Main description:

This book covers all the technical aspects of flow cytometry needed to set-up the instrument, solve problems encountered in daily work, or necessary for exam preparation. It provides the reader with an in-depth look at the device and its applications.

Each component and its function is described in an easy-to-understand manner, giving the reader a sound basic knowledge of this instrument. The practical examples given, simplify and enhance the learning process.

This book is a unique resource of knowledge for biomedical engineers and biotechnologists, flow cytometry operators, laboratory technicians and biomedical researchers, both biologists as well as medical doctors, and can also be a helpful tool for companies and manufacturers.

Contents:

TABLE OF CONTENTS FOREWORD PREFACE 1. GENERAL PRINCIPLES page x 1.1. Flow cytometers' general layout page x 1.2. Flow cytometers' features page x 1.3. Parameters and signals page x 1.4. Time page x 2. SIGNALS - SCATTER page x 2.1. Forward scatter (FSC) page x 2.2. Side Scatter (SSC) page x 2.3. Raman Scatter page x 2.4. Depolarized Scatter page x 3. SIGNALS - FLUORESCENCE, PHOSPHORESCENCE, IMPEDANCE, EXTINCTION page x 3.1. Fluorescence page x 3.1.1. Depolarized fluorescence page x .1.2. Autofluorescence page x .2. Phosphorescence page x 3.3. Impedance page x 3.4. Axial extinction page x 4. FLUIDICS page x 4.1. Overview on fluids page x 4.1.1. Laminar flow and turbulent flow page x 4.1.2. Hydrodynamic focusing page x 4.2. Cytometers' fluidics page x 4.2.1. Sheath and core page x 4.2.2. Flow system components page x 4.2.3. Flow rate control page x 4.2.4. Sample injection page x 4.2.4.1. Sample differential control page x 4.2.4.2. Absolute counts page x 4.2.5. Event-light Interaction - the interrogation point page x 4.2.5.1. In-cuvette interaction page x 4.2.5.2. Stream-in-air interaction page x 4.2.5.3. Interrogation on an open surface page x 4.2.5.4. Systems based on acoustic focusing page x 4.2.5.5. Sheathless systems page x 5. LIGHT SOURCES page x 5.1. Arc lamps page x 5.2. Lasers page x 5.2.1. Gas lasers page x 5.2.1.1. Argon ion lasers page x 5.2.1.2. Krypton ion lasers page x5.2.1.3. Mixed gas ion lasers (Argon/Krypton) page x 5.2.1.4. Helium-Neon atom lasers page x 5.2.1.5. Helium-Cadmium ion lasers page x 5.2.1.6. Helium-Silver and Neon-Copper metal vapor lasers page x 5.2.2.Solid-state lasers (SSLs) page x 5.2.2.1. Ultra violet (UV) emitting SSLs page x 5.2.2.2. Near ultraviolet (NUV) emitting SSLs page x 5.2.2.3. Violet emitting SSLs page x 5.2.2.4. Blue emitting SSLs page x 5.2.2.5. Green and green-yellow-emitting SSLs page x 5.2.2.6. Orange emitting SSLs page x 5.2.2.7. Red emitting SSLs page x 5.2.2.8. Infrared (IR) emitting SSLs page x 5.2.2.9. Supercontinuum white light emitting SSLs page x 5.2.3. Liquid state lasers (Dye lasers) page x 5.3. Light Emitting Diodes (LEDs) page x 6. OPTICAL BENCHES page x 6.1. From the light source(s) to the interrogation point(s) page x 6.2. From the interrogation point(s) to the detector(s) page x 6.3. Optical bench components page x 6.3.1.Absorption filters page x 6.3.2. Interference filters page x 