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Ratio-imaging : configuring the system

Ratio-imaging : configuring the system. Target : obtaining the best from the experiment. Why ratio imaging ? Is the experiment set correctly ? What’s the right set up for me ?. Calcium and Fura-2 as an example. Single wavelength excitation imaging. Dual wavelength excitation ratio imaging.

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Ratio-imaging : configuring the system

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  1. Ratio-imaging: configuring the system

  2. Target : obtaining the best from the experiment • Why ratio imaging ? • Is the experiment set correctly ? • What’s the right set up for me ? Calcium and Fura-2 as an example

  3. Single wavelength excitation imaging Dual wavelength excitation ratio imaging SINGLE WAVELENGHT IMAGING (Calcium Green) Vs RATIO IMAGING (Fura2) • Fluorescence intensity depends upon • optical path length through the sample • excitation intensity • fluorophore concentration • fluorescence quantum yield of the probe • calcium concentration • hidrolysis of loaded dye • Fluorescence ratio depends upon • calcium concentration • hidrolysis of the loaded dye

  4. How does excitation ratio imaging work? Fura-2 changes its spectral characteristics upon binding Calcium Fura-2-Ca 340 & 380 nm excitation Fura-2 Ca – Fura 2 Fura 2 Physiological range Fura-2 spectra from Molecular Probes’ website

  5. The Formula for in vivo calcium measurement [Ca++]=Kd*(R-Rmin)/(Rmax-R)*(F max 380/F min 380) Kd : dissociation constant for the chelator( Fura ) and ligand ( Calcium ) R : measured ratio (340/380) R min : 340/380 ratio at zero calcium concentration Rmax : 340/380 ratio at satured calcium conditions F : intensity of fluorescence at 380 , Fmax : satured calcium , Fmin : calcium free Grynkiewitz, Poenie and Tsien : J. Biol Chem 260, 3440 (1985)

  6. Equilibrium Equation Kd * [Ca-Fura -- ] = [ Ca ++ ] * [Fura ----] K dissociation=1/K affinity (high dissociation constants mean lower affinity between members of the equilibrium)

  7. What is Kd really? [Ca++] = Kd*(R-Rmin) / (Rmax-R) * (Fmax 380 / Fmin 380) • Choose denominator (wavelength 2) at isobestic point (360 nm), we get: • [Ca++]=Kd*(R-Rmin)/(Rmax-R) Further, since denominator is the isobestic value and same for all ratios: • [Ca++]=Kd*(F-Fmin)/(Fmax-F) • When F is midway between Fmin and Fmax , [Ca++]=KdHalf-saturation point !

  8. Kd = 0,15 um = [Ca++] at half saturation point In this case the sensitivity to calcium concentrations above 1uM is very limited.

  9. Fura-4F spectra from Molecular Probes’ website Choose the right low affinity calcium indicator for your application!

  10. Absolute ratio values of Fura-2 are equipments dependent • UV transmission of objectives • excitation filters’ transmission maximum • dichroic cube spectral properties • excitation, emission filter bandpasses • relative exposure times

  11. What is the right system ? • Microscope • CCD • Motorized stage xyz • Filter wheel or • monochromator • PC • Control and analysis software

  12. Which is the right CCD ? Speed : IPentaMAX Spatial resolution : MICROMAX 512BFT, COOLSNAP HQ Speed + spatial resolution : new CASCADE

  13. IPentaMax:RB GenII “Red/Blue” Gen II intensifier Moderate spatial resolution (45 lp/mm) ~20% QE throughout the UV “High QE” Gen III intensifier High spatial resolution (65 lp/mm) Sensitive from 475 nm to 900 nm Ideal for visible-wavelength imaging Minimum of 40% QE at 546 nm Highest available QE on the market “High Blue” Gen III intensifier High spatial resolution (65 lp/mm) Sensitive from 350 nm to 900 nm Ideal for visible-wavelength imaging ~35% QE at 520 nm Good QE in the green/blue region Fiberoptically coupled to CCD (1.5:1 taper ratio) Highest coupling efficiency available allows full use of 18-mm intensifier diameter 512 x 512 imaging array 15 x 15-µm pixels Frame-transfer readout 5-MHz A/D converter 15 fps at full resolution 1-MHz A/D converter 3 fps at lower noise I PentaMAX IPentaMax:HQ GenIII IPentaMax:HB GenIII

