Digital flow cytometry

Sri krishna jayadev. M

Department of life sciences
Shiv Nadar University
 Commercial Instruments
BC
BC

Luminex Bryte

Bay
Biosciences

Accuri

Millipore/Guava

BC

Amnis

BD BD Aria

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BD Influx Apogee
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12:44 PM ©1990-2012 J. Paul Robinson, Purdue University BMS 631
 Applications of Flow Cytometry.

• Cell size.
• Cytoplasmic granularity.
• Cell surface antigens (phenotyping).
• Apoptosis.
• Intracellular cytokine production.
• Intracellular signalling.
• Gene reporter (GFP).
• Cell cycle, DNA content, composition, synthesis.
• Bound and free calcium.
• Cell proliferation (BRDU and CFSE)
• Cell sorting, single cell cloning (clonecyt)
 Definition
Flow cytometry is a technology that simultaneously measures and
then analyses multiple physical and chemical characteristics of single
particles, usually cells, as they flow in a fluid stream through a beam
of light, usually a laser.
What can a flow cytometer tell us about a cell?

 Relative size (forward scatter-FSC)

 Relative granularity ( Side scatter-SSC)

 Relative flourescence intensity (FITC, PE, PerCP, APC etc)

Forward Scatter – Diffracted light
 Related to cell surface area

 Detection along axis of incident light in the forward direction

Side Scatter- reflected and refracted light

 Cell granularity and complexity

 Detection at 90 ° to the laser beam

 Light scattering
Scattering
(Mie, Rayleigh)
Particles

Absorption
Incident Beams

Emission
(Fluorescence)

The interaction of light with a particle in terms of scattering,

absorption, and emission.

J. Paul Robinson, Purdue university

Basic components of a flow cytometer
Flow cytometer

Fluidics Optics Electronics

It is to introduce and To generate and collect To convert the optical

focus cell for interrogation. light signals signals to digital signals

 Cells in suspension  Illuminate the sample

 Flow in single file through  Scatter light and emit fluorescence
 Light is filtered and collected
A cytometer consists of:

Electronics

Fluidics
Light detectors
FL3 PerCP, Cy5

FL2 PE
Computer

FL1 FITC

SSC

Laser
Light FSC

488nm
 Fluidics
The introduction of one smoothly flowing stream of
fluid (core) into the center of another smoothly
flowing stream (sheath fluid) in such a way that the
core becomes focused along a central axis is called
hydrodynamic focusing or sheath flow.

When conditions are right, sample fluid flows in a

central core at uniform velocity and position that
does not mix with the sheath fluid. This is laminar
flow.
 Sample Flow and Differential pressure

The flow characteristics of the central fluid can be estimated using Reynolds number (Re)

Re = pVD/μ
p= density of the fluid, V= mean velocity of the fluid, D= Diameter of the tube, μ= viscosity of
the fluid
 Optics
Optics is further classified into two categories

Excitation optics consists of :

lasers

Filters and mirrors that route the laser light to the fluidic stream.

Collection optics consists of :

Fiber optics that direct the emitted light to the appropriate emission block

Filters that direct the signals in the emission block to the appropriate detector i.e.
photomultiplier tube (PMT).
Excitation optics:
Lasers

Provides coherent single wavelength of light .

Output power ranges from mWatt to Watts of light

Regularly used lasers are

Blue ---488 nm

Red ---633 nm

UV ----355 nm

Violet---405 nm
 Collection optics
Collection / focusing lens that is optically coupled to the quartz flow cell to maximize
detection sensitivity.

Fixed alignment optical path to direct light from the flow cell to detector

Optical filters and dichroic mirrors provide separation of detector channels and wavelength
selection.

Light detectors

Photosensitive diodes
Photomultiplier tubes
Optical filters layout
Detectors and Filters
Stokes shift
Size and Granularity

Size
 Presenting and Interpreting Data

Single parameters can be displayed as a histogram.

Dual parameter data can be displayed in two dimensions using dot, density or
contour plots.
Gating:
Back-gating:
Available analysis software

• Cellquest
• WinList
• Flowjo
• WinMDI