6.3.3. Neutral density filters page x 6.3.4. Polarizing filters page x 6.3.5. Beam splitters page x 6.3.6. Prisms, gratings, CWDMs page x 6.3.7. Wavelength Division Multipliers (WDMs ) page x 6.4. Optical bench layouts page x 6.4.1. Transmission benches page x 6.4.2. Reflection benches page x6.4.3. Multilaser benches page x 6.4.3.1. Spaced lasers and separate pathways/detectors page x 6.4.3.2. Spaced lasers and shared pathwaysdetectors page x 6.4.3.3. Time delay page x 6.4.3.4. Continuous collinear lasers page x 6.4.3.5. Pulsed collinear lasers page x 6.4.4. Special solutions page x 6.4.4.1. Pie-shaped design page x 6.4.4.2. Bench for (de)polarized signals page x . DETECTORS AND ELECTRONICS page x 7.1. Photodetectors page x 7.1.1.Photodiodes (PDs) page x 7.1.2.Avalanche photodiodes (APDs) page x 7.1.3.Photomultipliers (PMTs) page x 7.1.4.Multi-anode photomultipliers page7.1.5.Charged-Coupled Devices (CCDs) page X 7.1.6. Transimpedance amplifiers (TIAs) page X 7.2. Circuitry page x 7.2.1. Analog model page x 7.2.1.1. Baseline restorers page x 7.2.1.2. Comparators and thresholds page x 7.2.1.3. Accessory circuits page x 7.2.1.4. Amplifiers page x 7.2.1.5. DC restorers page x 7.2.1.6. Peak detectors and integrators page x 7.2.1.7. Analog-to-digital converters (ADCs) page x 7.2.2. Digital model page x 7.2.2.1. Data acquisition boards (DAQs) page x 7.2.2.2. General considerations on the digital model page x 7.2.3. Hybrid model page x 8. THE CYTOMETRIC FILE page x 8.1. FCS format page x 8.2. Segments page x 8.2.1. Header segment page x 8.2.2. Text segment page x 8.2.3. Data segment page x 8.2.4. Analysis segment page x 8.2.5. Optional segments page x 8.3. Keywords page x 8.3.1. Standard keywords page x 8.3.1.1. Required keywords page x 8.3.1.2. Optional keywords page x 8.3.2. Non-standard keywords page x 8.3.3. Relationships between keywords and data representation page x 8.3.3.1. $PnE keyword page x 8.3.3.2. $PnD keyword page x 8.3.3.3. PnDISPLAY keyword page x 8.3.4. Relationships between keywords and compensation procedures page x 8.3.4.1. $DFCmn keyword page x 8.3.4.2. $DFCiTOj keyword page x 8.3.4.3. $COMP keyword page x 8.3.4.4. $Spillover keyword page x 8.3.4.5. Other non-standard keywords page x 9. SIGNAL ANALYSIS AND REPRESENTATION page x 9.1. The pulse page x 9.1.1. Pulse analysis in analog systems page x 9.1.2. Pulse analysis in digital systems page x 9.1.2.1. Window's Gate and Window's Extension page x 9.1.2.2. Area Scaling page x 9.1.3. Practical applications of pulse analysis page x9.1.3.1. FCS-A vs. FCS-H vs. FCS-W page X 9.1.3.2. FL-A vs. FL-W page x 9.2. Signal representation page x 9.2.1. Lin amplified signals page x 9.2.2. Log transformation page x 9.2.3. Other transformations page x 9.2.3.1. Log-like transformations page x 9.2.3.2. Polynomial transformation page x 9.3. Dynamic range of the signal page x 9.3.1. Effective resolution page x 9.3.2. Picket fence phenomenon page x 10. DATA REPRESENTATION AND ANALYSIS page x 10.1. Data representation page x 10.1.1. Concept of distribution page x 10.1.1.1. Log-normal distribution page x 10.1.1.2. Normal (Gaussian) distribution page x 10.1.1.3. Poisson