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
Non-radiative relaxation
Vibration
relaxation
Fluorescence
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
Highlighted groups
changed with pH
carboxy SNARF-1
Orgeon Green
Fluorescein
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.
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
Typical Liposome
Liposome containing Fluorescein
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