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

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
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.

5. 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

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

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

9. What Happens in a Flow Cytometer

11. optics
Fluidics
electronics
electronics

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

13. 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.

14. Interrogation point

16. The Flow Cell Sample Stream 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.

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

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

19. Cell cycle
Low pressure
High pressure

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

22. Optics system
Excitation
Collection

23. Optics system
Collection
Excitation

27. Light Amplification by Stimulated Emission of Radiation

28. 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

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

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

31. 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)
400nm
500nm
600nm
700nm
Transmittance

32. 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.
Dichroic
Filter
Detector 1
Detector 2 .

33. <670nm nm nm nm nm

37. Electronics System 1 Converts light signals into numerical data
Eliminate small signals 3 1 2
amplifier- מגבר
Current- זרם

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

41. Laser Delay

42. Treshold

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

44. 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-

45. G1
G2M