Professor Richard Berry D. Phil.

Abstract:

We present a transmission-mode digital holographic microscope that can switch easily between three different imaging modes: inline, dark field off-axis, and bright field off-axis. Our instrument can be used: to track through time in three dimensions microscopic dielectric objects, such as motile micro-organisms; localize brightly scattering nanoparticles, which cannot be seen under conventional bright field illumination; and recover topographic information and measure the refractive index and dry mass of samples via quantitative phase recovery. Holograms are captured on a digital camera capable of high-speed video recording of up to 2000 frames per second. The inline mode of operation can be easily configurable to a large range of magnifications. We demonstrate the efficacy of the inline mode in tracking motile bacteria in three dimensions in a 160 μm × 160 μm × 100 μm volume at 45× magnification. Through the use of a novel physical mask in a conjugate Fourier plane in the imaging path, we use our microscope for high magnification, dark field off-axis holography, demonstrated by localizing 100 nm gold nanoparticles at 225× magnification up to at least 16 μm from the imaging plane. Finally, the bright field off-axis mode facilitates quantitative phase microscopy, which we employ to measure the refractive index of a standard resolution test target and to measure the dry mass of human erythrocytes.

Abstract:

Over the past 50 years, protein complexes have been studied with techniques such as X-ray crystallography and electron microscopy, generating images which although detailed are static and homogeneous. More recently, limited application of in vivo fluorescence and other techniques has revealed that many complexes previously thought stable and compositionally uniform are dynamically variable, continually exchanging components with a freely circulating pool of “spares.” Here, we consider the purpose and prevalence of protein exchange, first reviewing the ongoing story of exchange in the bacterial flagella motor, before surveying reports of exchange in complexes across all domains of life, together highlighting great diversity in timescales and functions. Finally, we put this in the context of high-throughput proteomic studies which hint that exchange might be the norm, rather than an exception.

Abstract:

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents. Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol. Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo 3 -oxidase and F 1 F o ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to > 10 microns, as well as ATP production by this chain.