3. PRINCIPLE:
• The basic principles of flow cytometry is the
passage of cells in single file in front of a laser so
they can be
• detected
• counted
• sorted
• Cell components are fluorescently labelled and
then excited by a laser to emit light at varying
wavelengths. 3
5. MECHANISM:
1.Biological sample
2. label it with a fluorescent marker
3. cells move in a linear stream through a focused light source
(laser beam)
4. fluorescent molecule gets activated and emit light that is filtered
and detected by sensitive light detectors.
5. conversion of analog fluorescent signals to digital signals.
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6. A SORTER CAN BE DIVIDED INTO 3
SUBSYSTEMS
• .
Fluidics
Optics
electronics
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7. SUBUNITS
Fluidics:
to introduce and focus the cells for interrogation and
create a stable breakoff for sorting.
Optics:
to generate and collect the signal lights.
Electronics:
To convert the optical signals to proportional digital
signals process the signal and communicate with the
computer.
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8. FLUIDICS:
Sample is
* introduced into running sheath.
* hydrodynamically focused into core
stream.
* sheath fluid and sample do not mix.
* reduction of cross section.
* sample flow rate adjustable.
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9. TWO TYPES OF OPTICS:
Excitation optics:
it consists of * laser
* fibre optic cables
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10. COLLECTION OPTICS:
• * fibre optic cables that direct the emitted light
to appropriate emission filter block.
• * filter that direct the signals in the emission
block to the appropriate photon multiplier tube
(PMT).
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11. FSC
• Laser light is scattered in a
forward direction .
• the intensity of this signal
has been attributed to cell
size, refractive index
(membrane permeability).
• laser light is scattered at
900 to the axis of the laser
path.
• The intensity of this signal is
proportional to the amount
of cytosolic structure in the
cell (eg : granules ,cell
inclusions etc..,)
SSC
OPTICS:
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13. FLOW CYTOMETRY IN
BIOPROCESS:
• It is used for cell analysis and quantification
especially both bacteria and yeast in food,
drink and pharmaceutical industries.
• This technique is used in microbial
fermentation monitoring and control as well as
in development of more accurate kinetic
models directed to bioprocesses optimisation.
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14. APPLICATIONS OF FLOW
CYTOMETRY:
• Cell function
• DNA and RNA analysis
• Cell death
• Sorting and cell isolation
• Immunophenotyping
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15. FLOW CYTOMETRY
• Commonly found in research labs ,
user friendly.
• Cannot sort cells.
• Intuitive software interfaces.
• More personalized ,designed for
researchers.
• Sort heterogeneous mixture of cells
into different population.
• Automated features.
FACS
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COMPARISION:
16. FLUORESCENT ACTIVATED CELL
SORTING OF LIVE CELLS
• FACS is a particular form of cytometry that enables a
mixture of different cells to be sorted one by one by
one or more containers.
• cells are sorted according to their specific light
scattering and fluorescent characteristics.
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18. PROCESS OF FACS:
• Individual cells are interrogated by the laser as
in a normal flow cytometer.
• The machine is setup so that each individual
cell that enters a single droplet as it leaves the
nozzle tip.
• This drop is given an electronic charge ,
depending on the fluorescence of the cell inside
the drop.
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19. PROCESS OF FACS:
• Deflection plates attracts or repels the cells
accordingly into the collection tubes. For eg
• single FITC stained cell in a single droplet
would be given a positive charge and be
attracted to the right. Collection tube to the
right would collect all positively charged
FITC stained cell droplets.
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20. PROCESS OF FACS:
• A Single PE stained cell in a single
droplet would be given a negative
charge and be attracted to the left.
Collection tubes to the left would collect
all negatively charged PE stained cell
droplets.
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