Editorial Feature

Comparisons of Microscopy Techniques

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Microscopy is a technical field that employs instruments, called microscopes, to examine minute objects that are invisible to the naked eye, by magnifying or enlarging the image of the object1.

One of the most commonly used and well-known microscopy tools is the bright field light microscope, which utilizes visible light to magnify small objects2.

In a conventional bright field microscope, the light from the incandescent light source is aimed at the specimen through the light-focusing aperture located beneath the stage, which is known as the condenser2.

The illuminated specimen is then magnified by the objective lens and a second magnifying lens located near the eye called the ocular, or the eyepiece. Specimens in the light path will be seen as a result of the significant amount of light being absorbed by a colorless specimen which is thick enough, or by a colored specimen which may be due to a natural pigment or the stains used2.

The magnification of the image is calculated as the product of objective lens magnification and the ocular magnification. Modern light microscopes have a maximum total magnification of 1000x, which limits the structures that could be characterized by light microscopy.

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With recent advances in its instrumentation, bright field microscopes are becoming easier to use, especially for long periods of routine microscopy. Olympus has recently launched the Olympus’ CX3 series, which includes two new microscopes, the CX33 and CX433.

These microscopes have lowered frequently used controls, such as stage, stage controls, light intensity controller and coaxial focus knobs, which allow for a more natural position of user’s arms during operation, thereby reducing fatigue during prolonged periods of microscope use3.

This ergonomic design makes it easier to check or swap samples with just one hand, as well as access controls while the user’s forearms remains resting on the desk3.

These microscopes also have a low-positioned finger rest attached to the nosepiece, which can accommodate up to five objective lenses, allowing for quick changes in magnification while requiring minimum arm movement3.

The superior CX33 and CX43 microscopes are equipped with a new centering-free LED light source with a 60,000-hour lifetime to improve durability and reduce running costs. The ability of the new LED to provide uniform illumination and constant color temperature at any brightness levels allows CX33 and CX43 microscope users to experience a natural color representation of a variety of stains, which therefore allows for faster analysis with great confidence3.

While the CX33 has a built-in camera port, its camera has an optional trinocular observation head for digital imaging, along with a universal condenser that can support objectives from 2x to 100x3. The CX43 microscope allows the user to carry out a variety of observation methods, such as phase contrast and fluorescence microscopy, on a single microscope frame, enabling the users to switch to the appropriate microscopy method for their desired application3.

Aside from light microscopy, other techniques such as Fluorescence Microscopy (FM), Stochastic Optical Reconstruction Microscopy (STORM) or Photo Activated Localization Microscopy (PALM), Electron Microscopy (EM), which includes Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM), Focused Ion Beam (FIB) microscopy and FIB-SEM are also used in different fields of research.

In FM, the marker of interest is tagged or engineered with fluorophores such as green fluorescent protein (GFP). When the sample is illuminated with the light of a specific wavelength, the fluorophores absorb the light, and emit light of longer wavelengths to help the identification of the fluorophore-tagged marker of interest in a dense pool of unlabeled background, or against a counterstain of a different colored fluorophore5. Typical fluorescence microscopes have similar total magnification capabilities as compared to traditional light microscopes.

STORM employs the stochastic switching of a single molecule fluorescence signal to produce super resolution images6. Unlike in conventional FM, where all fluorophores present within the sample are fluorescent, the fluorescent probes used in STORM can switch between fluorescent and dark states6.

Several snapshots of a small and optically resolvable fraction of the fluorophores are detected, allowing for precise determination of the relative positions of the fluorescent spots from the center positions of the fluorescent spots6. The final image, with a super-resolution of about 25 nm, is reconstructed from the accumulated data gathered from the snapshots6.

Electron microscopes are incredibly complex scientific instruments that are used in research laboratories, universities, nanotechnology centers and companies around the world7. Used in a variety of industries including aeronautics, automotive manufacturing, clothing and apparel, machining, electronics, as well as in pharmacology and forensic sciences, electron microscopes are utilized for a wide range of applications including particle analysis, characterization of materials, industrial failure analysis and process control purposes7.

