základy princip $ pr - masaryk university · analog to digital converters (adc’s). • each...
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Základy princip pr tokovécytometrie
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Flow Cytometry
Flow cytometrie je technologický proces, který umož ujeindividuální stanovení fluorescencejednotlivých bun k.
Tento proces umož uje stanovit tisíce bun k za vte inu
Informace jsou d ležité z hlediska charakterizace bun k
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Historie• Flow cytometrie je vyvinuta z mikroskopické
techniky• F.T. Gucker (1947) poprvé sestrojil p ístroj pro
detekci bakterií v laminárním proudu vzduchu• L. Kamentsky (IBM Labs), a M. Fulwyler (Los
Alamos Nat. Lab.) dále experimentovali síd ním bun k podle jednotlivých charakterizací
• V roce 1972 L. Herzenberg (Stanford Univ.)vyvinul „t ídi “, který rozd loval bu ky obarvenéfluorescen zna enými Ab - FluorescenceActivated Cell Sorter (FACS).
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Aktiva ní proces fluorescence neboImunofluorescence
FITC FITC
FITC
FITC
FITC
FITC
Antibodies recognize specificmolecules in the surface ofsome cells
But not others
When the cells are analyzed by flowcytometry the cells expressing the markerfor which the antibody is specific willmanifest fluorescence. Cells who lack themarker will not manifest fluorescence
Antibodies are artificiallyconjugated to fluorochromes
Antibodies
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Bun né parametry, které mohou býteny pomocí FC
• No reagents or probesrequired (Structural)– Cell size(Forward Light
Scatter)– Cytoplasmic grabularity(90
degree Light Scatter)– Photsynthetic pigments
• Reagents are required.– Structural
• DNA content• DNA base ratios• RNA content
– Functional• Surface and intracellular
receptors.• DNA synthesis• DNA degradation
(apoptosis)• Cytoplasmic Ca++• Gene expression
Hlavní Vedlejší
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Aplikace FC
• Immunofluorescence• Kinetika bun ného cyklu• Bun ná kinetika• Genetika• Molekulární biologie• Mikrobiologie• Parasitologie
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• Velikosti bun k
• Cytoplazmatické granularity
• Povrchových antigen
• Apoptózy
• Intracelulární produkce cytokin
• Intracelulární signalizace
• Bun ného cyklu, množství DNA….
• Vázaného a volného kalcia
• Bun né proliferace
Tyto aplikace zahrnují zjišt ní:
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• Flow cytometrie v sob integrujeelektroniku, fluidiku, PC, optiku, softwarea laser technologie v relativnjednoduchém uspo ádání
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Laser optics
Laser Beam
Flowchamber
Sheath
SampleY
X
Z
Y Z
X
Hydrodynamickáfokusace
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PE FL
FITC FL
488nm Sct
Laminar Fluidic Sheath
CoreSheath
OuterSheath
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• Each cell generates a quanta of fluorescence
PE FL FITC FL 488nm Sct
Confocal LensDichroic Lenses
Photomultiplier Tubes
(PMT’s)
DiscriminatingFilters
ForwardLightScatteringDetector
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FlowCell
LaserBeam
FSSensor
FluorescencePickup Lens
SSSensor
FL1Sensor525BP
FL2Sensor575BP
FL3Sensor620BP
FL4Sensor675BP
488DL
488BK
550DL
600DL
645DL
Optical BenchSchematic
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From Fluorescence to Computer Display• Bun ná fluorescence je detekována r znými
detektory(PMT’s).• PMT’s convert light into electrical pulses.• These electrical signals are amplified and digitized using
Analog to Digital Converters (ADC’s).• Each event is designated a channel number (based on
the fluorescence intensity as originally detected by thePMT’s) on a 1 Parameter Histogram or 2 ParameterHistogram.
• All events are individually correlated for all theparameters collected.
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Light Scattering, 2 Parameter Histogram
Forward Light Scatter (FLS)
90 degreeLight Scatter
Bigger
MoreGranular
Live Cells
BiggerCells
DeadCells
ApoptoticCells
X Axis
Y Axis
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1 Parameter Histogram
1 2 3 4 6 7 150 160 170 .. 190
Channel Number
Positive
Negative
BrighterDimmerCount
1
4
6
Fluorescence picked up from the FITCPMT
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2 Parameter Histogram
FITC FL
PE FL
NegativePopulation
Single PositiveFITCPopulation
SinglePositive PIPopulation
Double PositivePopulation
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Flow Cytometry Data
SmallerRegion,Live cellsmostly
Larger Regionincludes all cells
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UltrasonicTransducer
488nm Formard Light Scatter Detector
Collimated Light Path ThroughDichroic and Band Pass Filters
SS FL2FL1
FL4
FL3
Pulse Height(0-10Volts)
Time(useconds)
Pressurized1XPBS(Sheath)
Pressurized CellSample
Analog Data
PMTs
Flow Cytometry and sorting
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ení trombocyt na analyzátoruSYSMEX XE-2100
U analyzátor využívající pr tokové cytometrie mohou býtvýsledky ovlivn ny zne išt ním a technickým stavem p ístroje a
ítomnosti jiných krevních element jedná se o metodu nep ímou.
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Stanovení trombocyt opticky• 4,5 l vzorku je na ed no 895,5 l Ret Search (II) diulentem• vzorek je injektován do RET reak ního kanálu, kde je ke vzorku p idáno
18 l barva, která proniká p es membránu do bun k a barví RNAretikulocyt a DNA/RNA v jaderných bu kách (reagencie Ret Search(II) dye)
• po 31 vte inách dochází k obarvení vzorku• vzorek je analyzován metodou pr tokové cytometrie za použití
semikonduktorového laseru
Vyhodnocení• zm ené výsledky jsou zobrazeny pro jednotlivé parametry na displeji
ístroje a na monitoru po íta e• analyzátor podává p esné informace i o po tech erytrocyt a
leukocyt