Editorial Feature

What Can We Discover with a 3-in-1 Microscope?

A recently conducted study by physicists from TU Delft in the Netherlands has demonstrated a sub-nanometer resolution, cryogenic electron tomography (cryo-ET) capable of in-situ observation of macromolecular complexes.

microscopes, microscopy, 3 in 1 microscope, three in one microscope

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The development of a 3-in-1 microscope that combines an ion beam, an electron beam and a light beam to precisely study thinly sliced biological samples is detailed in the publication eLife. An in-depth understanding of macromolecular complexes is essential for biomolecular research toward future pharmaceutical products.

To comprehend how complicated biological processes are at the molecular level, 3D reconstructions of biological molecules with high resolution in their almost natural cellular context are required. Cellular structures can be imaged with cryo-ET with previously unheard-of clarity and resolution.


Cryo-ET is an extension of Cryo-electron microscopes (Cryo-EM), which were first developed to image highly reactive samples. Cryo-EM, which was honored for its contributions with the 2017 Nobel Prize in Chemistry, allows for detailed, high-resolution investigation of soft samples that are protected from reactive interactions.

Using liquid nitrogen or ethane, the sample under inquiry is frozen to cryogenic temperatures in Cryo-EM. Samples are kept in their natural state with a thin covering of ice shielding them from the environment since the cooling procedure is carried out quickly. An image is created by firing electrons at the sample and detecting them using a camera.

The sample's molecules are arranged in various directions. Using computer software, all the molecules with the same orientation are grouped together and the images are sorted. For each sample, thousands of pictures are collected. To create a high-quality 3D structure of the sample, additional software editing combines the pictures. The sample must be extremely thin for high-resolution cryo-ET to work.

Sample Preparation

Among the various methods employed for milling thin samples, the ion-beam scanning electron microscope (FIB-SEM) has shown itself to be particularly effective. FIB-SEM is efficient for ablating the surplus bio-material surrounding the target region in order to produce sufficiently thin sections (lamellae) for high-resolution tomography.

The cryo-FIB milling procedure has undergone a number of changes recently, enhancing throughput, dependability, sample yield, and quality.

A critical first step in milling is the proper location of the region of interest. Unfortunately, neither the FIB nor the SEM offers a contrast mechanism for biomolecular composition, which might result in milling without knowing the exact location and possibly unintentional ablation of the target structure. This can also induce contamination in the sample area.

Previously, the material had to be examined under various microscopes, and the data from each microscope had to be superimposed. Or a collection of partitioned samples at random were analyzed hoping that one of them would contain the desired biomolecule.

This problem can be solved by using cryogenic correlative light and electron microscopy (cryo-CLEM). This method allows for targeted FIB milling by applying customized fluorescent labeling to target the location of particular objects or cellular regions for cryo-FIB milling.

One method to overcome the blind milling hurdle was the addition of an in-situ fluorescence microscope (FM) to a FIB-SEM. This technique streamlines the sample handling and lowers sample contamination risk by cutting down on the amount of cryo-transfer steps. However the sample still has to be moved within the vacuum chamber in order to switch between the FM and FIB-SEM imaging modalities.

3-in-1 Microscope

The application of cryo-CLEM for the cryo-ET workflow could be made easier by imaging with all three microscopes, which could reduce the necessity for fiducial markers and the likelihood of relocation errors. A geometry like this would enable FM imaging as well, enabling real-time milling process correction in the event of mistakes, drift, or other misalignments.

As described in detail in the journal report, various instrumental changes were implemented to construct the 3-in-1 microscope. A novel translation stage, a small cryogenic micro cooler, and a tilted objective lens within the vacuum chamber of the light microscope were some of the technical changes made. The latter is capable of performing widefield FM imaging while FIB milling, providing a coincident 3-beam cryo-CLEM system.

For in-situ manufacturing of lamellae in frozen conditions, this method can be coupled with standard systems to enable simultaneous, contemporaneous imaging with FM, SEM, and FIB.

The new 3-in-1 set-up developed at TU Delft provides the following benefits:

  • It is possible to coordinate the objective lens focal point with the FIB-SEM working point, enabling concurrent imaging with electron, ion, and photon beams.
  • It is possible to perform in-situ milling of frozen-hydrated lamella using FM-guided FIB without using any stage movements.
  • At every stage of the milling procedure, quality control of the lamella production process can be carried out using a light microscope (LM).
  • By centering and focusing the fluorescent region of interest in the LM picture after a single, initial objective lens alignment, multiple target regions on the sample can be processed.
  • High-resolution fluorescence localization is possible when using an OL with a numerical aperture (NA) of 0.85 in our situation.


In this study, for cryo-ET, a coincident three-beam microscope that enables direct light microscopy-focused lamella manufacturing without the use of fiducial markers or repositioning was developed. While milling, the target's integrity can be checked by keeping an eye on the lamella's fluorescence signal strength. Any misalignment can be seen and fixed right away.

The 3-in-1 method enables live, reflection and/or fluorescence image feeds throughout milling. This also creates the prospect of an automated milling process in which, before the sample is sent to the TEM, the fluorescent feature of interest in the sample is ensured to be inside the lamella.

More from AZoOptics: What is Quantum Microscopy?

References and Further Reading  

Daan B Boltje, Jacob P Hoogenboom, Arjen J Jakobi, Grant J Jensen, Caspar TH Jonker, Max J Kaag, Abraham J Koster, Mart GF Last, Cecilia de Agrela Pinto, Jürgen M Plitzko, Stefan Raunser, Sebastian Tacke, Zhexin Wang, Ernest B van der Wee, Roger Wepf, Sander den Hoedt (2022) A cryogenic, coincident fluorescence, electron and ion beam microscope eLife 11:e82891. https://doi.org/10.7554/eLife.82891

Sivarajah, I. (10 February 2022) How Cryogenic Electron Microscopy is Advancing Battery Design. [Online] AZoOptics.com. Available at: https://www.azooptics.com/Article.aspx?ArticleID=2144

Technology Networks  - Analysis & Separations. (7 December 2022) 3-in-1 Microscope Aids Biomolecule Structural Determinations. [Online] Technologynetworks.com. Available at: https://www.technologynetworks.com/analysis/news/3-in-1-microscope-aids-biomolecule-structural-determinations-368230

Disclaimer: The views expressed here are those of the author expressed in their private capacity and do not necessarily represent the views of AZoM.com Limited T/A AZoNetwork the owner and operator of this website. This disclaimer forms part of the Terms and conditions of use of this website.

Written by

Ilamaran Sivarajah

Ilamaran Sivarajah is an experimental atomic/molecular/optical physicist by training who works at the interface of quantum technology and business development.


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