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- 1. Vaseetharan.S Fluorescence Lifetime Imaging Microscopy(FLIM) Vaseetharan.S 1
- 2. Contents…….. Introduction MajorClassification Application Summary & final remarks 2
- 3. What is FLIM? Fluorescence-lifetimeimaging microscopy or FLIM is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. What is Fluorescence Life Time? Average time that molecules stay in their excited state E1 E0 Some Nano Seconds Excitation Emission 10-15S 10-12S 10-9S 3
- 4. Fluorescence Life TimeIntensity Time (ns) I/e τ It=I0 I0e-t/τ Exponential Decay Curve τ Time Constant (Life Time) Intensity α Photon No Fluorescence Decay Law 4
- 5. Fluorescence-lifetime imaging microscopy SomePhenomena affect fluorescence life time • Ion Imaging • Oxygen Imaging • Probing Micro Environment • Medical Diagnosis • FRET 5
- 6. FLIM Measurements Time Domain & FrequencyDomain Steps in Fluorescence lifetime imaging (FLIM) • Data acquisition Measurement of lifetime decay curves with spatial resolution • Exponential fit of decay curves in each pixel, calculate fluorescence lifetime in each pixel • Transformation of fluorescence lifetimes in color code 6
- 7. Time Domain FLIM Intensity Time(ns) Excitation Emission Methods • Short Excitation Pulses • Emission Detection In Time Windows • Excitation pulse width should be shorter than fluorescent lifetime. • Typical pulse widths < 10 ps 7
- 8. Time Domain FLIMContinues….. Intensity Time Integrated Intensities Average life time is a function of integrated intensities 8
- 9. Frequency Domain FILM Intensity ElapsedTime • Sample excited with intensity modulated light • Intensity of light is varied at high frequency. • Emission delayed relative to the excitation measured in phase shift Modulated Excitation – 20/80 MHz Requirements • Modulated Excitation • Modulation Detection CameraCommonly used with wide-field imaging techniques 9
- 10. Intensity Elapsed Time Frequency DomainFILM Continues….. 20ns Emission Long Life Time 10s Emission Short Life Time 1ns 10
- 11. Frequency Domain FILM DecreaseModulation Depth Phase Shift The life Time is calculated in every pixel of the Image From 11
- 12. Image Intensifier • Toextract phase shift and the decrease in modulation depth • Use of Image Intensifier as detector Microscope Camera Port Photon Photo Cathode -200-0V MCP Anode 0V 400-1000V 6kV Photons CCDCAMERA ē ē ē ē ē 12
- 13. MCP Anode 0V 400-1000V 6kV Photons CCDCAMERA ē ē ē ē ē ImageIntensifier • To extract phase shift and the decrease in modulation depth: The image intensifier is modulated in sensitivity Modulated Emission Signal Modulated Voltage Demodulation of signal DC Image Cathode 13
- 14. Frequency domain VsTime domain Frequency Domain Time Domain Theory Difficult Straight forward Average Life Time Determined by Decrease modulation depth and Phase Depth Function of Ratio of integrated intensities Excitation Emission 14
- 15. Frequency Domain TimeDomain Frequency Domain Vs Time Domain System Wide field >> at once >> Fast acquisition Scanning >>Pixel per Pixel Light Source Modulated :LED, Las er diode , laser >>Relatively cheap Pulsed Laser Required Intensities Moderate >>Less photo toxic High Excitation & Detection Simultaneously Pixel per Pixel Reduction of Auto fluorescence Multi frequency Delayed Gating 15
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- 17. CCD Camera MCP Intensifier Camera Objectives To computer Imagedate acquisition Emission Filter Dichroic Mirror Microscope Slide With sample Laser AOM External Repetitive Shutter Iris for Selecting Diffraction Spot Optical Fiber Inverted Microscope Objective The Diagramed Illustration Of FLIMicroscope 17
- 18. LED LED module asit is installed in standard microscope lamp housing Advantages of LED over Laser as a modulated light source • Inexpensive • Modulated over broad frequency range (10-100MHz) • No interference effect and speckles • Many wavelength available • 442nm (CFP) • 470nm (GFP) • 517nm (YFP) • 538nm (CY3) • 626nm (CY5) 18
- 19. Fluorescence Life TimeImaging Lifetimes are measured at each pixel and displayed as color contrast. It combines information about spatial distribution of a fluorescent molecule together with information about its microenvironment..Eg-PH Imaging modes wide-field Confocal Multi Photon 19
- 20. Types of FluorescenceMarkers • Auto fluorescence: NADH, Flavins, Chlorophyll • Fluorescent proteins: CFP, GFP, YFP • Fluorescent markers bound to antibodies: FITC • Ion indicators : Calcium, Sodium, pH (Fluo-3, Na-green, Oregon Green, DM-NERF, CI-NERF) 20
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- 22. Introduction The clinicalstrategic study of cancer requires understanding of signaling pathways in their pathophysiological contexts 2D-culture models lack the full dimensions of integrated local and systemic +ve and -ve feedback signals The in vivo environment contains 3 categories of factors that impose additional cell signaling on individual cells Neighboring cells Secreted soluble factors Non-cellular structural factors The routinely used methods are PCR Western Blotting 22
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- 28. The Schematic diagramof the time resolved fluorescence microscopy 28
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- 31. Final Remarks Lifetime independentof intensity caused by • Excitation not uniformity • Concentration Variations • Bleaching Frequency domain FLIM method offers • Speed up to real time FLIM • Robust modulated LED excitation • Stability • Easiness to install and to operate • Life cell analysis • Combination with spinning disc ,TIRF , Spectral 31
- 32. FILM APPLICATIONS Dyedifferential visualization. Energy transfer (FRET) for distance measurements Concentration measurements of ions (Ca2+ , Na+, pH), small ligands, oxygen Environmental studies (viscosity, refractive index, membrane potential) Protein studies (Proteomics) Intracellular signal transduction 32
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