L9 - Flow Cytometry II

Why not physical wall?

• Diameter of cells are different
• Wall may be too thin
• Choice of material

Scattering

Forward Scatter (FSC)

• $\propto$ cell size
• Obscuration bar is used to block light when there is no cell

Side Scatter (SSC)

• $\propto$ granularity and complexity
• Detected at $90^{\circ }$

2D Scatter Plot

Immunophenotyping

Flow cytometry can be used to distinguish between healthy and diseased cells. (e.g. myelomas, lymphomas, leukemias)

Fluorescence Imaging

Fluorochromes

Absorbs light energy $\to$ excitation
Re-emit at a longer wavelength $\to$ emission (in all direction)

The process of emission (fluorescence) is in the degree of $\mathrm{ns}(10^{-9})$

$E=hf\propto \frac{1}{\lambda }$
By conservation of energy, the emitted photons must have a lower energy.
In order words, lower frequency, longer wavelength).
e.g. $488\mathrm{nm}$ blue light to $520\mathrm{nm}$ green light

Excitation and Emission Spectra

Each fluorochrome has a unique excitation and emission spectra.

The emission spectra is always fixed.

Cellular Markers

• Voltage mathrmlse $\propto$ amount of fluorescence

Optical Filters

Pass through	Dichroic
Absorb	Reflect

Short pass	Long pass	Band pass
e.g. $<650\mathrm{nm}$	e.g. $>550\mathrm{nm}$	e.g. $550-650\mathrm{nm}$

Gating

1. Direct the analysis software to consider only certain cell type.
2. Produce additional histograms and dot plots to dissect subpomathrmlations, which have been labeled with fluorescent antibodies. (functional phenotyping)

Power of Cytometry

• Better understanding of immune mechanisms
• Correlation of cell analysis to genomics and proteomics
• $N$ fluorophores + FSC + SSC = $N+2$ parameters
• routine clinical use
  • $50000cells/s$
  • $7-8$ colors