The high-quality imaging combined with an extraordinary resolution in nanometer scale that is obtained by these microscopes makes them especially useful in life sciences7. Within this field its applications include exploring the molecular mechanism(s) of diseases, visualizing the 3D structure of tissues and cells, determining the structure of various proteins and complexes, and even observing individual viruses and macromolecular complexes7. These remarkable scientific instruments are furthering our understanding of the structure-property-function relationships of a wide range of materials and processes7.

EM utilizes a beam of electrons to illuminate the sample. As compared to optical microscopes that use visible light, TEM utilizes an electron beam to map the local electron density differences within a sample to visualize objects8. When the electron beam hits the ultra-thin specimen in vacuum, the accelerated electrons are either absorbed or scattered.

A sophisticated system of electromagnetic lenses form an image based on the electron scattering pattern. The images are then focused onto a fluorescent screen for viewing, or onto a computer screen for capturing the image through monochromatic camera8.

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TEM is often operated to obtain images with a magnificent resolution of up to 5 nm, and a final magnification of 1,000x to 250,000x8. As impressive as it sounds, transmission electron microscopes are highly expensive, the labeling of specific proteins is particularly difficult, and TEM samples often require tedious processing time, therefore limiting its accessibility and practical use to only a few research facilities8.

In SEM, the specimen is placed in a sputter coater and is subjected to sputtering, a process of coating the specimen with a conducting material, which will typically be a metal such as gold (Au), platinum (Pt), palladium (Pd)9, as well as carbon (C).

When the electrons beam is focused on the specimen, secondary electrons produced on the metal-coated specimens, which is sensed by the detector to reveal the image on the screen9. SEM has a much higher resolution and a larger depth of field, which describes the amount of specimen that is focused at one time, as compared to the traditional light microscopes9,10.

While advantageous in these aspects, the use of SEM is limited by the cost of the equipment and the possibility of artifacts9,10.

For example, Keysight’s U9320B 8500B Field Emission Scanning Electron Micoscope (FE-SEM) and Phenom World’s Phenom ProX are both advancing technologies that have allowed SEMs to become much more compact in their design23,24.

As a major distributor of SEC Series Tabletop SEMs for both the United States and Canada, NanoImages LLC provides consumers with SEM systems that exhibit exemplary capabilities at a more convenient size as compared to most full-size systems. The two Tabletop SEMs offered by NanoImages include the SNE-3200M and SNE-4500M Tabletop SEM. While both SEMs contain dual SE/BSE imaging detectors, the versatile SNE-3200M is equipped with a 3-axis stage control capable of 15 nm resolution, whereas the SNE-4500M is equipped with a 5-axis stage control that displays a 5nm resolution.

When the electrons beam is focused on the specimen, secondary electrons produced on the metal-coated specimens, which is sensed by the detector to reveal the image on the screen9. SEM has a much higher resolution and a larger depth of field, which describes the amount of specimen that is focused at one time, as compared to the traditional light microscopes9,10.

While advantageous in these aspects, the use of SEM is limited by the size of its equipment, cost, the possibility of artifacts and the suitability to only solid samples9,10.

FIB instruments are similar to the SEM, however, FIB systems use ions rather than electrons to focus11. While electrons are used in both SEM and TEM, there are other charged particles that can be accelerated and focused using electric and magnetic fields11.

The charged particles used in FIB are ions, which are atoms or molecules in which the total number of electrons is not equal to the number of protons, resulting in a net positive or net negative electric charge11. The FIB systems are generally used to modify, or “mill,” the specimen surface with nanometer precision by utilizing the sputtering process11.

Electrons have a relatively low mass as compared to ions, therefore, in SEM, the electrons will be interacting with the sample non-destructively in order to produce secondary electrons that will sensed by the detectors to form a high resolution down that is to a sub-micrometer range11. On the other hand, in FIB systems, the utilized ions are relatively heavier than electrons11.

To put this difference into perspective, the lightest ion is measured to be at least 2000 times heavier than the electrons. Therefore, by carefully controlling the energy and intensity of the ion beam composed of heavier ions, it is possible with the FIB systems to either perform a very precise nano-machining to produce minute components, or to remove unwanted material from the specimen11.

