Flow Cytometry and Confocal Microscopy (2019) PDF

László Mátyus Flow cytometry and cell sorting Confocal microscopy Debrecen, November 2019 THE BEGINNING THE COULTER BROTHERS  DEFINITIONS Flow Cytometry – Measuring properties of cells in flow Flow Sorting – Sorting (separating) cells based on properties...

László Mátyus Flow cytometry and cell sorting Confocal microscopy Debrecen, November 2019 THE BEGINNING THE COULTER BROTHERS  DEFINITIONS Flow Cytometry – Measuring properties of cells in flow Flow Sorting – Sorting (separating) cells based on properties measured in flow – Also called Fluorescence- Activated Cell Sorting (FACS)  WHAT CAN FLOW CYTOMETRY DO? Enumerate particles in suspension Determine “biologicals” from “non- biologicals” Separate “live” from “dead” particles Evaluate 105 to 106 particles in less than 1 minute Measure particle-scatter as well as fluorescence Sort single particles for subsequent analysis  WHAT ARE THE PRINCIPLES? Light scattering by particles (laser or arc lamp as light sources) Specific fluorescence detection Hydrodynamically focused stream of particles Electrostatic particle separation for sorting Multivariate data analysis capability  BASICS OF FLOW CYTOMETRY Cells in suspension Fluidics flow in single-file through an illuminated volume where they Optics scatter light and emit fluorescence that is collected, filtered and converted to digital values Electronics that are stored on a computer HYDRODYNAMIC SYSTEMS Sample in Sheath Piezoelectric crystal oscillator Fluorescence Sensors Laser beam Scatter Sensor Sheath Core FLUID NOZZLES  OPTICS Need to have a light source focused on the same point where cells have been focused (the illumination volume) Two types of light sources – Lasers – Arc-lamps  OPTICS - LIGHT SOURCES 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  LASER LINES - FLUORESCENT DYES Common300 nm 350 400 nm 457 488 514 500 nm 610 632 600 nm 700 nm Laser Lines PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid OPTICAL CONFIGURATION DOUBLE BEAM INSTRUMENT OPTICS - OPTICAL CHANNELS An optical channel is a path that light can follow from the illuminated volume to a detector Optical elements provide separation of channels and wavelength selection FORWARD ANGLE LIGHT SCATTER (FALS) FALS detector  OPTICS - FORWARD SCATTER CHANNEL 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 The intensity of forward scatter is proportional to the size, shape and optical homogeneity of cells (or other particles)  o 90 LIGHT SCATTERING  OPTICS - SIDE SCATTER CHANNEL 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 90o scatter channel The intensity of side scatter is proportional to the size, shape and optical homogeneity of cells (or other particles)  OPTICS - LIGHT SCATTER Forward scatter tends to be more sensitive to surface properties of particles (e.g., cell ruffling) than side scatter – distinguishes live from dead cells Side scatter tends to be more sensitive to inclusions within cells than forward scatter – distinguishes granulated cells from non-granulated cells  TYPES OF OPTICAL FILTERS Wavelengths LONG (700nm) Short Pass Long Pass Band Pass Pass Through Filters 650 Short Pass 550 Long Pass 600/100 Band Pass SHORT (500nm) (600SP) (650LP) (600/100) Transmitted 550 nm 550 - 650 nm (600±50) Blocked >650 nm 650 nm