An optical diagnostic technique used to measure
physical and chemical characteristics of cells
Applied in both research and clinical laboratories
for disease profiling
Applications
- Rapid and sensitive screening for biological
contamination (e.g., bioterrorism).
- Agricultural and livestock research.
- Clinical diagnostics and biomedical research.
Flow Cytometry = Analysis of cells or particles in suspension passing single-file through a laser beam.
Optical Detection
Measures light scattering and fluorescence emission.
Fluorescence typically enhanced using fluorochrome-labeled antibodies or dyes.
Generates multiparametric data (size, granularity, fluorescence intensity).
Key Advantages
Analyzes up to 75,000 cells/sec.
Enables cell sorting (FACS) by optical and electrical gating.
Provides real-time, high-throughput cellular analysis.
Significance
Flow cytometry bridges biology, optics, and informatics —providing rapid, quantitative insight into the
structure and function of living cells.
Light Source: Stable, low-noise lasers; single/multi-excitation lines. Trend from gas lasers (Ar⁺, HeNe) to
compact DPSS/diode lasers (405, 488, 532, 635–670 nm); NIR (700–800 nm) reduces autofluorescence.
Flow Cell (Hydrodynamic Focusing): Core–sheath coaxial laminar flow; single-file cell alignment; velocity ≳1
m/s; core diameter ≪ beam width; syringe/pressure control; trade-offs via sheath pressure & ΔP.
Illumination Optics: Beam shaping to elliptical spot (≈10–20 μm × ≈60 μm); minimizes multi-cell illumination;
tolerates lateral drift; multi-laser co- or sequential-interrogation; critical alignment at interrogation point.
Collection Optics: FSC (low-angle), SSC (90°), multi-band fluorescence via dichroics + bandpass filters +
pinholes; optional dispersive optics (prism/grating) for polychromatic detection.
Detectors & Electronics: PMTs for fluorescence; photodiode for FSC; preamp with DC offset; log vs linear
amplification; APDs/small PMTs emerging; ADC (≈1024 channels), thresholding & software gating.
Cell Sorting:
Electrostatic: Drop delay, charge assignment, E-field deflection; up to ~50k cells/s.
Mechanical Gate: Gentle but slower (~300 cells/s).
Signals at Interrogation Point
FSC (low-angle, same λ as laser): correlates with cell size/cross-section/volume; also depends on refractive
index mismatch (dead cells often yield reduced FSC).
SSC (≈90°, same λ): reports internal complexity/granularity and surface texture (granulocytes >>
lymphocytes/RBCs).
Fluorescence (Stokes-shifted): from cellular components or fluorochrome staining; encodes
structure/function/phenotype.
Immunofluorescence Labeling
Direct IF: fluorochrome-conjugated primary Ab; one-step, fast.
Indirect IF: unlabeled primary + fluorochrome-conjugated
secondary; cheaper primaries, signal amplification, but more time
and higher nonspecific risk.
CD (Cluster of Differentiation): monoclonal Ab panels against CD
antigens (CD1 → >CD200) define cell subsets.
Additional Optical Parameters
Polarization of scattered light → birefringence (eosinophil granules,
etc.).
Pulse-shape analysis → cell shape/morphology cues.
Selection Drivers
Application-specific + available laser lines (e.g., 488 nm argon line).
Prefer antibody-conjugatable dyes for phenotyping
Single-Laser, Multi-Color
Use dye pairs with different Stokes shifts so emissions are well-
separated even with 488 nm only.
Classic dual-color: FITC (Ex 488 / Em ~520) + PE (Ex 488 / Em ~575).
Tandem Dyes (FRET-based)
PerCP (carotenoid→chlorophyll): Ex 488 → Em ~670.
PE-Cy5: Ex 488 via PE → Em ~670 (Cy5).
Typical 3-color @488 nm: FITC / PE / PE-Cy5
Nucleic Acid Dyes
PI: Ex 488; stains ds regions of DNA & RNA, poor live-cell permeability;
popular for DNA content.
DAPI/Hoechst: UV excitation; base-pair preferences (AT/GC).
Acridine Orange: Green (~530 nm) with dsDNA, Red (~640 nm) with
ssRNA/denatured DNA → DNA/RNA discrimination, cell-cycle studies.
Purpose
Classify cell types, detect tumors, study cell cycle and drug/gene effects.
Combine DNA quantification with antigen expression analysis.
Principle
Normal diploid (2C) DNA content; exceptions: meiosis, mitosis, apoptosis.
Abnormal (aneuploid) cells show multiple peaks in PI histograms.
Staining
Propidium Iodide (PI): penetrates fixed/permeabilized cells; fluorescence ∝ DNA content;
RNase treatment removes RNA signal.
G₀/G₁ (2C), S (2C–4C), G₂/M (4C).
BrdU incorporation: labels newly synthesized DNA; FITC-anti-BrdU + PI → two-parameter plot.
Cell Death Assay
Annexin V-FITC / PI staining:
Live: FITC⁻ / PI⁻
Apoptotic: FITC⁺ / PI⁻
Necrotic: FITC⁺ / PI⁺
Applications
Tumor classification, proliferation studies, apoptosis detection.
Rapid, quantitative cell cycle profiling.