Femtosecond stimulated Raman spectroscopy (FSRS) is an ultrafast vibrational technique which allows rapid collection of Raman spectra with simultaneously high temporal and spectral resolution. With the recent development of FSRS methodology, three FSRS techniques (conventional, tunable, and anti-Stokes) have been implemented in our laboratory to dissect the excited state structural...
Intracellular pH (pH(i)) imaging is of paramount importance for life sciences. In this work, we implement the ultrafast electronic and stimulated Raman spectroscopies to unravel the fluorescence mechanism of an excitation-ratiometric pH(i) indicator in basic aqueous solution. After photoexcitation of the pH(i) indicator HPTS, a hidden charge-transfer (CT) state following...
Biosensors have become an indispensable tool set in life sciences. Among them, fluorescent protein-based biosensors have great biocompatibility and tunable emission properties but their development is largely on trial and error. To facilitate a rational design, we implement tunable femtosecond stimulated Raman spectroscopy, aided by transient absorption and quantum calculations,...
Tracking vibrational motions during a photochemical or photophysical process has gained momentum, due to its sensitivity to the progression of reaction and change of environment. In this work, we implemented an advanced ultrafast vibrational technique, femtosecond-stimulated Raman spectroscopy (FSRS), to monitor the excited state structural evolution of an engineered green...
Imaging Ca²⁺ dynamics in living systems holds great
potential to advance neuroscience and cellular biology. G-GECO1.1
is an intensiometric fluorescent protein Ca²⁺-biosensor with a Thr-Tyr-Gly chromophore. The protonated chromophore emits green
upon photoexcitation via excited-state proton transfer (ESPT). Upon
Ca²⁺ binding, a significant population of the chromophores becomes
deprotonated. It...
Fluorescent proteins (FPs) are luminescent biomolecules that emit characteristic hues upon irradiation. A group of calmodulin (CaM)-green FP (GFP) chimeras have been previously engineered to enable the optical detection of calcium ions (Ca²⁺). We investigate one of these genetically encoded Ca²⁺ biosensors for optical imaging (GECOs), GEM-GECO1, which fluoresces green...
Fluorescent proteins (FPs) have played a pivotal role in bioimaging and advancing biomedicine. The versatile fluorescence from engineered, genetically encodable FP variants greatly enhances cellular imaging capabilities, which are dictated by excited state structural dynamics of the embedded chromophore inside the protein pocket. Visualization of the molecular choreography of the...