distribution page x 10.1.2. Histograms page x 10.1.2.1. Location measurements page x 10.1.2.2. Spread measurements page x 10.1.3. Cytograms page x 0.1.3.1. Representation by dots (Dot plot) page x 10.1.3.2. Representation by contours (Contour plot) page x 10.1.3.3. Representation by false colors or gray tones page x 10.1.3.4. Pseudo-three-dimensional representation page x 10.1.3.5. Three-dimensional representation page x 10.2. Data analysis page x 10.2.1. Immunofluorescence measurements page x 10.2.1.1. Histograms page x 10.2.1.2. Cytograms page x 10.2.1.3. Weak positivity in immunofluorescence page x 10.2.2. DNA content measurements page x 10.2.3. Concept of gate and region page x 10.2.4. Boolean approach to the combined use of regions and gates page x 10.2.4.1. In the determination of the hematopoietic precursors (HSC) page x 10.2.4.2. In the determination of the minimal residual disease (MRD) page x 10.2.4.3. In the augmentation of the dimensionality in cell subset analysis page x 10.2.5. Advanced tools and future perspectives page x 10.2.5.1. Pre-processing programs page x 10.2.5.2. Data processing programs page x 11. STANDARDS, SET-UP, CALIBRATION, AND CONTROL TECHNIQUES page x 11.1. Standards in flow cytometry page x 11.1.1. Natural standards page x 11.1.2. Artificial standards page x 11.1.2.1. Type 0 artificial standards page x 11.1.2.2. Type I artificial standards page x 11.1.2.3. Type II artificial standards page x11.1.2.4. Type III artificial standards page x 11.1.3. Standard use in quality procedures page x 11.1.3.1. Internal quality controls (IQCs) page x 11.1.3.2. External quality assessment (EQAs) page x 11.2. Optical bench setup page x 11.3. Photodetectors' setup page x 11.3.1. SDEn (electronic noise standard deviation) page x 11.3.2. PMT set-up page x 11.3.3. APD set-up page x 11.4. Calibration page x 11.4.1. Calibration in ERF page x 11.4.2. Calibration in MESF page x 11.4.3. Calibration in ABC page x 11.4.3.1.1. With conjugated monoclonal antibodies page x 11.4.3.1.2. With unconjugated monoclonal antibodies page x 11.4.4. Calibration in FLU page x 11.4.5. Calibration in nanometers page x 11.4.6. MFI (Mean Fluorescence Intensity) page x 11.4.7. RFI (Relative Fluorescence Intensity) page x 11.4.8. Primary performance parameters page x 11.5. Instrument performance and its control page x 11.5.1. Linearity page x 11.5.2. Accuracy page x 11.5.2.1. Carry-over page x 11.5.2.2. Count inaccuracy at high speeds page x 11.5.3. Resolution page x 11.5.4. Sensitivity page x 11.5.4.1. Background page x 11.5.4.2. Q, Qr, Stain Index and other indexes page x 11.5.4.3. Antibody titration page x 11.5.4.4. Control procedures page x 11.5.5. Limits of Blank (LOB), Detection (LOD), and Quantitation (LOQ) page x 11.5.5.1. LOB, LOD and LOQ in the detection of weak signals page x 11.5.5.2. LOB, LOD and LOQ in rare event analysis page x 11.5.6. Precision page x 11.5.7. Specificity page x 12. FLUOROCHROMES - OVERVIEW page x 12.1. Spectral behavior of fluorescent molecules page x 12.2. Relationships with the environment page x 12.2.1. Spectral effects page x 12.2.1.1. Bathochromic effect page x 12.2.1.2. Hypsochromic