Cryofixation during live-imaging enables millisecond time-correlated light and electron microscopy.

Correlating live-cell imaging with electron microscopy is among the most promising approaches to relate dynamic functions of cells or small organisms to their underlying ultrastructure. The time correlation between light and electron micrographs, however, is limited by the sample handling and fixation required for electron microscopy. Current cryofixation methods require a sample transfer step from the light microscope to a dedicated instrument for cryofixation. This transfer step introduces a time lapse of one second or more between live imaging and the fixed state, which is studied by electron microscopy. In this work, we cryofix Caenorhabditis elegans directly within the light microscope field of view, enabling millisecond time-correlated live imaging and electron microscopy. With our approach, the time-correlation is limited only by the sample cooling rate. C. elegans was used as a model system to establish compatibility of in situ cryofixation and subsequent transmission electron microscopy (TEM). TEM images of in situ cryofixed C. elegans show that the ultrastructure of the sample was well preserved with this method. We expect that the ability to correlate live imaging and electron microscopy at the millisecond scale will enable new paradigms to study biological processes across length scales based on real-time selection and arrest of a desired state. LAY DESCRIPTION: Researchers seek to link cellular functions to their smallest structural components. Currently this requires correlation of two imaging techniques, live imaging and electron microscopy. Current correlative methods, however, have limited time resolution due to the sample preparation procedures for electron microscopy. Following live imaging, samples are transferred from the light microscope to a cryofixation, or ultra-fast freezing, instrument. The biological process progresses until the sample freezes, 1 second or more after the last live image. In this work, samples are cryofixed directly within the light microscope field of view. By eliminating the transfer step, time correlation between light and electron microscopy images of our samples is limited only by the freezing rate to the order of milliseconds rather than seconds.

Asymmetric nanofluidic grating detector for differential refractive index measurement and biosensing.

Measuring small changes in refractive index can provide both sensitive and contactless information on molecule concentration or process conditions for a wide range of applications. However, refractive index measurements are easily perturbed by non-specific background signals, such as temperature changes or non-specific binding. Here, we present an optofluidic device for measuring refractive index with direct background subtraction within a single measurement. The device is comprised of two interdigitated arrays of nanofluidic channels designed to form an optical grating. Optical path differences between the two sets of channels can be measured directly via an intensity ratio within the diffraction pattern that forms when the grating is illuminated by a collimated laser beam. Our results show that no calibration or biasing is required if the unit cell of the grating is designed with an appropriate built-in asymmetry. In proof-of-concept experiments we attained a noise level equivalent to ∼10-5 refractive index units (30 Hz sampling rate, 4 min measurement interval). Furthermore, we show that the accumulation of biomolecules on the surface of the nanochannels can be measured in real-time. Because of its simplicity and robustness, we expect that this inherently differential measurement concept will find many applications in ultra-low volume analytical systems, biosensors, and portable devices.

X-ray scattering experiments with high-flux X-ray source coupled rapid mixing microchannel device and their potential for high-flux neutron scattering investigations.

Small-angle X-ray scattering provides global, shape-sensitive structural information about macromolecules in solution. Its extension to time dimension in the form of time-resolved SAXS investigations and combination with other time-resolved biophysical methods contributes immensely to the study of protein dynamics. TR-SAXS can also provide unique information about the global structures of transient intermediates during protein dynamics. An experimental set-up with low protein consumption is essential for an extensive use of TR-SAXS experiments on protein dynamics. In this direction, a newly developed 20-microchannel microfluidic continuous-flow mixer was combined with SAXS. With this set-up, we demonstrate ubiquitin unfolding dynamics after rapid mixing with the chaotropic agent Guanidinium-HCl within milliseconds using only ∼ 40 nanoliters of the protein sample per scattering image. It is suggested that, in the future, this new TR-SAXS platform will help to increase the use of time-resolved small-angle X-ray scattering, wide-angle X-ray scattering and neutron scattering experiments for studying protein dynamics in the early millisecond regime. The potential research field for this set-up includes protein folding, protein misfolding, aggregation in amyloidogenic diseases, function of intrinsically disordered proteins and various protein-ligand interactions.

Suspended microchannel resonators with piezoresistive sensors.

Precision frequency detection has enabled the suspended microchannel resonator (SMR) to weigh single living cells, single nanoparticles, and adsorbed protein layers in fluid. To date, the SMR resonance frequency has been determined optically, which requires the use of an external laser and photodiode and cannot be easily arrayed for multiplexed measurements. Here we demonstrate the first electronic detection of SMR resonance frequency by fabricating piezoresistive sensors using ion implantation into single crystal silicon resonators. To validate the piezoresistive SMR, buoyant mass histograms of budding yeast cells and a mixture of 1.6, 2.0, 2.5, and 3.0 µm diameter polystyrene beads are measured. For SMRs designed to weigh micron-sized particles and cells, the mass resolution achieved with piezoresistive detection (∼3.4 fg in a 1 kHz bandwidth) is comparable to what can be achieved by the conventional optical-lever detector. Eliminating the need for expensive and delicate optical components will enable new uses for the SMR in both multiplexed and field deployable applications.