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Biomedical and Organic Sample Preparation

In bio application, there’re many things including nano-drugs, blood, cell, tissue till even medical devices need to be observed or analyzed. As one may know, biological samples are usually high water content with low contrast under TEM. So, a proper sample preparation for keeping the sample with the original feature will be the key and the main challenge.

 

MA-tek can provide solutions for bio-sample preparation, such as Native staining, CPD, unltr-microtome, cryo-treatment, and also a unique wet cell for liquid TEM. 

 

  • Negative Staining
  • Resin Embedding and Ultramicrotome
  • Critical Point Drying
  • Cryo-transfer system
  • K-kit for Liquid TEM
  • Wet & Dried Mode of K-kit

 

 

 

Technical Concept

Negative Staining

Any organic or biological samples are composed of light atoms (such as carbon, hydrogen, oxygen etc), thus result in low contrast in their EM images; negative staining with heavy elements (metals) could enhance the contrast for these samples; this is called “negative staining”. 

 

Biomaterial samples | PLGA nanoparticles

Biomaterial samples | Liposomes

 

Biomaterial samples | Influenza Virus

Biomedical samples | Abraxane

 

 

Resin Embedding and Ultramicrotome

Biological samples with high water content, such as cells, bacteria, tissues, organs, as well as many synthetic biomaterials have to be undergoing the procedure of fixation, dehydration, resin embedding and ultramicrotomy; then the slices can be collected and placed on copper grid for TEM observation.

 

Biological samples | Muscle

Biological samples | Bacteria

 

Biological samples | Rat Liver

Biological samples | Leukocytes

 

Biomaterials | Facial mask

Gold nanoparticles on the fiber surface can be clearly visualized. The first figure shows gold nanoparticles on the surface of the facial mask fiber edge; the second figure shows EDX result from the marked position in the first figure. 

 

Biomaterials | cosmetics

Ultrathin slice of cosmetic cake under TEM, particle morphology including the aggregation/agglomeration can be observed, and the elements can be identified by EDX.

 

 

  • Ultramicrotome, Leica EM UC7 

In ultramicrotome, samples can be sliced with a diamond blade into thin sections no more than 100 nm, which is necessary for TEM observation.

Biological tissue, cells, bacteria and some medical materials can be sliced with ultramicrotome after embedding, freezing or chemical fixation.

 

  • Cryo-Ultramicrotome, Leica EM FC7 

Some materials are only suitable to be sliced at low (cryo-) temperatures, our Leica EM FC7 cryo-ultramicrotome can operate at -15℃~-185℃, suitable for polymers, rubber and biological tissues.

 

 

Critical Point Drying

Biological samples with high water content would need to be fixed (by glutaraldehyde), dehydrated (by ethanol), critical point dried (with carbon dioxide supercritical fluids), sputter coated with Pt or Au prior to be observed with SEM.

 

Supercritical drying is a process to remove liquid from specimen without subject to surface tension, which is usually detrimental to the structure. The liquid is first completely replaced by CO2 in liquid phase, at high pressure. Then, CO2 is brought beyond critical point and then allowed to lower pressure, converting to gas phase. The specimen is dried preserving original morphology.

 


Cells are being fixed, dehydrated, and coated with Pt thin film and imaged by the SEM; gold nanoparticles of ~ 200 nm can be visualized.


Critical Point Drier K850 

 

 

Cryo-transfer System

Cryo-transfer holders are used in applications that require low temperature transfer and observation of frozen hydrated specimens for cryo-electron microscopy (cryo-EM).

 

With cryo TEM, solutions can be rapidly frozen and kept below sublimation temperatures to be observed under TEM. The morphology, size distribution of nano-objects can be analyzed with great resolution. The dispersiveness was also “frozen” in the process, allowing the studying of aggregation and agglomeration state much like the original liquid state.

 

Liposome, showing bi-layer structure

 

 

 

K-kit for Liquid TEM

K-kits are sample holders designed to facilitate convenient TEM observation of liquid samples, allowing nanoobjects, aggregates, and agglomerates (NOAAs) in liquid samples to be characterized; with vacuum compatible sealing of liquids in electron-transmitting thickness, K-kits are micro reaction chambers for countless experiments in materials, chemical and biological research.

 

 

K-kit is carried with a 3mm TEM grid so that it is compatible with all kinds of TEM holders.

