1) Flow cytometry works by passing cells in suspension through a laser or light beam which causes them to scatter light. Optical and electronic systems then collect and analyze these light signals. 2) Key components include fluidics to hydrodynamically focus cells, optics to illuminate cells and collect scattered light, and electronics to convert light signals to digital data. 3) Imaging flow cytometry enhances conventional flow cytometry by incorporating microscopy imaging of cells as they flow, allowing analysis of intracellular structures and distributions.

1. OPTICS
Anthony Schaeve
Spring 2017

2. OVERVIEW OF FLOW CYTOMETRY
The basic function of Flow Cytometry
technology is to measure and analyze
particles as they stream through a
beam of light. In this case these
“particles” are biological cells.
[1]
• Flow: The motion characteristics of
fluids
• Cyto: combining form of cells or
cells
• Metry: measurement

3. OVERVIEW OF FLOW CYTOMETRY
In order to accomplish
the system consists of
three main
components:
Fluidics
Optics
Electronical
components
[3]

4. FLUIDICS
The central core is subjected to
a focusing region where a
sheathing fluid is moving faster
than the central core. With this
action the cells are forced into
a single file line. Then they are
transported to an inspection
point.
[4]
After the cells are placed into a suspension it is then pumped into a small holding area called a
central core.

5. OPTICS SYSTEM: LIGHT SCATTER
[5]
When the light hits the cell,
forward scattered light and
side scattered light is
produced.
Cells are transported to the inspection or excitation point via the sheathing fluid. At this point an arc
lamp or laser is used to illuminate the cell.

6. OPTICS SYSTEM: CELL CHARACTERISTIC
Scatters represent different
characteristics of each cell.
In addition, a fluorescent
dye can be used to identify
a particular cell type.
A simplified set-up is shown
here
[3]

7. OPTICS SYSTEM
The BD FACS Vantage SE is a
more complicated set-up but
reflects a more realistic optics
system.
Here 3 lasers are used for
excitation instead of arc lamps.
[1]

8. ELECTRONICS SYSTEM: SIGNAL DETECTION
• The electronic system is used to convert light signals into electrical signals.
Light signals are generated as cells pass through the
laser beam. These light signals are converted to
electronic signals by photodetectors.
[2]
The peak of the pulse occurs when the cell is in the
exact center of the beam. This when the maximum
amount of scatter occurs.

9. ELECTRONICS SYSTEM: PHOTODETECTORS
• Semiconductors can be used to detect photons
• These types of semiconductors are called photodetectors
• They transform optical signals into electrical signals
[8]
A phototube is a device that contains a
chamber and is either filled with gas or is a
vacuum so that light passes through it.
Phototubes are extremely sensitive and
detect ultraviolet, visible, and infrared light.

10. ELECTRONICS SYSTEM: SIGNAL CONVERSION
• Analog-to-Digital converters (ADC) translate analog signals such as voltage
or light intensity into a digital representation of that signal.
• This digital representation can then be processed, computed, and stored.
[9]

11. ELECTRONICS SYSTEM: DATA ANALYSIS
• Flow cytometric data is filed and stored in a standard
format which was developed by the Society for
Analytical Cytology.
• This includes the description of the sample, instrument
in which the data was collected, the data set, and
results of analysis.

12. DATA ANALYSIS: DENSITY PLOT
[13]
Forward Scatter (FSC)
FSC is a rough measure of size and is influenced by the wavelength of light and
the angle, among others.
Side Scatter (SSC)
SSC is a measure of light scattered at an angle of
90º (orthogonal).
This gives a view of the complexity of the cell’s
internal structures.
The more ‘granular’ a cell is the higher its SSC will
be.
Here, a neutrophil is much more granular than a
lymphocyte.

13. DATA ANALYSIS: HISTOGRAMS
The histogram shows the total number of cells in
a sample that possess certain physical properties
selected for or which express the marker of
interest. Cells with the desired characteristics are
known as the positive dataset.
Single-parameter histogram displays a single
measurement parameter such as fluorescence
or light scatter intensity on the x-axis and the
cell count on the y-axis.
[12]

14. DATA ANALYSIS: GATING
An important principle of flow cytometry data analysis is to selectively
visualize the cells of interest while eliminating results from unwanted
particles e.g. dead cells and debris. This procedure is called gating.
Gating helps to prevent
data being collected
when two cells stick
together or pass through
the intercept point too
closely.
[16]

15. DATA ANALYSIS: GATING
Plotting a time vs a scatter plot
to see how even the flow was
during the run will help eliminate
artifacts caused by poor flow.
Time Gating
[16]
If an unstable flow stream is established, data results will be questionable.