The FIB-SEM systems are dual beam systems, where the SEM and FIB systems are combined to make an even more powerful system. In these dual beam systems, the electron beam and the ion beam intersect at a 52º angle at the point of coincidence near the sample surface, allowing for a high resolution imaging of the FIB-milled surface11. The FIB-SEM systems provide the benefits of both complimentary imaging and beam chemistry capabilities as a result of combining both the FIB and SEM systems11.

Zeiss recently launched a new generation FIB-SEM called Zeiss Crossbeam 550, which favors a significant increase in resolution for imaging to obtain best quality images, both in 2D and 3D. Characterization of nanostructures such as composites, metals, biomaterials and semiconductors can be performed by Crossbeam 550 model of Zeiss by utilizing the analytical and imaging methods simultaneously12.

This FIB-SEM features simultaneous monitoring and modification of samples, allowing for fast sample preparation and high throughput for a variety of processes such as cross-sectioning, TEM lamella preparation or nano-patterning12.

The Zeiss Crossbeam FIB-SEM is equipped with new software and hardware advancements to facilitate high-end applications in research and industry. The pioneering Gemini II electron optics allows users to obtain optimum resolution at a low voltage and high probe current simultaneously12.

High precision and much more efficient material processing and imaging is possible with this FEB-SEM due to its FIB column’s ability to combine the highest available FIB current of 100 nA with the new FastMill mode12.

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The new Tandem decel mode in the Crossbeam 550 model enables for enhanced resolution, while also maximizing the image contrast at low landing energies12. The Crossbeam 550 FEB-SEM’s new process for automated emission recovery optimizes the FIB column to obtain reproducible results, along with improving the user-friendliness12.

The workstation of this new FIB-SEM can be optimally integrated into correlative workflows, as well as combined with light, X-ray or ion beam microscopy12. The extraordinary 3D analytical properties made possible with the 3D EDS analyses with Zeiss Atlas 5, combined with the improved resolution at low voltages and outstanding stability for long term 3D tomography, provides a variety of uses for the Zeiss Crossbeam 550 FIB-SEM in various research fields such as material sciences and life sciences12.

By combining both light and electron microscopy, Correlative Light and Electron Microscopy (CLEM) is a valuable imaging technique that allows for a precise view and measurement of both the structure and function of a given sample.

To do so, either fluorescence or ultra resolution images are initially taken, in which these samples are then taken to be imaged under the electron microscope. By superimposing images taken from both microscopes, users are provided with both the high-resolution image that provides information about the structure, as well as information on where the signal that represents a biological molecule of interest, such as a protein, is present13.

What is especially advantageous about the CLEM approach is the ability of this technique to integrate two significant aspects of the specimen at a nanometer scaled resolution.

As one of the pioneers of this technology, ZEISS offers consumers several different microscopes that are capable of bridging the previously existing gap between structural and functional recorded images. These products include Shuttle & Find microscopes, with specific applications for both Life Sciences and Material Analysis purposes.

Similarly, the ZEISS ZEN CLEM for Serial Sections performs large-scale microscopy imaging of sections through an automatic computer process that outlines the specific region(s) of interest (ROI)14. What is particularly impressive about this image is the ability to align and reconstruct recorded images from both the light and electron microscopes in order to produce an incredibly accurate 3D image.

Since its introduction into the market, researchers have found CLEM to be a spectacular research tool that has increased the ability to distinguish important structural elements in a variety of scientific fields, especially cell biology.

By combining distinct images of the different membrane bound compartments present within the eukaryotic cell with fluorescently tagged molecules, such as enzymes, proteins and cytoskeletal components, researchers have found this imaging technique to supersede any other tool of its kind. Researchers around the world have utilized CLEM in addition to a number of other techniques to target specific areas of interest within their specimen.

For example, in 2012, shortly after CLEM was originally introduced, researchers from the University of Texas Southwestern Medical Center developed a microinjection CLEM procedure to introduce specific quantities of moles into the eukaryotic cell cytoplasm to understand its effects at the nanometer level.

