Optical Imaging of the Nanoscale Structure and Dynamics of Biological Membranes

Biological membranes serve as the fundamental unit of life, allowing the compartmentalization of cellular contents into subunits with specific functions. The bilayer structure, consisting of lipids, proteins, small molecules, and sugars, also serves many other complex functions in addition to maintaining the relative stability of the inner compartments. Signal transduction, regulation of solute exchange, active transport, and energy transduction through ion gradients all take place at biological membranes, primarily with the assistance of membrane proteins. For these functions, membrane structure is often critical. The fluid-mosaic model introduced by Singer and Nicolson in 1972 evokes the dynamic and fluid nature of biological membranes.(1) According to this model, integral and peripheral proteins are oriented in a viscous phospholipid bilayer. Both proteins and lipids can diffuse laterally through the two-dimensional structure. Modern experimental evidence has shown, however, that the structure of the membrane is considerably more complex; various domains in the biological membranes, such as lipid rafts and confinement regions, form a more complicated molecular organization. The proper organization and dynamics of the membrane components are critical for the function of the entire cell. For example, cell signaling is often initiated at biological membranes and requires receptors to diffuse and assemble into complexes and clusters, and the resulting downstream events have consequences throughout the cell. Revealing the molecular level details of these signaling events is the foundation to understanding numerous unsolved questions regarding cellular life

A Photoactivatable BODIPY Probe for Localization‐based Super‐Resolution Cellular Imaging

The synthesis and application of a photoactivatable boron‐alkylated BODIPY probe for localization‐based super‐resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously‐unknown boron‐photodealkylation reaction with a single low‐power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization‐based microscopy of biological systems. The probe can be easily linked to nucleophile‐containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super‐resolution imaging of microtubules is demonstrated. This is the first example of single molecule localization‐based super‐resolution microscopy using a visible‐light activated BODIPY compound as a fluorescent probe

Efficient Far-Red/Near-IR Absorbing BODIPY Photocages by Blocking Unproductive Conical Intersections

Photocages are light-sensitive chemical protecting groups that give investigators control over activation of biomolecules using targeted light irradiation. A compelling application of far-red/near-IR absorbing photocages is their potential for deep tissue activation of biomolecules and phototherapeutics. Towards this goal, we recently reported BODIPY photocages that absorb near-IR light. However, these photocages have reduced photorelease efficiencies compared to shorter-wavelength absorbing photocages, which has hindered their application. Because photochemistry is a zero-sum competition of rates, improving the quantum yield of a photoreaction can be achieved either by making the desired photoreaction more efficient or by hobbling competitive decay channels. This latter strategy of inhibiting unproductive decay channels was pursued to improve the release efficiency of long-wavelength absorbing BODIPY photocages by synthesizing structures that block access to unproductive singlet internal conversion conical intersections, which have recently been located for simple BODIPY structures from excited state dynamic simulations. This strategy led to the synthesis of new conformationally-restrained boron-methylated BODIPY photocages that absorb light strongly around 700 nm. In the best case, a photocage was identified with an extinction coefficient of 124,000 M-1cm-1, a quantum yield of photorelease of 3.8%, and an overall quantum efficiency of 4650 M-1cm-1 at 680 nm. This derivative has a quantum efficiency that is 50-fold higher than the best known BODIPY photocages absorbing \u3e600 nm, validating the effectiveness of a strategy for designing efficient photoreactions by thwarting competitive excited state decay channels. Furthermore, 1,7-diaryl substitutions were found to improve the quantum yields of photorelease by excited state participation and blocking ion pair recombination by internal nucleophilic trapping. No cellular toxicity (trypan blue exclusion) was observed at 20 μM, and photoactivation was demonstrated in HeLa cells using red light

Direct Photorelease of Alcohols from Boron-Alkylated BODIPY Photocages

BODIPY photocages allow release of substrates us-ing visible light irradiation. They have the drawback of requiring reasonably good leaving groups for photorelease. Photorelease of alcohols is often accomplished by attachment with carbonate linkages, which upon photorelease liberate CO2 and gen-erate the alcohol. Here, we show that boron-alkylated BODIPY photocages are capable of directly photoreleasing both aliphatic alcohols and phenols upon irradiation via photocleavage of ether linkages. Direct photorelease of a hydroxycoumarin dye was demonstrated in living HeLa cells