  14. I PentaMAX QE

  15. Virtual chip Virtual chip mode is a special fast acquisition technique that allows frame rates in excess of 100fps to be obtained region ms / frame fps 160 x 160 7.07 141 98 x 98 3.08 324 89 x 89 2.72 367 68 x 68 1.74 574 56 x 56 1.39 719 51 x 51 1.14 877 41 x 41 0.92 1087 38 x 38 0.78 1282 32 x 32 0.68 1470 32 binned x 5120.52 1923 Virtual Chip speeds determined from scan code calculator assuming exposure equals readout time, for an IPentaMax.

  16. MICROMAX 512BFT and CoolSNAP HQ MicroMax 512BFT CoolSNAP HQ

  17. New CASCADE ROI size Frames per second 653 x 492 25 256 x 256 48 128 x 128 92 64 x 64 172 32 x 32 307 16 x 16 510

  18. What is on-chip multiplication gain? It’s the on-chip multiplication of charge (electrons) generated by incident photons in each pixel.

  19. Standard CCD architecture Active array Masked array Preamplifier Serial register Output node ADC (Frame-transfer CCD)

  20. Active array Masked array Preamplifier Extended multiplication register Output node ADC CCD with on-chip multiplication gain Serial register

  21. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  22. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  23. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  24. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  25. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  26. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  27. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  28. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process Serial register

  29. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  30. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  31. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  32. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  33. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  34. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  35. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  36. Active array Masked array Serial register Preamplifier Extended multiplication register Output node ADC On-chip multiplication gain process

  37. Why is it so revolutionary? • Amplifies the signal above the read noise right on the CCD (without using external photocathodes) • Challenges traditional tradeoff between speed (frame rate) and sensitivity in the world of low-light imaging • On-chip multiplication process is easily controlled by varying the applied voltage

  38. Cascade I-PentaMAX Fluorescence from single molecules of perylene diimide in polymethylmethacrylate gel; 100 msec; 535 nm; 60x; NA 1.3 Experiment required 4x magnifier in front of I-PentaMAX to resolve single molecules.

  39. Cascade CoolSNAPHQ Fluorescence from single molecules of perylene diimide in polymethylmethacrylate gel; 100 msec; 535 nm; 60x; NA 1.3

  40. Excitation wavelenghts devices • 55 msec shift • High throughput • Fixed bandpass and intensity • 1,2 msec shift • High throughput • Fixed bandpass and variable intensity • 3 msec shift • 1 nm resolution • Low throughput • Variable bandpass and intensity Filter wheel Galvanometric wavelenght switcher Galvanometric monochromator

  41. Comparison filters/monochromator

  42. Comparison filters/monochromator RATIO imaging + FISH, Colocalization, GFP imaging, FRET: filters and /or fast monochromator Only RATIO imaging ( BCECF, Fura2.. ) : fast monochromator

  43. SOFTWARE CONTROL MAIN FEATURES • # of wavelenghts controlled • # of ratios controlled • experiment playback for offline analysis • macro RATIO -Average of ratios Vs Ratio of averages 4 pixel : 1w1 0 2w1 50 3w1 100 4w1 0; 1w2 50 2w2 0 3w2 50 4w2 80 ratio of average (scartando pixel con valore 0 o saturati ) : w1 : 75, w2: 60, R: 75/60 =1.25 average of ratio : ratio 3=2

  44. # wavelenghts and ratio controlled GFP + Fura 2

  45. Complete experiment

  46. Roberto Becattini Biology apps specialist

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