effect page x 12.2.1.3. Hyperchromic effect page x 12.2.1.4. Hypochromic effect page x 12.2.1.5. Solvatochromic effect page x 12.2.2. Other effects page x 12.2.2.1. Extinction or Quenching page x 12.2.2.2. Photodestruction or Photobleaching page x 12.2.2.3. Non-radiative transfer of energy (FRET) page x 12.3. Accessory groups page x 13. FLUOROCHROME SUITABLE FOR ANTIBODY CONJUGATION page x 13.1. Large protein molecules page x 13.1.1. Phycobiliproteins page x 13.1.1.1. R-Phycoerythrin (PE) page x 13.1.1.2. Allophycocianin (APC) page x 13.1.1.3. B-Phycoerythrin (PE-B) page x 13.1.1.4. C-Phycocyanin (PC) page x 13.1.1.5. Criptofluor (TM) molecules page x 13.1.2. Peridinin-Chlorophyll-Protein (PerCP) complex page x 13.1.3. Amcyan and Amcyan 100 page x 13.2. Small organic molecules page x 13.2.1. Pyrene derivatives page x 13.2.2. Pyridyl-oxazole derivatives page x 13.2.3. Coumarin derivatives page x 13.2.4. Xanthene derivatives page x 13.2.4.1. Blue excited Xanthenes (FITC and others) page x 13.2.4.2. Green and yellow-green excited Xanthenes (TRITC and others) page x 13.2.4.3. Red and NIR excited Xanthenes (Vita Blue and others) page x 13.2.5. Cyanines page x 13.2.5.1. Cyanine 2 (CY2) and Cyanines CY2-like page x 13.2.5.2. Cyanine 3 (CY3) and Cyanines CY3-like page x 13.2.5.3. Cyanine 3.5 (CY3.5) and Cyanines CY3.5-like page x 13.2.5.4. Cyanine 5 (CY5) and Cyanines CY5-like page x 13.2.5.5. Cyanine 5.5 (CY5.5) and Cyanines CY5.5-like page x 13.2.5.6. Cyanine 7 (CY7) and Cyanines CY7-like page x 13.2.5.7. Near Infrared (NIR) emitting Cyanines page x 13.2.6. Proprietary molecules page x 13.2.6.1. Alexa series page x 13.2.6.2. Other series page x 13.2.6.3. Miscellany page x 13.3. conjugated organic polymers (Brilliant Violet and others) page x 13.3.1. Organic polymers-based tandems page x 13.3.2. P-Dots pagine x 13.4. Nanocrystals page x 13.4.1. Quantum Dots (Qdots) page x 13.4.2. Upconverting nanoparticles page x 13.5. Tandem fluorochromes page x 13.5.1. UV excited tandems page x 13.5.1.1. DUV series page x 13.5.1.2. BUV series page x 13.5.2. Violet excited tandems page x 13.5.2.1. BV421-based tandems page13.5.2.2. Super Bright 436-based tandems page x 13.5.2.3. Supernova V428 based tandems page x 13.5.3. Blue excited tandems page x 13.5.3.1. PE-CF594 page x 13.5.3.2. PE-Dazzle 594 page x 13.5.3.3. PE-Texas Red page x 13.5.3.4. PE-CY5 page x 13.5.3.5. PE-CY5.5 page x 13.5.3.6. PerCP-CY5.5 page x 13.5.3.7. PE-CY7 page x 13.5.3.8. Other PE-based NIR-emitting tandems page x 13.5.4. Green-Yellow excited tandems page x 13.5.5. Red excited tandems page x 13.5.5.1. APC-CY5.5 page x 13.5.5.2. APC-AF700 page x 13.5.5.3. APC-CY7 and APC-H7 page x 13.5.5.4. Other APC-based NIR-emitting tandems page x 13.5.6. New solutions page x 13.5.6.1. Nanoparticle-encapsulated fluorophores page x 13.5.6.2. Backbone-linked fluorophores page x 14. FLUOROCHROMES THAT BIND TO NUCLEIC ACID page x 14.1. Triaryl derivatives page x 14.2. Stilbene derivatives page x 14.3. Phenylindole derivatives (DAPI, DIPI) page x 14.4. Bisbenzymidazole derivatives page x 14.4.1. HO33258 page x 14.4.1.1. HO33258 in chromosome analysis page x 14.4.2. HO33342 page