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Understanding Flow Cytometry: Applications and Technology

Learn about the principles, applications, and components of flow cytometry, a powerful tool for analyzing properties of cells in fluid motion, with a comparison to traditional cytometry and detailed insights into optics, fluidics, and electronics.

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Understanding Flow Cytometry: Applications and Technology

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  1. 2015 היחידה להפרדת תאים ד"ר דבי איצקוביץ' 054-7204868

  2. What Is Flow Cytometry? • Flow ~ cells in motion- זרימה • Cyto ~ cell- תאים • Metry ~ measure- מדידה • Measuring properties of cells while in a fluid stream

  3. Cytometry vs. Flow Cytometry Cytometry • Localization of antigen is possible • Poor enumeration of cell subtypes • Limiting number of simultaneous measurements Flow Cytometry. • Cannot tell you where antigen is. • Can analyze many cells in a short time frame. • Can look at numerous parameters at once.

  4. אפליקציות בשימוש ה-FACS • It can be used for… • Immunophenotyping • DNA cell cycle/tumor ploidy • Membrane potential • Ion flux • Cell viability • Intracellular protein staining • pH changes • Cell tracking and proliferation • Sorting • Redox state • Chromatin structure • Total protein • Lipids • Surface charge • Membrane fusion/runover • Enzyme activity • Oxidative metabolism • Sulfhydryl groups/glutathione • DNA synthesis • DNA degradation • Gene expression

  5. The use of flow in research has boomed since the mid-1980s

  6. גודל יחסי של התא • גרגור יחסי של התא • עוצמת פלואורסנציה יחסית בתא • פעילות מכשיר ה-FACS מורכבת משלוש מערכות: • זרם- • אופטיקה • אלקטרוניקה

  7. What Happens in a Flow Cytometer

  8. optics Fluidics electronics electronics

  9. הגדרת המושג Flow Cytometry הגדרת הדוגמאות המתאימות למכשיר ה-FACS הגדרת המאפיינים שהשיטה מודדת הגדרת שלוש המערכות שעובדות במכשיר הFACS.

  10. How The Flow Cell Works • The cells from the sample tube are injected into the sheath stream • Flow in a flow cell is laminar. • Hydrodynamic focusing pushes the cells to line up single file along their long axis. • The shape of the flow cell provides the means for hydrodynamic focusing.

  11. Interrogation point

  12. SampleStream Cell The Flow Cell Sheath 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.

  13. Sample Sample Sheath Sheath Sheath Sample Core Stream Laser Focal Point Incoming Laser Low Differential High Differential

  14. 10 psi 10 psi 10 psi 10.2 psi 10.4 psi 10.8 psi Sample Differential

  15. Cell cycle Low pressure High pressure

  16. מה תפקיד מערכת הזרימה מהו hydrodynamic focusing כיצד קצב הריצה של הדוגמא והלחץ במערכת משפיעים על התוצאה.

  17. Optics system Collection Excitation

  18. Light Amplification by Stimulated Emission of Radiation

  19. Filters • Many wavelengths of light will be scattered from a cell, we need a way to split the light into its specific wavelengths in order to detect them independently. This is done with filters • Optical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting other. • 3 types of filters • Long Pass filter • Short Pass filter • Band Pass filter

  20. Transmittance 400nm 700nm 600nm 500nm Long Pass Filters • Transmit all wavelengths greater than specified wavelength • Example: 500LP will transmit all wavelengths greater than 500nm

  21. Transmittance 400nm 700nm 600nm 500nm Short Pass Filter • Transmits all wavelengths less than specified wavelength • Example: 600SP will transmit all wavelengths less than 600nm.

  22. Transmittance 400nm 700nm 600nm 500nm Band Pass Filter • Transmits a specific band of wavelengths • Example: 550/20BP Filter will transmit wavelengths of light between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20)

  23. Detector 1 Detector 2 Dichroic Filter Dichroic Filters • Can be a long pass or short pass filter • Filter is placed at a 45º angle to the incident light • Part of the light is reflected at 90º to the incident light, and part of the light is transmitted and continues on.

  24. <670nm 564-606nm 483-493nm 515-545nm 750-810nm

  25. Electronics System • Converts light signals into numerical data • Eliminate small signals 1 3 2

  26. Photodetectors Photodiode (FSC) PMT- photomultiplier tube How Do PMTs Work?

  27. Laser Delay

  28. Treshold

  29. Time  Photons/Detector (V) The Pulse

  30. What are doublet & clumps Doublet: Two cells stuck together Clumps: More than two cells stuck cells stuck together Why do we need to gate out doublets? GFP+ GFP- GFP- GFP- GFP+ GFP- GFP+ GFP+

  31. G1 G2M

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