Fluorescence imaging of cellular organelles and membrane dynamics

Fluorescence microscopy is a versatile technique for studying biological structures in their native environment. The live cell imaging ability of this technique enables the study of dynamic processes in biological systems. Fluorophores play an important role in fluorescence-based cellular imaging. Development of cell permeable, non-toxic, small organic molecules with specific targeting ability is advantageous for imaging of live cellular organelles. Two coumarin-based compounds were developed to selectively target the endoplasmic reticulum (ER), which is the largest organelle in most mammalian cell types. Unlike the commercially available ER targeting probes, the coumarin-based compounds can be used to image the ER in both live and fixed cells. The simple synthetic procedure, bright emission in the blue region, narrow emission profile, low toxicity, and the specificity contribute to the utility of these probes for imaging the dynamic events of the ER. The spatial resolution the traditional optical microscopy is limited by the diffraction of light. Subcellular organelles are in the nanometer size range, and the majority of the biological phenomena happen in the nanoscale. Therefore traditional light microscopic techniques have limited capability of studying these systems. Super-resolution microscopic techniques that were developed in the past decade to overcome this issue. Single molecule localization microscopy (SMLM) is based on the activation and excitation of subsets of fluorophores, followed by their localization with nanometer spatial resolution to generate a super-resolution image. Traditional SMLM fluorophores have the drawbacks of requiring two high power lasers including lasers emitting in the ultra-violet range. Also, the requirement of harsh chemicals in the imaging medium limits the ability to image live cells. A BODIPY-based photoactivatable probe is developed as a promising probe for SMLM. A single low-power visible laser is utilized without requiring harsh conditions in the imaging medium to make this probe exceptionally useful for imaging in biological systems. The probe can be simply linked to nucleophile-containing targeting groups to image specific cellular components. Super-resolution images of in vitro and in vivo microtubules are generated using paclitaxel attached probe. Membrane receptors are one of the major components that contribute to the control of cellular function. Interactions of receptor proteins with extracellular ligands, other membrane constituents, and intracellular components result in the activation of intracellular signaling mechanisms. These dynamic interactions are governed by the lateral diffusion of membrane components. RAGE is a multi-ligand receptor responsible for various pathological diseases. Amyloid beta 1-40 and 1-42 peptides react with RAGE protein to stimulate neuronal dysfunction and are also responsible for Alzheimer's disease. The effects of these peptides on RAGE diffusion is not yet known. The fluorescence-based single particle tracking method is used to measure the effect of amyloid beta ligands for the lateral diffusion of the receptor for advanced glycation endproducts (RAGE) at the single receptor level. Both of the peptides altered the RAGE membrane diffusion, but to a different extent. Activation of the p38 MAPK pathway is observed for the treatment of RAGE with amyloid beta 1-42 ligand. The effect of different oligomeric forms of these two peptides on the RAGE diffusion and signaling is further studied.</p

Optical Imaging of the Nanoscale Structure and Dynamics of Biological Membranes

Biological membranes serve as the fundamental unit of life, allowing the compartmentalization of cellular contents into subunits with specific functions. The bilayer structure, consisting of lipids, proteins, small molecules, and sugars, also serves many other complex functions in addition to maintaining the relative stability of the inner compartments. Signal transduction, regulation of solute exchange, active transport, and energy transduction through ion gradients all take place at biological membranes, primarily with the assistance of membrane proteins. For these functions, membrane structure is often critical. The fluid-mosaic model introduced by Singer and Nicolson in 1972 evokes the dynamic and fluid nature of biological membranes.(1) According to this model, integral and peripheral proteins are oriented in a viscous phospholipid bilayer. Both proteins and lipids can diffuse laterally through the two-dimensional structure. Modern experimental evidence has shown, however, that the structure of the membrane is considerably more complex; various domains in the biological membranes, such as lipid rafts and confinement regions, form a more complicated molecular organization. The proper organization and dynamics of the membrane components are critical for the function of the entire cell. For example, cell signaling is often initiated at biological membranes and requires receptors to diffuse and assemble into complexes and clusters, and the resulting downstream events have consequences throughout the cell. Revealing the molecular level details of these signaling events is the foundation to understanding numerous unsolved questions regarding cellular life.This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in Analytical Chemistry, copyright © American Chemical Society after peer review. To access the final edited and published work see DOI:10.1021/acs.analchem.8b04755. Posted with permission.</p

Inorganic Semiconductor Quantum Dots as a Saturated Excitation (SAX) Probe for Sub‐Diffraction Imaging

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