the cuvette (not the target), so that the target is close to the camera-
side cuvette wall.

• Move the camera so that the target is on focus. Then stop the live stream.

• Calibrate the target by clicking on “Calculate Center” and following the

instructions. Make sure that the target center is correctly identified by a
red cross.

• Press the “MTF” button to calculate the MTF for the current image.

• Press “Lock spectrum” to save the current MTF in the graph for
comparison.

• Move the target back to the illumination side of the cuvette and calculate
the MTF. The camera must be re-focused at the new location.

• Continuously updating the MTF while Live streaming might help with
focusing. To enable this, press the MTF button while in Live mode.

Q4: What does MTF measure and how is it calculated?

Q5: Why does the MTF change with the object’s position in the solution?

Exercise 3) Fourier filtering

An aperture placed in the focal plane of an imaging lens can be used as an optical Fourier filter.
Investigate how the filtering affects the contrast/MTF.

• Leave the target at the last position from the previous task and illuminate at 671 nm.

• Start Live mode (video and MTF) and press “Lock spectrum”.

• Remove the cover cloth on the camera objective and slowly close the aperture inside.

Q6: Why does optical Fourier filtering improve the contrast? What is the trade-off?
Exercise 4) Structured Illumination
Structured illumination (or periodic shadowing) can be used to “tag” non-scattered light and
subsequently remove it in post-processing. Typically, a sinusoidal stripe patten is used for which
three phase-shifted images is needed to perform the filtering. The same three images can also be used
to create a regular unfiltered image. Investigate how structured illumination is performed and how
this filtering affects the contrast/MTF.

• Open the aperture again to stop the Fourier filtering and put the cloth back on.

• Flip the sinusoidal grating into the beamline.

• Start Live mode and press “1D phases”. You will now see the horizontal profile of the image
in instead of an MTF graph.

• Press “Phase 1” to save your current image as the first phase-shifted image.

• Use the small traverse to move the sinusoidal stripe pattern 1/3 period across the image, then
save “Phase 2” image. Repeat for “Phase 3”.

• Stop the live stream and then press “Calculate SI” to compute the regular and structured
illumination image.

• Use the MTF tool to compare the contrast of the two images.

Q7: Why does structured illumination improve the contrast? What is the trade-off?

• Flip the sinusoidal grating out of the beamline and try to achieve the same contrast using
Fourier filtering. You may need to check the “lock” box next to “Calculate Center” while doing
this.

• Try illuminating at 532 nm and use your new filtering skills to improve the contrast.

Exercise 5) Phantom solution 2 – Polystyrene spheres

Now you are going to look through the solution with monodisperse polystyrene spheres. Turn off the
lasers and lift-up the target (upper knob).

• Slide the cuvettes so that the cuvette containing the polystyrene spheres is in the beam path.
Then lower the target into the solution.

• Start with the 671 nm laser and illuminate the sample. What do you see?

• Try with the other lasers.

Q8: Which wavelength gives the best visibility and what is likely the size of the polystyrene spheres in
solution 2? (Hint: look at Figure 3 showing the extinction efficiency)
TASK 3 - SCATTERING PHASE FUNCTIONS
Exercise 6) Scattering phase function as function of size and wavelength
Small cuvettes labeled A-D are available which contains dilute dispersions. One contains intralipids
solution and the other three contain polystyrene spheres that are either 2 µm, 5 µm or 10 µm in
diameter. Your goal is to deduce which is in each of the 4 cuvettes by observing the scattering pattern
formed on the white screen.

• Switch the optical fibers by carefully rotating the wheel in front of the laser system.
Light is now directed into a second beam path.

• Choose the 532 nm laser and compare the scattering pattern from each of the four samples.

• Examine how the pattern changes when you change wavelength for at least one of the
samples.

• Try illuminating with all three lasers at once. Adjust the laser powers to get white light.

Q9: What is the origin of the ring patterns and how they vary with particle size and wavelength?

Q10: Pair the 4 cuvettes (A-D) with the correct content (intralipids, 2 µm, 5 µm or 10 µm spheres).

Exercise 7) Blood cells

The 5th small cuvette contains a diluted blood sample.

• Illuminate the blood sample with a laser of your choosing and compare the pattern to those
of the other cuvettes.

Q11: What conclusions can you draw about the content of blood based on scattering pattern?