Transmission Electron Microscopy Services

What is transmission electron microscopy?

Transmission electron microscopy (TEM) is a powerful imaging method in which electrons, pass through ultra-thin specimens, and are recorded as 2D projection images.  These images can be analyzed to provide detailed views of nanoparticles at magnifications and resolutions much higher than possible using visible light. 


Used in both biological and material sciences, TEM methods provide valuable morphological and structural information for a wide variety of nanoparticles and are a critical aspect of particle characterization in biologics and drug development. 


While TEM has been used for decades, the more recent so called "Resolution Revolution" (Kuhlbrandt, 2014) has firmly established TEM as  method capable of solving protein structures to near-atomic resolution, putting it on a par with the long standing methods of X-ray crystallography and NMR.


Why choose NIS for transmission electron microscopy services?

Since 2007, NIS has completed over 2000 TEM-enabled projects for both negative stain TEM and cryoEM (aka cryogenic TEM) workflows. We provide complete sample-to-analysis transmission electron microscopy services that meet the evolving imaging, analysis and documentation requirements of the pharmaceutical, biotechnology and nanotechnology industries. 


Our automated methods provide reliable high throughput image acquisition that can be used to particles size and size range, shape, lamellarity, and sub-structure features like presence of internal cargo or surface characteristics (smooth or spiked). With our in-house state of the art high-end microscopes and computational infrastructure we also provide a complete pipeline for cryoEM (single particle reconstruction), cryo-electron tomography, and microED.

Negative Stain

Negative stain is a rapid and straightforward method that can be used to obtain nanometer resolution images of biological macromolecules (from isolated organelles to viruses to proteins). Alone, or as an entry point for the cryoEM (single particle analysis) workflow, negative stain EM studies provide information on particle size, symmetry, morphology, degree of aggregation, conformational and compositional heterogeneity, antibody interactions and higher order structure. 

Sample preparation
Data Acquisition
Data Analysis & Reporting
Sample preparation

Negative stain samples are typically prepared by (i) depositing a 1-3 ul of sample solution onto  a TEM  grid, (ii) removing most of  the excess buffer by wicking it away into filter paper, (iii) depositing 1-3 ul of a liquid heavy metal salt solution (e.g. uranium, tungsten or molybdenum) onto the still wet sample, (iv) removing  excess stain by again wicking it away, and (v) allowing the sample  to dry.  This process encases nanoparticles in a heavy metal shell and results in very high contrast images in the TEM.  Negative stain can be used to either take a "quick-look" at a sample to assess particle behavior, aggregation, etc. or can be "optimized" to provide the highest quality images that be subsequently be used to extract characterization metrics or analyzed by 2D classification.

Data Acquisition

Images are collected at room temperature using a low voltage (80-120 KeV) microscope. Out Tecnai T12 instrument is ideally suited for this work. Imaging protocols include low magnification images to provide large fields of view for assessment of aggregation and particle distribution. Additional imaging at optimized magnifications is included to provide the higher resolution characterization possible for particle morphology. 

Data Analysis & Reporting
Clients received detailed reports prepared by senior scientists that document sample receipt, preparation, imaging, analysis, and interpretation efforts. The summary provides detailed observations and key conclusions. Representative images are provided within the report with all additional image data made available through the NIS website portal. If negative stain studies are combined with more detailed analytical services, such as 2D classification, fraction counting or size distribution analysis, these results are included in a single integrated study report. We also provide supplemental and potentially critical data for demonstrating procedures to ensure stability, batch-to-batch uniformity, and overall quality for IND and NDA submissions.  We provide qualified evidence that established protocols and specifications for production processes, as well as demonstration of bioequivalence, when manufacturing protocols are modified or improved.

CryoTEM

Cryogenic Electron Microscopy (cryoTEM or cryoEM) is an electron microscopy technique in which biological samples flash to trap them into a thin layer of vitrified buffer. The method is ideal for examining a wider range of biological samples, including  proteins, viruses and lipid nanoparticles

Recent advances in hardware and software technology and algorithms have allowed for the determination of biomolecular structures at near-atomic resolution, establishing cryoEM as a viable alternative to X-ray crystallography or NMR spectroscopy for macromolecular structure determination via cryoEM.

Sample preparation
Data Acquisition
Data Analysis & Reporting
Sample preparation

CryoEM samples are typically prepared by (i) depositing a 1-3 ul of sample solution onto  a TEM  grid, (ii) removing most of  the excess buffer by wicking it away into filter paper, (iii) rapidly plunging the TEM grid into a good cryogen (typically ethane or ethane/propane mix).  The thin layer of liquid sample is snapped from a liquid to a solid (vitreous) state in less than a millisecond preserving the native hydrated state of the sample. Sample substrate and dilution conditions are optimized as required and benefit from a highly successful proprietary workflow developed at NIS from the extensive know-how of our microscopy team. In addition to standard plunge-freezing methods, NIS also provides access to the SPTL chameleon instrument when samples are especially challenging with regard to available quantities or issues with air-water interface effects.

Data Acquisition

The grid is loaded on the microscope and images are generated using a focused electron beam and a series of electromagnetic lenses.   The images are collected using highly sensitive detectors and computationally analyzed. All microscopes at NIS are equipped for cryoEM data acquisition. For lower resolution studies supporting biologics and drug development, most work is carried out on the Tecnai T12 and or the TFS Glacios microscopes. High resolution studies of proteins and viruses for single particle analysis are performed using the TFS Glacios and Titan Krios microscopes. 

Data Analysis & Reporting

NIS provides project-specific analysis and reporting modes for cryoEM studies. The details of these reports vary depending on the research question posed and the completed study. Regardless of workflow, detailed reports with scientific interpretation by senior scientists are provided for all work performed, often complemented with follow-up conference calls to delve more deeply into the observations.  We also provide supplemental and potentially critical data for demonstrating procedures to ensure stability, batch-to-batch uniformity, and overall quality for IND and NDA submissions. We provide qualified evidence of established protocols and specifications for production process, as well as demonstration of bioequivalence, when manufacturing protocols are modified or improved.

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