Cryogenic Correlative Single-Particle Photoluminescence Spectroscopy and Electron Tomography for Investigation of Nanomaterials.

Cryogenic single-particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complementary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single-particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in-plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases, the onset of the splitting corresponded to changes in the in-plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.

Cryo-electron microscopy and X-ray crystallography: complementary approaches to structural biology and drug discovery.

The invention of the electron microscope has greatly enhanced the view scientists have of small structural details. Since its implementation, this technology has undergone considerable evolution and the resolution that can be obtained for biological objects has been extended. In addition, the latest generation of cryo-electron microscopes equipped with direct electron detectors and software for the automated collection of images, in combination with the use of advanced image-analysis methods, has dramatically improved the performance of this technique in terms of resolution. While calculating a sub-10 Šresolution structure was an accomplishment less than a decade ago, it is now common to generate structures at sub-5 Šresolution and even better. It is becoming possible to relatively quickly obtain high-resolution structures of biological molecules, in particular large ones (>500 kDa) which, in some cases, have resisted more conventional methods such as X-ray crystallography or nuclear magnetic resonance (NMR). Such newly resolved structures may, for the first time, shed light on the precise mechanisms that are essential for cellular physiological processes. The ability to attain atomic resolution may support the development of new drugs that target these proteins, allowing medicinal chemists to understand the intimacy of the relationship between their molecules and targets. In addition, recent developments in cryo-electron microscopy combined with image analysis can provide unique information on the conformational variability of macromolecular complexes. Conformational flexibility of macromolecular complexes can be investigated using cryo-electron microscopy and multiconformation reconstruction methods. However, the biochemical quality of the sample remains the major bottleneck to routine cryo-electron microscopy-based determination of structures at very high resolution.

Phosphorus detection in vitrified bacteria by cryo-STEM annular dark-field analysis.

Bacterial cells often contain dense granules. Among these, polyphosphate bodies (PPBs) store inorganic phosphate for a variety of essential functions. Identification of PPBs has until now been accomplished by analytical methods that required drying or chemically fixing the cells. These methods entail large electron doses that are incompatible with low-dose imaging of cryogenic specimens. We show here that Scanning Transmission Electron Microscopy (STEM) of fully hydrated, intact, vitrified bacteria provides a simple means for mapping of phosphorus-containing dense granules based on quantitative sensitivity of the electron scattering to atomic number. A coarse resolution of the scattering angles distinguishes phosphorus from the abundant lighter atoms: carbon, nitrogen and oxygen. The theoretical basis is similar to Z contrast of materials science. EDX provides a positive identification of phosphorus, but importantly, the method need not involve a more severe electron dose than that required for imaging. The approach should prove useful in general for mapping of heavy elements in cryopreserved specimens when the element identity is known from the biological context.

New controlled environment vitrification system for preparing wet samples for cryo-SEM.

A new controlled environment vitrification system (CEVS) has been designed and constructed to facilitate examination by cryogenic scanning electron microscopy (Cryo-SEM) of initial suspension state and of microstructure development in latex, latex-composite and other coatings while they still contain solvent. The new system has a main chamber with provisions for coating as well as drying, and for well-controlled plunging into cryogen. An added subsidiary chamber holds samples for drying or annealing over minutes to days before they are returned to the main chamber and plunged from it. In the main chamber, samples are blade-coated on 5 x 7 mm pieces of silicon wafer and held at selected temperature and humidity for successively longer times, either there or after transfer along a rail into the subsidiary chamber. They are then placed in the sample holder mounted on the plunge rod, so as to permit adjustment of the sample's attitude when it plunges, at controlled speed, into liquid ethane at its freezing point, to a chosen depth, in order to solidify the sample without significant shear or freezing artifacts. The entries of plunging samples and related sample holders into liquid ethane were recorded with a high-speed, high-resolution Photron digital camera. The data were interpreted with a new hypothesis about the width of the band of extremely rapid cooling by deeply subcooled nucleate boiling below the line of entry. Complementary cryo-SEM images revealed that the freezing rate and surface shearing of a sample need to be balanced by adjusting the plunging attitude.