aspect, mKeima exhibits high resistance to lysosomal enzyme-mediated degradation; this inspired and resulted in the modification of mKeima as an autophagy
sensor, specifically to detect the event of conversion of autophagosomes to
autolysosomes. In the fluorescent pH sensor, mKeima was fused with light chain
3 (LC3) of microtubule-associated protein [72]. Under starvation-induced
autophagy, LC3 is cleaved and allows recruitment of phosphatidylethanolamine to
the outer and inner membranes of autophagosome, which then fuses with the
lysosomes leading to the degradation of packaged cargo under a highly acidic
environment [73]. The mKeima-LC3 probe enabled visualization of these
autolysosomal maturation events by detection of the acidification-induced color
change of mKeima and provided a cumulative fluorescent readout of autophagic
activity, with the deduction of the hallmark event of autophagy, i.e., LC3 localization. Another variant of mKeima, pH-Red, was developed with a specific purpose of
achieving pH-dependent readout in near-infrared region. This property can provide
the advantage of light emission with higher penetration and less light scattering in
biological tissues and thus offer advantage for deep tissue pH monitoring across a
broad pH range. pHRed with an apparent pKa of 6.6 demonstrated nearly tenfold
change in ratio of fluorescence emission upon excitation with a wavelength of
585 nm [74].
To date, various GES for monitoring pH have been generated in such as way they
can respond with either changes in fluorescent brightness of a single fluorescence
peak or ratiometric changes with two excitation peaks [75]. The latter type of pH
sensors imply ratiometric measurement of fluorescence brightness excited at two
different wavelengths and hence are free from artifacts that arise owing to variable
protein concentrations, cell thickness associated signal attenuation, cell movement,
or excitation intensity since measurement in one peak can normalize the other peak
that was used for pH measurement [75]. Ratiometric pH sensors commonly respond
with a change in the ratio of excitation efficiency at 400 nm versus 480 nm owing to
a shift in the protonated/deprotonated chromophore ratio [52]. An internal control of
overall signal stability can be the intensity of fluorescence excitation at the isosbestic
point between the two excitation peaks, at $430 nm. Another type of ratiometric
GES that utilizes the pH-sensitive efficiency of excited state proton transfer (ESPT)
from the protonated GFP chromophore excited at 400 nm. These sensors are excited
at 400 nm and respond with a change of fluorescence ratio between 450 nm and
510 nm [62]. Ratiometric pH sensors can also be developed by fusing pH-stable and
pH-sensitive fluorescent protein variants of different colors. In this case, changes in
the fluorescence brightness ratio of the two fused FPs can be measured along with
FRET efficiency between the two FPs, as discussed in detail below.
Fluorescent protein-based sensors for measuring metal ions in living cells can be
categorized into intensiometric sensors which change in fluorescence intensity when
the chromophore bound to a metal ion and ratiometric biosensors that exhibit shift in
the absorption or emission spectra when the chromophore bound to a metal ion. The
intensiometric fluorescent sensors are the preferred option for developing quantitative assays, since their fluorescence intensity has been determined by the sensor
concentration in each cell and the path length in addition to the ion concentration.
Applications of Fluorescent Protein-Based Sensors in Bioimaging
159
sensor, specifically to detect the event of conversion of autophagosomes to
autolysosomes. In the fluorescent pH sensor, mKeima was fused with light chain
3 (LC3) of microtubule-associated protein [72]. Under starvation-induced
autophagy, LC3 is cleaved and allows recruitment of phosphatidylethanolamine to
the outer and inner membranes of autophagosome, which then fuses with the
lysosomes leading to the degradation of packaged cargo under a highly acidic
environment [73]. The mKeima-LC3 probe enabled visualization of these
autolysosomal maturation events by detection of the acidification-induced color
change of mKeima and provided a cumulative fluorescent readout of autophagic
activity, with the deduction of the hallmark event of autophagy, i.e., LC3 localization. Another variant of mKeima, pH-Red, was developed with a specific purpose of
achieving pH-dependent readout in near-infrared region. This property can provide
the advantage of light emission with higher penetration and less light scattering in
biological tissues and thus offer advantage for deep tissue pH monitoring across a
broad pH range. pHRed with an apparent pKa of 6.6 demonstrated nearly tenfold
change in ratio of fluorescence emission upon excitation with a wavelength of
585 nm [74].
To date, various GES for monitoring pH have been generated in such as way they
can respond with either changes in fluorescent brightness of a single fluorescence
peak or ratiometric changes with two excitation peaks [75]. The latter type of pH
sensors imply ratiometric measurement of fluorescence brightness excited at two
different wavelengths and hence are free from artifacts that arise owing to variable
protein concentrations, cell thickness associated signal attenuation, cell movement,
or excitation intensity since measurement in one peak can normalize the other peak
that was used for pH measurement [75]. Ratiometric pH sensors commonly respond
with a change in the ratio of excitation efficiency at 400 nm versus 480 nm owing to
a shift in the protonated/deprotonated chromophore ratio [52]. An internal control of
overall signal stability can be the intensity of fluorescence excitation at the isosbestic
point between the two excitation peaks, at $430 nm. Another type of ratiometric
GES that utilizes the pH-sensitive efficiency of excited state proton transfer (ESPT)
from the protonated GFP chromophore excited at 400 nm. These sensors are excited
at 400 nm and respond with a change of fluorescence ratio between 450 nm and
510 nm [62]. Ratiometric pH sensors can also be developed by fusing pH-stable and
pH-sensitive fluorescent protein variants of different colors. In this case, changes in
the fluorescence brightness ratio of the two fused FPs can be measured along with
FRET efficiency between the two FPs, as discussed in detail below.
Fluorescent protein-based sensors for measuring metal ions in living cells can be
categorized into intensiometric sensors which change in fluorescence intensity when
the chromophore bound to a metal ion and ratiometric biosensors that exhibit shift in
the absorption or emission spectra when the chromophore bound to a metal ion. The
intensiometric fluorescent sensors are the preferred option for developing quantitative assays, since their fluorescence intensity has been determined by the sensor
concentration in each cell and the path length in addition to the ion concentration.
Applications of Fluorescent Protein-Based Sensors in Bioimaging
159