Download - Fluoresecent pH-dependent Lipobeads in vivo
Fluoresecent pH-dependent Lipobeads in vivo
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
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!
Fluorescence
Vibration relaxation
Fluo
resc
ence
Non
-rad
iativ
e re
laxa
tion
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
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
Structures of pH-sensitive Dyes
carboxy SNARF-1Orgeon Green
Fluorescein
Highlighted groups changed with pH
pH Insensitive Dyes
pH insensitive dyes, such as tetramethylrhodamine, are used as a control
Tetramethylrhodamine
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.
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
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
Cellular Data
Mice macrophages loaded with fluorescent lipobeads under bright field (left) and fluorescent imaging (right) under x40.
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.
Advantages & Disadvantages
• Advantages– No leaking like other methods (polymer matrix)– High chemical stability in solution– Protection of dye from quenching species
• Disadvantges– Biocompatability/Cytotoxicity