Principles of flow cytometry: optics, fluorescent excitation/emission &

compensation

Shoreline Community College BIOL 288

Components of a Flow Cytometer

Fluidics Optics Electronics

Sheath Sample

Light source (lasers) Optical path (filters & mirrors) Light collection (detectors)

Detector signal processing Computer interface Data storage & output

• The optics are the heart of the cytometer.

There are three major components to the optical system of a cytometer.

Lasers – Excitation Light Source

Mirrors and Filters – Direct and focus light to the interrogation point and to the light collection point

Detectors – collect the light energy

Optics

Optics

Laser sources

Steering Optics

BD LSR II

Optics

Beam focusing

Flow Cell

Optics

Flow Cell

Fluorescence & Side scatter

Sheath fluid

Focused Laser Beam

Forward scatter

Sample Injector Tip

Fluidics Optics

Flow Cell

Optics

Fluorescence and Side Scatter (SSC)

Forward Scatter (FSC)

Optics

Laser sources

Steering Optics

BD LSR II

Fiber Optic Launch

Optics

Fiber optic cables

Flow Cytometer

488nm Blue laser

633nm Red laser FL1

FL2 FL3

FL4

Fluorescent detectors

Flow Cell

Sample Intake Probe (SIT)

Sheath fluid Cleaning and Waste

Optical System

Fluidic System

Detectors or Photomultiplier Tubes (PMT’s) collect light energy not colors.

Filters

The filters and mirrors direct specified wavelength of light to the detector with the final filter in front of the detector the most selective and determines what “color” is seen

530/30 nm

530 +/- 15 {515 – 545} nm range

Filters

488nm SP

Wavelength above 488nm

Wavelength below 488nm

Bandpass Filter

Shortpass Filter

Longpass Filter

650nm LP

Wavelength below 650nm

Wavelength above 650nm

Optics

Fiber optic cable

Absorption

Excitation

Emission

The excitation wavelength is always a higher energy than the emission wavelength

Lower energy=longer wavelength Higher energy=shorter wavelength

?? What is the difference between Absorption and Excitation??

Common Fluorescent Markers

Dye or Protein

Excitation Maximum

Emission Maximum

Pacific Blue 405 455

Pacific Orange 405 551

BV605 405 605

PE 480;565 578

PE-Cy7 conjugates 480;565 767

PE-Texas Red 480;565 613

FITC 495 519

PerCP-Cy5.5 490 694

EGFP 484 507

EYFP 514 527

mCherry 587 610

APC 650 660

APC-Cy7 conjugates 650;755 767

Alexa Fluor 680 679 702

Spectral Spillover

Compensation

uncompensated

How does the software calculate Compensation?

Compensation

Single stained controls

Compensation

Compensation

FSC Sensor

Compensation

Compensated data will have spreading error

The use of FMO’s helps for gating on dim positive events

Panel Design Pair the dimmest marker to your brightest fluorochrome. Utilize the Staining Index (SI) to determine brightness of dyes.

But remember minimizing spillover especially when trying to detect a dim population in combination with other highly expressed ones may require alternate dye choices