Over the past 70 years fluorescence has evolved from a scientific curiosity into an indispensable tool for examining biological phenomena. Today fluorescent molecules are used extensively as labels for biomolecules, substrates for enzymes, indicators for ions, and stains for subcellular domains. Small organic molecules constitute an important subclass of useful fluorophores, and countless small-molecule probes are available either commercially or through de novo design and synthesis. Attachment of different reactive groups, substrate moieties, chelating components, and other chemical functionalities to a fairly small set of dye scaffolds gives rise to the large collection of fluorescent probes now available. A panel of 30 common fluorescent dyes spanning the ultraviolet, visible, and near-infrared spectra regions is shown in the figure.
My laboratory uses organic chemistry to build new fluorescent probes from these foundational fluorescent molecules. In particular, we are interested in developing probes in which the chemical and photophysical properties can be "masked" through attachment of assorted molecular functionalities and then unmasked by a specific chemical reaction using light, enzymatic activity, or other environmental changes. This chemical masking suppresses unwanted fluorescence signals in various applications, thereby functioning as a quasi-filter for bioimaging and other experiments. By combining this strategy with advances in instrumentation, protein engineering, and genetic techniques, we can devise sophisticated ways to illuminate biological systems.
Photoactivation of Fluorophores
Light can provide exquisite spatiotemporal control over chemical structure. Photochemical uncaging of small-molecule agonists, fluorescent dyes, and fluorescent proteins can be used in sophisticated experiments to deliver biologically active molecules or to facilitate complex imaging. An exciting technology that uses photoactivatable fluorophores (PAFs) is photoactivated localization microscopy (PALM), developed by Eric Betzig and Harold Hess. This technique involves iterative activation and measurement of caged fluorophores within a sample, allowing construction of a super-resolution miscopy image. The current palette of small molecule- and protein-based PAFs remains sparse. We have developed facile and efficient synthetic chemistry strategies to bolster the current collection of photoactivatable small-molecule fluorophores. Our probes allow new super-resolution microscopy experiments, and further refinement of these molecules will enable detailed, multicolored mapping of biological structures.
We are also exploring strategies for the attachment of small-molecule dyes to biomolecules in a specific fashion. Traditional strategies use biological nucleophiles, but recent advances allow labeling of small molecules inside living cells through genetic incorporation of proteinic reactive tags that react in an orthogonal fashion with small-molecule ligands. In collaboration with Loren Looger and colleagues in the industrial sector, we are expanding these chemistries to photoactivatable dyes.
Enzymatic Activation of Fluorophores
In addition to light, enzymatic activity can be used to control fluorophore properties. For example, endogenous enzymes facilitate the delivery of various fluorescent molecules to biological systems. In a common example, polar moieties on the dye are cloaked as simple esters to allow efficient passage of the molecule through lipid bilayers. Once inside the cell, nonspecific esterases catalyze the hydrolysis of the masking ester bonds, releasing and trapping the fluorescent molecule.
While this strategy works well for general molecular delivery, it does not allow for targeted delivery of the compounds to specific cellular populations. Together with Scott Sternson and Loren Looger, we use fluorogenic enzyme substrates to interrogate the specificity of the cellular "enzome" and identify selective enzyme-substrate pairs that can unmask molecules in genetically defined cell types. Delivery of fluorophores is useful for a variety of imaging applications, including determining gap junction connectivity in cells and tissue. Moreover, these strategies can be applied to other small-molecule tools, such as pharmacological agents, thereby combining the molecular and temporal specificity of small molecules with the cellular specificity of genetics.
Fluorophores that are sensitive to calcium ions enable important biological experiments. Calcium ion is an important second messenger in cells and calcium indicators are used in a variety of cellular assays including high-throughput screening. Calcium ion concentration also changes when neurons fire, making calcium imaging a useful technique to measure activity in the brain. Together with Karel Svoboda, we are updating the current crop of calcium indicators, making molecules that are brighter, loadable in tissue, able to exhibit a range of spectral properties, and able to permanently mark active cells. We are also applying our enzyme-mediated targeting strategies to deliver these improved probes to genetically defined cellular populations.
Expanding Dye Synthesis
As toolmakers, our overall goal is developing novel molecules to advance bioresearch. Along the way, however, we develop innovative paths to the synthesis of fluorescent dyes and their derivatives. Our projects take advantage of the ability of synthetic organic chemistry to tune small-molecule fluorophores to a specific function. Increasing the repertoire of chemical reactions that can construct and modify dye structures is therefore important to any effort involving the synthesis of new probes. Rather than be satisfied with the current portfolio of chemical transformations used in dye research (some reactions date back to the 19th century) we are exploring new synthesis techniques and applying them to the creation of novel dye derivatives. This effort enables the synthesis of known and novel fluorophores that are tailored for specific biological experiments.
Our close interactions with engineers and biologists at Janelia Farm provide a fertile environment for discovering new applications for organic chemistry. The unique power of the chemical discipline is the capacity to construct molecules of definite structure. As chemists at Janelia, we have the opportunity to ply our craft in concert with scientists from other disciplines to shed light on complex problems in neurobiology and beyond.