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Lavis Lab / Research
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brightness vs. absorption

Over the last 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 fluorophores, and countless small-molecule probes are available either commercially or through de novo design and synthesis. This plethora of probes bears an inherent modularity—attachment of different reactive groups, substrate moieties, chelators, and other chemical functionalities to a somewhat diminutive set of dye scaffolds gives rise to the extant collection of fluorescent probes. A panel of 30 common fluorescent dyes spanning the ultraviolet, visible, and near-infrared spectra regions can be seen in in the above image. The compounds are plotted according to their brightness (the product of the extinction coefficient and quantum yield) versus absorption maximum and the structures are colored according to their emission maximum.
Our laboratory focuses on the design and synthesis of new fluorescent probes by “tailoring” these foundational fluorescent molecules using organic chemistry. We search for general strategies to enhance the brightness and photostability of chemical dyes to enable sensitive fluorescence microscopy in living cells. We also develop probes where the chemical and photophysical properties can be masked by assorted molecular functionalities and then unmasked by a user-designated process involving light, enzymatic activity, or other environmental changes. This chemical masking can suppress unwanted fluorescence signals in various applications, thereby functioning as a filter for bioimaging and other experiments. By combining this strategy with advances in instrumentation, protein engineering, and genetic manipulation, we can devise sophisticated ways to illuminate biological systems.

Brighter Dyes

Many of the small molecule fluorophore scaffolds were first discovered over a century ago. This antiquity is a testament to the fantastic chemical and spectroscopic properties of these molecules—they have been distilled by history. From a synthetic chemistry perspective, however, the age of these dyes often means the chemistry we use to build these molecules is also old. Rather than remain complacent about the current portfolio of chemical transformations used in dye research, we explore new synthesis techniques and apply them to the creation of novel dye derivatives. In particular, we have developed new divergent synthetic methodologies using chemical reactions that are typically reserved for drug discovery, allowing the efficient construction of fluorescent dyes. This effort has resulted in the discovery of a new auxochrome for small molecule fluorophores, azetidine, which gives substantial increases in brightness and photostability through a diminutive change in structure. These "Janelia Fluor" dyes enable new and improved single-molecule imaging experiments and we work with the labs of Robert Tjian, Eric Betzig, and James Liu, along with the Transcription Imaging Consortium (TIC) and the Applied Physics and Instrumentation Group (APIG), to push the boundaries of fluorescence microscopy. 

Photoactivatable Fluorophores

Light can provide exquisite spatiotemporal control over chemical structure. Photochemical activation of small-molecule agonists, fluorescent dyes, and fluorescent proteins can be used in sophisticated experiments to deliver biologically active molecules or facilitate advanced imaging experiments. Photoactivatable “caged” small-molecule fluorophores have certain advantages over photoactivatable fluorescent proteins including smaller size, faster diffusion times, higher brightness, and an expanded color palette. Despite these advantages, the utility of caged small molecules has languished due to the difficult organic chemistry required to synthesize these compounds. We have developed efficient, general syntheses of highly soluble caged rhodamine and fluorescein dyes to supplement (or supplant) the proteinous caged probes. We collaborate with Eric Betzig and Harald Hess to apply these compounds to the Nobel Prize winning idea of photoactivated localization microscopy (PALM). This technique involves iterative activation and measurement of caged fluorophores within a sample, allowing construction of an "super-resolution" image. We also collaborate with Josh Dudman to develop caged fluorophores for neural tracing experiments and to mark highly active subcellular regions.

Enzyme-Activated Fluorophores

In addition to light, enzymatic activity can be used to activate fluorescence. We can use fluorogenic enzyme substrates to examine the specificity of endogenous esterases in vitro or in live cells to better understand the activity of these enzymes and develop improved delivery strategies for small molecules. In addition, we use fluorogenic compounds to discover and evaluate orthogonal enzyme–substrate pairs for targeted cellular delivery. We work with Loren Looger and Scott Sternson to find enzyme–substrate pairs that allow pharmacological agents to be targeted to genetically defined cellular populations in complex biological environments.


The opportunity to interact closely with physicists, engineers, and biologists at Janelia Research Campus provides a fertile place for applying organic chemistry to biological problems. The unique power of chemistry is the capacity to construct molecules of definite structure. As chemists at Janelia, we ply our craft in concert with scientists from other disciplines to shed light on complex problems in neurobiology and beyond.