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

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

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.

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

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

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

What Happens in a Flow Cytometer

optics Fluidics electronics electronics

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

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.

Interrogation point

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.

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

Sample Differential 10 psi 10.2 psi 10 psi 10.4 psi 10 psi 10.8 psi

Cell cycle Low pressure High pressure

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

Optics system Excitation Collection

Optics system Collection Excitation

Light Amplification by Stimulated Emission of Radiation

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

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

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

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

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 .

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

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

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

Laser Delay

Treshold

The Pulse Time  Photons/Detector (V)

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-

G1 G2M