 

K-kits are Si-based microchannel devices with silicon nitride windows that allow TEM observation. The seemingly irregular shape is a result of KOH anisotropic wet etching, which is also responsible for forming the rectangular observation window in the middle of the device. The liquid channel is parallel to the window, with openings at both ends.

 

 

 

Wet & Dried Mode of K-kit

The K-kit users may select a proper option per their observation purposes, either by dried mode (Thin-layer Mode) or by wet mode for the sample preparation. For example, if one would like to just observe the shapes, sizes, or aggregates and agglomerates (NOAAs) of the particles in liquid, making a dried mode of K-kit sample will be our suggestion, due to it usually with very good image quality at dried state. If someone would like to study kinds of chemical reactions in liquid, such as Au reduction process in AuCl4 solution by TEM electron beam energy, it should be considered to adopt the wet mode K-kit, which with liquid preserved inside. 

In addition, if the liquid sample essentially being very easy to react with the electron beam energy of TEM, by mostly bringing about a serious bubbling or disturbance from the liquid to hinder the TEM observation, the customers may consider applying the dried mode of K-kit as the priority solution.

 

As stated above, there’re two modes of sample preparation Wet and Dried (also called Thin Layer) available for K-kit. Generally, by using K-kits in larger gap heights (like 2um ones), it will be easy to result in the inner conditions in either fully dried without sealing both ends of the channel or with a thin liquid layer preserved on the inner walls as gluing sealed the openings within a few seconds after the liquid loading. Basically, it can be with relatively slight reaction, if less amount of liquid remained in the channel. Being able to reduce the liquid bubbling effect by the dried mode, it’s also a unique feature of K-kit that better than the other products in the market.

 

  • Wet Mode:The loaded liquid sample is sealed and imaged using TEM in the native liquid environment. (Acceptable image quality with liquid inside the K-kit)
  • Dried or Thin Layer Mode:A patented liquid drying protocol preserves the original morphology and physical state of nanomaterials with improved imaging resolution. (Very good image quality, due to the channel being dried out)

 

The loaded liquid sample is sealed and imaged using TEM in the native liquid environment.  

 

 

 

A patented liquid drying protocol preserves the original morphology and physical state of nanomaterials with improved imaging resolution.  

 

The channel gaps of K-kit make big difference. Basically, we use 2um gap K-kits for dry mode and 0.2um gap for wet mode. It’s because the liquid loaded by a larger gap height of K-kit can be much easier to be evaporated or pumped out at room temperature than by a smaller one in gap height. And, to ensure that the sample liquid could be well dried in K-kit when making a dried mode, it’s essential to keep both ends of the channel open to the atmosphere, no need to do glue sealed step.

 

Mechanism of Thin Layer Mode

  1. Filled with liquid sample
  2. Dried out by vacuum pumping
  3. By the dried process, a thin liquid layer would be formed on walls, which liquid layer trapping the particles to remain in situ.
  4. The particles would also stay in place, as the thin liquid dried out. 

 

A thin layer mode result of gap 2um K-kit loaded with polystyrene bead solution.

 

Using the dried mode of K-kit, one can prevent the nanoparticles from externally induced aggregates and keep them to remain in situ as the look suspended in liquid; the result will be almost the same as it observed by the wet mode. 

 

Dried on Cu grid

Dried Mode of K-kit

By the dried mode of K-kit, one can get the images of the nanoparticles that spread uniformly, keeping them to remain in situ after dried; if drying on Cu grid, the particles will be aggregated. (CMP Slurry)

 

 

The Liquid Layer Observations in Thin Layer Mode of K-kit

 

The quantitative counting and EELS data showed the water layer by thin layer mode of K-kit being with a persistent thickness.

 

TEM images of Cu pattern, which formed by an electron beam direct writing on a K-kit loaded with AuCl4 in a thin layer mode.

 

 

 

Equipment

 

 

 

Reference

  • US 9384942 B2, " Speciment Preparation for Transmission Electron Microscopy,” July 2016.
  • “Stable Water Layers on Solid Surfaces,” Phys. Chem. Chem. Phys., 18, 5905-5909, 2016.
  • “Direct-Writing of Cu Nano-Patterns with an Electron Beam,” Microsc. Microanal. 21, 1639–1643, 2015.
  • “Electron Beam Manipulation of Gold Nanoparticles External to the Beam,” RSC Adv., 4, 31652–31656, 2014. 

 

 

Contact

Taiwan Lab

Mr. Chiang

: +886-3-6116678 ext:3257

: +886-922-301-636

: ms@ma-tek.com