Kevan T. Samiee

Cell investigation of nanostructures: zero-mode waveguides for plasma membrane studies with single molecule resolution

Plasma membranes are highly dynamic structures, with key molecular interactions underlying their functionality occurring at nanometre scales. A?fundamental challenge in biology is to observe these interactions in living cells. Although fluorescence microscopy has enabled advances in characterizing molecular distributions in cells, optical techniques are restricted by the diffraction limit. We address this limitation with an approach based on zero-mode waveguides (ZMWs), which are optical nanostructures that confine fluorescence excitation to sub-diffraction volumes. Successful use of ZMWs with cell membranes is reported in this paper. We demonstrate that plasma membranes from live cells penetrate these nanostructures. Cellular exploration of the nanoapertures depends heavily on actin filaments but not on microtubules. Thus, membranes enter the confined excitation volume, and diffusion of individual fluorescent lipids can be monitored. Through fluorescence correlation spectroscopy, we compared DiIC12 and DiIC16 fluorescent labels incorporated into plasma membranes and found distinctive diffusion behaviours. These results show that the use of optical nanostructures enables the measurement of membrane events with single molecule resolution in sub-diffraction volumes.

Single-molecule mobility and spectral measurements in submicrometer fluidic channels

Electrophoretic mobility differences of biological molecules are frequently exploited to physically separate and subsequently identify the components of a mixture. We present a method to rapidly identify single molecules by measuring both their mobility and fluorescence emission under continuous flow without separation. Submicrometer fluidic channels were used to detect individual nucleic-acid-engineered fluorescent labels driven electrokinetically in free solution. Two separate focal volumes along the length of the fluidic channel collected spectral, spatial, and temporal information from the passage of fluorescent labels through the channel. One focal volume was defined by a focused 488-nm-wavelength laser and the other by a focused 568-nm laser. The subfemtoliter focal volumes resulted in signal-to-noise ratios sufficient for single-fluorophore detection, and the two excitation wavelengths enabled detection of multicolor fluorescent labels and discrimination of single-color detection events. Each fluor...

Nanodevices for single molecule studies

During the last two decades, biotechnology research has resulted in progress in fields as diverse as the life sciences, agriculture and healthcare. While existing technology enables the analysis of a variety of biological systems, new tools are needed for increasing the efficiency of current methods, and for developing new ones altogether. Interest has grown in single molecule analysis for these reasons. The ability to detect single molecules provides a number of advantages in biomolecular analysis [1–10]. One benefit is an increase of quantification accuracy, as analysis occurs at the ultimate resolution limit. Single molecule techniques also consume less reagent than conventional techniques, and reduce analysis times. Mass production of micro-total-analysis-systems with the ability to analyze single molecules could increase the scope of otherwise prohibitively expensive and protracted processes, such as genomic sequencing and drug discovery. In addition to increasing the efficiency of existing technologies, single molecule analysis grants access to information that is otherwise unobtainable. The characteristics of biomolecular reactions are of interest in this regard. Molecular biologists have used conventional methods to study the ensemble characteristics of many systems. While this approach yields important information regarding the average behavior of a system, it tells little about the specific behavior of single molecules. This includes the time evolution and statistical distribution of parameters obscured by traditional techniques. A variety of nanofabricated structures have emerged as potential tools for single molecule analysis. Several nanostructures have been developed for enhanced optical detection, including quantum dots [11–13], metallic nanobarcodes [14], and nanometric slits [15]. Two optical structures in particular have demonstrated their utility for single molecule analysis – fluidic channels with submicrometer and nanometer dimensions, and optical nanostructures known as zero mode waveguides. Fluidic channels provide controlled transport of analytes through a subfemtoliter focal volume, while zero mode waveguides have

Modulation of transmission through isolated subwavelength apertures by dielectric filling and its implications for use in biophysical research

Use of finite element method to study transmission through dielectric-filled subwavelength apertures shows that a small change in the filling refractive index can induce a large change in light transmission for certain subwavelength aperture radii.