Flow Cytometry
~Priyanka, Roll no 25, Second Year
B.Pharm.~
internal complexity,
size
shape
number
structure
• sorts cells based on their
size
shape
color of light they fluoresce
• detector measures changes in light:
Scattered (Shape, size)
Absorbed (Shape, Size)
Emitted (nature of cells, kind of fluorescent tag they are
having)
• Based on these measurements, specific types of cells are
recognized and sorted by the sorter by manipulating their
charge.
Most commonly analyzed materials are:
– blood
– bone marrow aspirate
– lymph node suspensions.
• 0.2–150 micrometers
• HAS 3 KEY SYSTEMS:
FLUIDICS-Cells in suspension are brought in single file…
OPTICS-…past a focused laser which scatter light and emit
fluorescence that is filtered and collected.
ELECTRONICS-Emitted light is converted to digitized values
that are stored in a file for analysis
FLUIDICS
•PRINCIPLE OF HYDRODYNAMIC FOCUSING.
•'Only one cell or particle can pass through the laser
beam at a given moment.'
•The flow of sheath fluid accelerates the particles and
restricts them to the center of the sample core. This
process is known as hydrodynamic focusing.
•The sample pressure is always higher than the sheath
fluid pressure, ensuring a high flow rate allowing more
cells to enter the stream at a given moment.
through which
the sample is
injected.
contains
faster
flowing
fluid
(0.9%
Saline)
enclosing
the central
core.
OPTICS
• Light Source:
1. Laser (common)
2. Arc lamp
• Why Lasers more common?
highly coherent + uniform.
can be easily focused on a very small area (like a sample
stream).
monochromatic, emitting single wavelengths of light.
– argon ion laser(blue to blue-green) commonly used
because the 488-nm light that it emits excites more than
one fluorochrome.
#1 Light Scattering
Forward Scatter
Side Scatter
• Factors that affect light scattering :
cell membrane
nucleus
any granular material inside cell
cell shape
surface topography
SSC
FSC
Forward-scattered channel (FSC) |cell-surface area or size
Side-scatter channel (SSC) |cell shape & amt of cytosolic
struct. (eg. granules, cell inclusions)
#2 EMISSION/FLUOROSCENCE
The amount of fluorescent signal
detected is proportional to the
number of fluorochrome
molecules on the particle.
Commonly used Fluorochromes
FLUOROCHROMES EMISSION MAXIMUM
Fluorescein Isothiocynate (FITC) 530nm
Phycoerythrin (PE) 576nm
Peridin-chlorophyll alpha complex (PerCP) 680nm
Allophycocyanin (APC) 660nm
Texas red 620nm
ECD( PE - Texas Red Tandem) 615nm
PC5 (PE - cyanin 5 dye tandem) 667nm
Photodetectors
• convert photons to electrical impulses.
– Photodiode -forward scatter signals
– Photomultiplier tube (PMT) -side scatter and fluorescent
signals.
• Amplifies pulse & digitalises the amplified
pulse
FILTERS
Dichroic Filters- Long pass or short pass
filters. Type of beam splitters .
ELECTRONICS- Creation of a Voltage
Pulse
• Photons { PMT or Photodiode
electrons current}{Amplifier Voltage
Pulse}
• ParticlelaserFilterPhotodetector
AmplifierDETECTORCOMPUTER DATA
DATA ANALYSIS
• graph of INTENSITY
versus NUMBER OF CELLS
• indicates the
fluorescence intensity
detected against the
number of times it was
detected.
Plot Types
Histogram
number
of events
(cells)
counted
intensity of the detected signal
Contour Plot Density Plot
Greyscale Density Dot Plot
DATA COLLECTION AND STORAGE
• Flow cytometric data is stored according to
the flow cytometry standard (FCS) format,
developed by the Society for Analytical
Cytology.
PROS OF FCM
OVER CONVENTIONAL METHODS
• Enable measurements in excess of 100,000 cells.
• increased sample size means acquisition of
statistically significant results and the detection
of rare cell types.
• Measurement of single cells, identification of sub
populations
• Efficient and fast
• can detect and sort multiple kinds of
characteristics of a single type of cell.
CONS OF FCM
Cannot tell intracellular location and distribution of
proteins
Pre treatment of cells for fluorescent staining is time
consuming
Expensive and needs highly trained technicians
Aggregates or debris can give false results
CLINICAL APPLICATIONS OF FCM
1) Positive selection and isolation of clones
2) Diagnosis of diseased state
3) Toxicity profile monitoring,
4) Detection of pollutants and their effects
REFERENCES
• http://www.d.umn.edu/~biomed/flowcytometry/introflowcytomet
ry.pdf
• http://www.slideshare.net/tashagarwal/flow-cytometry-
46618943?next_slideshow=1
• https://www.youtube.com/watch?v=sv_5h7GjZL
• https://www.youtube.com/watch?v=ZBqg0ShradQ
• http://www.slideshare.net/annasilvina/flowcytometry-basics
THANK YOU!