x 14.4.2.1. HO33342 in the analysis of the "side population" page x 14.4.3. HO34580 page x 14.4.4. DyeCycle Violet (DCV) page x 14.5. Phenantridine derivatives page x 14.5.1. Propidium iodide page x 14.5.1.1. Propidium iodide in DNA content evaluation page x 14.5.1.2. Propidium iodide in membrane permeability evaluation page x 14.5.2. Ethidium bromide page x 14.5.3. Benzophenanthridine alkaloids page x 14.6. Cyanines page x 14.6.1. TOTO series molecules page x 14.6.2. TO-PRO series molecules page x 14.6.2.1. YO-PRO-1 page x 14.6.2.2. TO-PRO-3 page x 14.6.2.3. TO-PRO-5 page x 14.6.3. SYTOX series molecules page x 14.6.4. SYTO series molecules page x 14.6.5. SYBR series molecules page x 14.7. Aromatic molecules, heterocyclic page x 14.7.1. Acridines page x 14.7.1.1. Acridine Orange page x 14.7.1.2. Proflavine page x 14.7.1.3. Quinacrine page x 14.7.2. Oxazines page x 14.7.3. Thiazole Orange (TO) and its analogues (DETC) page x 14.7.4. 7-Amino-Actinomycin D page x 14.7.5. Pironin Y page x 14.7.6. LDS-751 page x 14.7.7. Berberine (BRB) page x 14.8. Aromatic molecules, non heterocyclic page x 14.8.1. Tricyclic antibiotics page x 14.8.2. Antraquinones page x 14.8.2.1. DRAQ molecules page x 14.8.2.2. CyTRAK Orange page x 15. FLUOROCHROMES FOR THE STUDY OF THE CELL FEATURES page x 15.1. Protein content page x 15.2. Nucleic acid content and chromatin organization page x 15.3. Cell viability page x 15.3.1. DNA impermeant probes page x 15.3.2. Amine Reactive Dyes page x 15.3.3. Fluorescein derivatives page x 15.3.3.1. Carboxyfluorescein esters page x 15.3.3.2. Calcein page x 15.3.4. Calcofluor White page x 15.3.5. Trypan Blue page x 15.4. Membrane potential page x 15.4.1. Carbocyanines page x 15.4.2. Oxonol derivatives page x 15.4.3. Xanthene derivatives page x 15.5. Mitochondrial membrane potential page x 15.5.1. JC-1 page x 15.6. Mitochondrial mass page x 15.6.1. Nonyl-Acridine Orange (NAO) page x 15.6.2. Mitotracker molecules page x 15.6.3. Mitofluor molecules page x 15.7. Intracellular pH page x 15.7.1. DCH page x 15.7.2. BCECF- acetoxymethyl ester page x 15.7.3. SNARF- acetoxymethyl ester page x 15.8. Lysosomal mass and lysosomal pH page x 15.8.1. Lysotracker and Lysohunt molecules page x 15.8.2. Lysosensor molecules page x 15.8.3. Acridine Orange page x 15.9. Free radicals (oxidative burst) page x 15.9.1. Diidroethidium (DHE) page15.9.2. Dichlorofluorescein diacetate (DCF-DA) page x 15.9.3. Dihydrorhodamine 123 (DHR) page x 15.9.4. Tetrazolium derivatives (NBT e CTC) page x 15.10. Calcium content page x 15.10.1. Indo-1 acetoxymethyl ester page x 15.10.2. Xanthenes (Fluo molecules and others) page x 15.10.3. Fura molecules page x 15.10.4. BTC page x 15.11. Sodium content page x 15.11.1. SBFI page x 15.11.2. Sodium Green page x 15.12. Potassium content page x 15.12.1. PBFI page x 15.12.2. BCECF page x 15.13. Chloride content page x 15.14. Magnesium content page x 15.15. Glutathione content page x 15.16. Heavy metals content page x 15.17. Cell proliferation page x 15.17.1. Fluorescein esters (CFSE) page x 15.17.2. PKH molecules page x 15.17.2.1. PKH 2 page x 15.17.2.2. PKH 26 page x 15.17.2.3. PKH 67 page x 