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Live Cell Imaging

Live Cell Imaging. Toxicity Phototoxicity Compartmentalization Compatibility Interference (buffer / sink) Temporal Constraints Permeability. Permeability. 1. Endocytosis 2. Injection / infusion 3. Esterification acetate acetoxymethyl (AM). Ion Selective Dyes.

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Live Cell Imaging

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  1. Live Cell Imaging Toxicity Phototoxicity Compartmentalization Compatibility Interference (buffer / sink) Temporal Constraints Permeability

  2. Permeability 1. Endocytosis 2. Injection / infusion 3. Esterification acetate acetoxymethyl (AM)

  3. Ion Selective Dyes Calcium / Magnesium / Zinc ions Protons (pH) Sodium & Potassium ions Chloride / Halide ions

  4. Ion Selective Dyes Magnesium Green (APTRA) 5-carboxy-2’,7’-chlorofluorescein Sodium Green (diaza crown ether) Calcium Green (BAPTA) 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid

  5. Ion Selective Dyes Spectral compatibility Stoichiometry Specificity Affinity (KD) Dynamic range Permeability Optical response

  6. Optical Response • 1. Change (+/-) in Quantum Yield with little change in spectra • e.g., fluo-3, Calcium Green, SPQ • 2. Blue shift of absorption (excitation) spectrum with little change in maximum emission • e.g., fura-2, Fura Red, SBFI, PBFI, BCECF • Blue shift in both absorption and emission spectra • e.g., indo-1, SNARF & SNAFL

  7. Calcium Dyes - Issues Qualitative vs quantitative (calibration) KD for calcium Compartmentalization pH effects Interaction with other ions

  8. Calcium Green

  9. Calcium Green

  10. Calcium Green

  11. Dynamic Range

  12. Calcium Green

  13. Fluo Dyes Fluo 4

  14. Fluo-3

  15. Fluo Calcium Indicators

  16. Optical Response • 1. Change (+/-) in Quantum Yield with little change in spectra • e.g., fluo-3, Calcium Green, SPQ • 2. Blue shift of absorption (excitation) spectrum with little change in maximum emission • e.g., fura-2, Fura Red, SBFI, PBFI, BCECF • Blue shift in both absorption and emission spectra • e.g., indo-1, SNARF & SNAFL

  17. Fura-2 & Indo-1 Fura-2 Indo-1

  18. Fura-2 Isosbestic point

  19. H. Ma et al., Science 287, 1647 -1651 (2000) Published by AAAS

  20. No Caption Found M. A. Takasu et al., Science 295, 491 -495 (2002) Published by AAAS

  21. Optical Response • 1. Change (+/-) in Quantum Yield with little change in spectra • e.g., fluo-3, Calcium Green, SPQ • 2. Blue shift of absorption (excitation) spectrum with little change in maximum emission • e.g., fura-2, Fura Red, SBFI, PBFI, BCECF • Blue shift in both absorption and emission spectra • e.g., indo-1, SNARF & SNAFL

  22. Fura-2 & Indo-1 Fura-2 Indo-1

  23. Indicator Kdin vitro * Kdin situ Cell/Tissue Type fluo-3 390 nM 2570 nM Frog skeletal muscle fura-2 145 nM 371 nM U373-MG astrocytoma cell fura-2 145 nM 350 nM Rabbit gastric gland indo-1 230 nM 844 nM Rabbit cardiac myocyte Calcium indicators - affinity Table 20.8 Comparison of in vitro and in situ Kd values for fluo-3, fura-2 and indo-1.

  24. Photo-activated “Caged” compounds GABA ATP uncaging requires violet - UV irradiation

  25. Red calcium indicators Rhod-2 X-rhod-1

  26. Rhod-2 & X-Rhod-1 Rhod-2 X-Rhod-1

  27. Red calcium indicators Fura Red Calcium Orange

  28. Fura Red

  29. Fura-2

  30. More Furas fura-5F fura-4F fura-FF fura-6F

  31. Fura-2 C18

  32. Indo-1

  33. Other Ratiometric Dyes Quin-2AM benzothiaza-1

  34. Ion Selective Dyes Calcium / Magnesium / Zinc ions Protons (pH) Sodium & Potassium ions Chloride / Halide ions

  35. pH Sensitive DyesFluoroscein Diacetate (FDA)

  36. Protonation of FDA

  37. Fluorescein Diacetate

  38. Permeability of pH-sensitive dyes 1) calcein AM 2) BCECF AM 3) TFFDA 4) FDA 5) HFFDA 6) CFDA 7) CMFDA

  39. BCECF-AM

  40. BCECF-AM Fluorescence Spectrum

  41. BCECF-AM Spectra

  42. SNARF & SNAFL Carboxy-SNAFL-1 AM SemiNAphthoRhodaFluors& SemiNAphthoFLuoresceins

  43. Indo / SNARF-1 Fluorescence from the dual-emission Ca2+ indicator, indo-1 AM, is shown at 405 and 475 nm (left panels). Fluorescence from the dual-emission pH indicator, SNARF-1 AM, is shown at 575 and 640 nm (right panels)

  44. Applications of pH selective dyes • Multidrug resistance • Cell volume changes • Cytosolic pH regulation in osteoblasts and osteoclasts • pH in lateral intercellular spaces of epithelial cell monolayers and interstitial spaces of normal and neoplastic tissue • Phagocytosis • Cl–/HCO3– exchange • K+/H+ exchange • Lactate transport and metabolism • Na+/H+ exchange • Na+/Ca2+ exchange • NH4+ transport • Apoptosis • Cytotoxicity

  45. Reactive Dyes Chloromethyl-SNARF-1

  46. Reactive Dyes Chloromethylfluorescein Diacetate

  47. “Cell Tracker” Dyes

  48. Ion Selective Dyes Calcium / Magnesium / Zinc ions Protons (pH) Sodium & Potassium ions Chloride / Halide ions

  49. Sodium & Potassium Selective Dyes PBFI SBFI benzofuranyl fluorophores

  50. SBFI Excitation Spectra

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