16. FLOW CYTOMETRY & COMPONENTS

17. FLUORESCENTS
Fluorescent probes enable researchers to detect particular components of
complex biomolecular assemblies
A basic principle in fluorescence is the specific
visualization of cellular components with the
help of a fluorescing agent.
[10]

18. FLUROPHORES
[11]
• Ground State: Before Absorption
• Excited state
• Emission of lower energy photons
Flurophores such as Ethidium Bromide
can be used to visual the protein of
interest.
This emission of photons can be detected through the
previous mentioned photodetectors

19. FLUORESCENTS
• The X-axis is the amount of red fluorescence
• The Y-axis is the amount of blue fluorescence
The more red fluorescence a cell emits, the
farther to the right the cell data will appear on
the histogram. The more blue fluorescence a
cell emits, the cell data will appear closer to the
top on the histogram.
[14]
Resulting Graph Example

20. BUILDING ON THE CONCEPTS OF FLOW CYTOMETRY
IMAGING FLOW CYTOMETRY
[20]
• Recent advances in imaging technologies,
electronics, and digital computing have enabled
imaging flow cytometry (IFC).
• As an integration of fluorescence microscopy
and conventional flow cytometry, IFC combines
flow cytometry's cell identification and high
throughput with microscopy's cell image
acquisition.

21. IMAGING FLOW CYTOMETRY:
TEMPORALLY CODED EXCITATION
[19]
Camera-based Imaging Flow Cytometry
Motion blur occurs when the exposure
time is longer than the time it takes the
flowing cell to move a minimum
resolvable distance. This technique
exploits temporally coded excitation
which effectively eliminates motion
blur for fluorescence imaging of
flowing cells.

22. TEMPORALLY CODED EXCITATION
[20]
Reconstruction Technique

23. TEMPORALLY CODED EXCITATION
[20]
Wiener Filter
Another image reconstruction technique

24. TEMPORALLY CODED EXCITATION
[20]
Limitations:
Wiener filters are unable to reconstruct frequency components which
have been degraded by noise. They can only suppress them.
Wiener Filter
Another image reconstruction technique

25. IMAGING FLOW CYTOMETRY
The Image Stream Mark II
Imaging Flow Cytometer is a
commercially available
product for researchers and the
medical community.
[17]
Camera-based Imaging Flow Cytometry

26. IMAGING FLOW CYTOMETRY
[17]
Camera-based Imaging Flow Cytometry
ImageStream uses high-speed CCD
cameras with a time delay technique that
can detect fluorescence signals without
motion blur caused by an increase in
exposure time.

27. IMAGING FLOW CYTOMETRY
[17]
This type of CCD provides higher sensitivity by having multiple rows of sensors which shift
their partial measurements to the adjacent row synchronously with the motion of the moving
cell image across the array of sensors.

28. IMAGING FLOW CYTOMETRY
[18]
Instead of reading fluorescence using
a photomultiplier, the signals from
these cells are imaged as the cells
traverse the light source.
These images show the
inhomogeneous distribution of the
label within the cells.

29. IMAGING FLOW CYTOMETRY
[17]

30. IMAGING FLOW CYTOMETRY
• Flow cytometry is finding increasing use in clinical laboratories for the
diagnosis and monitoring of disease.
• Applications of imaging flow cytometry can be used for the assessment of
acute leukemia.
• Imaging flow cytometry has been used to study erythrocytes erythroid cell
maturation, sickle cell disease, and infectious diseases such as malaria.

31. REFERENCE
[1] Flow Cytometry Jahan-Tigh, Richard R. et al. Journal of Investigative
Dermatology , Volume 132 , Issue 10 , 1 - 6
[2] Introduction to Flow Cytometry: A Learning Guide, Becton, Dickenson and
Company
[3] Flow Cytometry Andrew Wetzel 22 March 2011
https://prezi.com/4wqeuvsd9qff/cell-separationflow-cytometry/ )
[4] https://www.bio-rad-antibodies.com/flow-cytometry-fluidics-system.html
[5] https://www.youtube.com/watch?v=EQXPJ7eeesQ
[6] Photodetectors, Engr Syed Absar Kazmi :
https://www.slideshare.net/engrabsarkazmi1/photodetectors-46504354
[7] Ge Transimpedance Amplified Photodetectors
https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=947
[8] Photodetector http://www.tech-faq.com/photodetector.html
[9] https://wiki.analog.com/university/courses/electronics/text/chapter-20

32. REFERENCE
[10] http://www.leica-microsystems.com/science-lab/fluorescent-dyes/
[11] https://www.youtube.com/watch?v=SGFlr1jFNBM
[12] http://www.assay-protocol.com/cell-biology/flow-cytometry/flowcytometry-
diagrams
[13] https://www.slideshare.net/richardhastings589/kumc-introduction-to-flow-
cytometry
[14] http://unsolvedmysteries.oregonstate.edu/flow_07
[15] https://expertcytometry.com/3-flow-cytometry-gates-that-will-improve-the-
accuracy-of-your-facs-data-analysis/
[16] https://expertcytometry.com/how-to-create-flow-cytometry-gates/
[17] https://www.youtube.com/watch?v=5SD7Q-RtPb4
[18] http://www.sharpedgelabs.com/imaging-cytometry/

33. REFERENCE
[19] http://pubs.rsc.org/EN/content/articlehtml/2016/lc/c6lc01063f
[20] http://www.bostonphotonics.org/files/seminars/20130313-ESchonbrun.pdf