15.17.3. CellVue (R) series molecules page x 15.18. Multidrug Resistance page x 15.18.1. Calcein acetoxymethyl ester page x 15.18.2. Rhodamine 123 page x 15.19. Membrane fluidity page x 16. FLUORESCENT PROTEINS page x 16.1. Fluorescent proteins and Flow Cytometry page x 16.1.1. Green fluorescent proteins (GFP) page x 16.1.2. Blue fluorescent proteins (BFP) page x 16.1.3. Cyan fluorescent proteins (CFP) page x 16.1.4. Yellow fluorescent proteins (YFP) page x 16.1.5. Red fluorescent proteins (RFP) page x 16.1.5.1. Orange fluorescent proteins page x 16.1.5.2. "Red" fluorescent proteins page x 16.1.5.3. "Long red" fluorescent proteins page x 16.1.5.4. "Long long red" fluorescent proteins page x 16.1.6. Infrared fluorescent proteins (iRFP) page X 17. SPILLOVER AND COMPENSATION page x 17.1. Spillover page x 17.1.1. Intra-laser spillover page x 17.1.1.1. Intra-laser spillover to the right page x 17.1.1.2. Intra-laser spillover to the left page x 17.1.2. Inter-laser spillover page x 17.1.3. Spillover matrix and compensation matrix page x 17.2. Compensation page x 17.2.1. Paradoxical effects page x 17.2.1.1. Post-compensation spreading page x 17.2.1.2. Perturbation of the negative cluster's distribution page x 17.2.2. Negative data and their management page x 17.2.3. Compensation by hardware page x 17.2.4. Compensation by software page x 17.2.4.1. In digital instruments page x 17.2.4.2. In analog instruments page x 18. ARTIFACTS 18.1. Escapees page x 18.2. Debris page x 18.3. Other artifacts page x 18.3.1. Due to anticoagulants page x 18.3.2. Due to fluorochromes page x 18.3.2.1. Influence on MoAb binding capacity page x 18.3.2.2. Interaction with Fc freceptors page x 18.3.2.3. Interaction with the instrument page x 18.3.3. Due to serum facors page x 18.3.4. Anecdotal reports page x 19. CELL SORTING page x 19.1. Fluid switching sorters page x 19.1.1. Pros and cons of fluid switching sorters page x 19.2. Electrostatic sorters page x 19.2.1. Sorting procedures page x 19.2.2. Sorting modalities page x 19.2.3. High pressure systems page x 19.3. Pneumatic sorters page x 20. "NON CONVENTIONAL" FLOW CYTOMETRY page x 20.1. Imaging flow cytometry page x 20.1.1. Optical bench in imaging flow cytometry page x 20.1.2. Compensation issues in imaging flow cytometry page x 20.1.3. Format issues in imaging flow cytometry page x 20.1.4. Pros and cons in imaging flow cytometry page x 20.2. Multispectral flow cytometry page x 20.2.1. Optical bench in multispectral flow cytometry page x 20.2.2. Spectral unmixing in multispectral flow cytometry page x 20.2.3. Format issues in multispectral flow cytometry page x 20.2.4. Pros and cons in multispectral flow cytometry PAGE X 20.3. Mass cytometry page x 20.3.1. Pros and cons in mass cytometry page x 20.4. Lifetime cytometry page x 20.4.1. Features of lifetime cytometry page x 20.5. Raman cytometry page x 20.5.1. Features of Raman cytometry page x 20.6. Microfluidic devices (Labs on Chips) page x 20.6.1. Hydraulic issues in microfluidic devices page x 20.6.2. Sorting procedures in microfluidic devices page x 21. NOTES page x 22. ABBREVIATIONS page x 23. ANALYTICAL INDEX page x 24. REFERENCES page