Flow Cytometry Principles practice of Fluorescence Spectroscopy in

Flow Cytometry Principles & practice of “Fluorescence Spectroscopy in Biological Diagnosis & Research” Dr. Hekmatimoghaddam Assistant professor of pathology

Definitions n Flow Cytometry – Measuring properties of cells in flow n Flow Sorting – Sorting (separating) cells based on properties measured in flow – Also called Fluorescence-Activated Cell Sorting (FACS)

Basics of Flow Cytometry Fluidics • Cells in suspension • flow in single-file through • an illuminated volume where they Optics • scatter light and emit fluorescence • that is collected, filtered and Electronics • converted to digital values • that are stored on a computer

Fluidics n Need to have cells in suspension flow in single file through an illuminated volume n In most instruments, accomplished by injecting sample into a sheath fluid as it passes through a small (50 -300 µm) orifice

Flow Cell Injector Tip Fluorescence signals Focused laser beam Sheath fluid

Fluidics n When conditions are right, sample fluid flows in a central core that does not mix with the sheath fluid n This is termed Laminar flow

Fluidics n The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing

Fluidics - Differential Pressure System n Use air (or other gas) to pressurize sample and sheath containers n Use pressure regulators to control pressure on each container separately

Fluidics - Differential Pressure System n Sheath pressure will set the sheath volume flow rate (assuming sample flow is negligible) n Difference in pressure between sample and sheath will control sample volume flow rate n Control is not absolute - changes in friction cause changes in sample volume flow rate

Fluidics - Differential Pressure System C. Göttlinger, B. Mechtold, and A. Radbruch

Fluidics - Particle Orientation and Deformation “a: Native human erythrocytes near the margin of the core stream of a short tube (orifice). The cells are uniformly oriented and elongated by the hydrodynamic forces of the inlet flow. b: In the turbulent flow near the tube wall, the cells are deformed and disoriented in a very individual way. v>3 m/s. ” V. Kachel, et al. - MLM Chapt. 3

Fluidics - Flow Chambers Flow through cuvette (sense in quartz) H. B. Steen - MLM Chapt. 2

Flow Cell Injector Tip Fluorescence signals Focused laser beam Sheath fluid

Optics n Need to have a light source focused on the same point where cells have been focused (the illumination volume) n Two types of light sources – Lasers – Arc-lamps

Optics - Light Sources n Lasers – can provide a single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths – can provide from milliwatts to watts of light – can be inexpensive, air-cooled units or expensive, water-cooled units – provide coherent light

Optics - Light Sources n Arc-lamps – provide mixture of wavelengths that must be filtered to select desired wavelengths – provide milliwatts of light – inexpensive, air-cooled units – provide incoherent light

Optics - Forward Scatter Channel n When a laser light source is used, the amount of light scattered in the forward direction (along the same axis that the laser light is traveling) is detected in the forward scatter channel n The intensity of forward scatter is proportional to the size, shape and optical homogeneity of cells (or other particles)

Forward Angle Light Scatter Laser FALS Sensor

Optics - Side Scatter Channel n When a laser light source is used, the amount of light scattered to the side (perpendicular to the axis that the laser light is traveling) is detected in the side or 90 o scatter channel n The intensity of side scatter is proportional to the internal structure and granularity of cells (or other particles)

90 Degree Light Scatter Laser FALS Sensor 90 LS Sensor

Optics - Light Scatter n Forward scatter tends to be more sensitive to surface properties of particles (e. g. , cell ruffling) than side scatter – can be used to distinguish live from dead cells n Side scatter tends to be more sensitive to inclusions within cells than forward scatter – can be used to distinguish granulated cells from non-granulated cells

Fluorescence Detectors Laser FALS Sensor Fluorescence detector (PMT 3, PMT 4 etc. )

Optics - Filter Properties n Long pass filters transmit wavelengths above a cut-on wavelength n Short pass filters transmit wavelengths below a cut-off wavelength n Band pass filters transmit wavelengths in a narrow range around a specified wavelength – Band width can be specified

Standard Long Pass Filters Light Source 520 nm Long Pass Filter Transmitted Light >520 nm Light Standard Short Pass Filters Light Source 575 nm Short Pass Filter Transmitted Light <575 nm Light

Standard Band Pass Filters 630 nm Band. Pass Filter White Light Source Transmitted Light 620 -640 nm Light

Optics - Filter Properties n When a filter is placed at a 45 o angle to a light source, light which would have been transmitted by that filter is still transmitted but light that would have been blocked is reflected (at a 90 o angle) n Used this way, a filter is called a dichroic filter or dichroic mirror

Dichroic Filter/Mirror Filter placed at 45 o Light Source Transmitted Light Reflected light

Optics - Filter Layout n To simultaneously measure more than one scatter or fluorescence from each cell, we typically use multiple channels (multiple detectors) n Design of multiple channel layout must consider – spectral properties of fluorochromes being used – proper order of filters and mirrors

Common Laser Lines 350 300 nm 457 488 514 400 nm 500 nm 610 632 600 nm 700 nm PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid

Example Channel Layout for PMT Laser-based Flow Cytometry 4 Flow cell PMT Dichroic Filters 3 PMT 2 Bandpass Filters PMT 1 Laser

Optics - Detectors n Two common detector types – Photodiode § used for strong signals when saturation is a potential problem (e. g. , forward scatter detector) – Photomultiplier tube (PMT) § more sensitive than photodiode but can be destroyed by exposure to too much light

Summary of Part 1 Fluidics • Cells in suspension • flow in single-file through • an illuminated volume where they Optics • scatter light and emit fluorescence • that is collected, filtered and Electronics • converted to digital values • that are stored on a computer

Typical Research Cytometer (Coulter 753) (1980 s) $200 -300, 000 Detectors Lasers Fluidics Computers Laser Power Supply

Typical Clinical Cytometer Computer System Detector & Mechanical $90 -120, 000 Fluidics

Clinical Applications Of Flow Cytometric Analysis Flow. Cytometric(immunophenotypic) Classification Of Leukemias

Immunophenotyping CD 4 CD 2

Immunophenotyping . 1 1 10 Log FITC 1000

CD 4/CD 8 Quadstats 1 2 45% 2% 27% 3 . 1 1 26% 10 100 Log FITC Fluorescence (CD 8) 4 1000

The Cell Cycle G 2 S M G 1 G 0 Quiescent cells

Normal Cell Cycle G 2 M G 0 DNA Analysis G 1 s G 0 G 1 C o u n t s 0 200 400 G 2 M 600 4 N 2 N DNA content 800 1000

DNA Analysis DNA index 1. 21 Aneuploid peak 0 200 400 600 PI Fluorescence 800 1000

Reticulocyte Analysis RMI = 34 RMI = 0 R 4 R 3 R 2 R 1 . 1 1 10 100 log Thiazole Orange R 4 R 3 R 2 R 1 1000 . 1 1 10 1000 log Thiazole Orange

Light Scatter Gating Side Scatter Projection Neutrophils Scale 1000 200 100 50 40 Monocytes 30 20 15 Lymphocytes 8 0 200 400 600 800 90 Degree Scatter 1000