1. Fluoresecent pH-dependent Lipobeads in vivo

2. pH changes in biology pH changes are important to several biological processes: muscle contraction, endocytosis, cell proliferation, apoptosis, ion transport Biological enzymes function at an optimal pH Folded protein is stabilized by the specific ions Protein enzyme ATPase uses a proton gradient for energy production

3. Standard pH meter pH electrode – glass electrode filled with electrolyte and Ag/AgCl reference electrode – thin glass membrane in contact with solution – Potential difference builds up over the thin glass membrane due to differences in H + concentration – Potential measured against reference electrodes and pH calculated Too big to be implanted in a cell!

4. Fluorescence Vibration relaxation Fluorescence Non-radiative relaxation 1.Absorption excites the molecule to excited state some molecules may be in vibrationally or rotationally excited states 2. Vibrational relaxation molecule transitions to lowest energy excited state 3.Fluorescence molecule returns to ground state by emitting a photon 4. Non-radiative relaxation molecule returns to ground state but does not emit radiation

5. pH sensitive fluorescent dyes Fluorophores are aromatic or conjugated with delocalized electrons pH sensitive fluorophores – emission differs at different pH values Fluorescein and tetramethylrhodamine (pH insensitive) are used frequently in cellular applications – high absorbance and emission wavelength in the visible light range

6. Structures of pH-sensitive Dyes carboxy SNARF-1 Orgeon Green Fluorescein Highlighted groups changed with pH

7. pH Insensitive Dyes pH insensitive dyes, such as tetramethylrhodamine, are used as a control Tetramethylrhodamine

8. Experimental Results Fluorescence spectra of individual lipobeads containing fluorescein at varying pH levels: (a )pH = 5, (b) pH = 6 (c) pH = 7, (d) pH = 8.

9. Liposome containing Fluorescein Typical Liposome Dye Delivery Fluorescent dyes can be encapsulated by phospholipid bilayer vesicles (liposomes). – Water soluble while retaining same reactivity – Protect dye from quenchers Liposome vesicle fuses with cellular lipid bilayer and delivers dye inside cell for reactions – Also used for drug or gene delivery

10. Cellular data Mice macrophages were incubated with fluorescent pH-sensing lipobeads – Lipobead is a membrane on a polystyrene bead – Lipobeads filled with fluorescein and tetramethylrhodamine Dyes allowed to interact endocytosed allowing dyes to interact with intracellular environment Cells were analyzed with fluorescence-imaging microscopy – Exposure to detection light Intracellular pH is determined from emission peaks

11. Cellular Data Mice macrophages loaded with fluorescent lipobeads under bright field (left) and fluorescent imaging (right) under x40.

12. Cellular Data pH change of a single liposome of fluorescent marker: A sharp drop in fluorsecence is observed (t=9 sec) when the cell ingests the dye into a more acidic environment. The more acidic environment causes fluorescein fluorescence to decrease.

13. Advantages & Disadvantages Advantages – No leaking like other methods (polymer matrix) – High chemical stability in solution – Protection of dye from quenching species Disadvantges – Biocompatability/Cytotoxicity