By applying the CLEM technology, the researchers were able to determine the precise subcellular location of the microinjected molecules, as well as any other morphological and ultra structural changes that could have been induced as a result of the microinjection15.

Atomic Force Microscopes (AFM) allow users to visualize biological materials at the nanoscale by relying on the forces that are present between the tip of the applied cantilever and the sample to be studied. Until the cantilever approaches the sample, van der Waals forces between these two objects will attract the extremely sharp point of the cantilever to bend towards the surface16.

However, if the cantilever is brought too close to the surface, it will be repelled due to the interatomic repulsive forces present between the atoms of the cantilever and those present on the surface of the specimen. AFMs are equipped with an XY scanner that will move the sample back and forth, whereas a Z scanner is connected to the cantilever to move it up and down.

These simultaneous movements are recorded by a position sensor that tracks the movements of a laser beam that is reflected off of the flat surface of the cantilever. Any bending or movement of the cantilever will cause a change in the reflected laser beam17, which will allow the topography of the sample surface to be recorded by the position sensor, generating an accurate image mapping a given sample’s surface.

There are two types of AFM techniques that can be used, of which include contact and noncontact AFM. In contact AFM, the cantilever is in direct contact with the surface of the sample, which causes strong repulsive forces to bend the cantilever over the features of the sample’s surface.

While the contact AFM is simpler in its design, there is a greatly possibility of causing damage to the sample, as well as the tip of the cantilever, which can lead to expensive maintenance costs17. In noncontact AFM, the cantilever oscillates just above the surface without ever coming into direct contact with the sample due to the presence of a precise feedback loop. Advantages of the noncontact AFM include a preserved sample surface, longer lasting lifetimes of the cantilever, which in turn can reduce the overall operating cost of the AFM system.

Asylum Research, an Oxford Instruments Company, offers the Cypher AFM, one of the highest resolution fast scanning AFMs available on the market. Through their SpotOnTM automated laser alignment, the Cypher AFM utilizes a fully motorized laser that can be activated by a mouse click in order to perfectly align the laser to the tip of the cantilever.

With the smallest laser spot size, measuring at 3 μm, available in the industry today, the Cypher utilizes sub-picoNewton force measurements that allow for much faster image production at the highest possible resolution18.

Bruker Nano Surfaces also offers AFM products that are powered by PeakForce Tapping19. PeakForce Tapping, a technology that is exclusive to Bruker products, provides researchers with precise control over how the point of the cantilever interacts with the sample at a piconewton (pN) force sensitivity20. With some of the most cited products in use all over the globe. Bruker’s AFMs can be specific for large samples, small sample, biological samples, nanochemicals and industrial applications.

As compared to conventional optical micoroscopy technologies, confocal microscopes are used to overcome its disadvantages such as out-of-focus glare, while also being capable of imaging living tissues.

By focusing a beam of light into the specimen, dichroic mirrors are used to focus the reflected light through a confocal pinhole that is then detected to produce the final image. The advantages of a confocal microscope is the ability to control the amount of light that passes through the pinhole, which eliminates the possibility of out of focus or thin light of the specimen from obscuring the sharpness of the final image.

Fluorescence imaging is often used in conjunction with this type of microscopy, as it allows for specific biological molecules or structural regions to be visualized under the microscope21. In fact, this microscope can also create the construction of 3D images, as multiple imaging planes, which can range from 2-200 planes, are taken and combined by a computer program to construct the 3D image.

A third impressive advantage of confocal microscopes is the ability to visualize and record live specimens, in which images are taken over a period of time to create a time-lapse video.

X-ray microscopes expose specimens to soft x-rays, in which the amount of radiation that is absorbed by the sample will be detected by a charge-coupled device (CCD). Some determining factors of the final image produced by x-ray microscopy include sample composition and thickness of the specimen22.

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References:

  1. “Medical Definition of Microscopy” – MedicineNet.com
  2. “Light Microscopy” – Rice University
  3. “Olympus’ CX3 Series – Get Comfortable in Your Routine” – Microscopy News
  4. “CX33 Biological Microscope” – Olympus
  5. “Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision.” W. Kukulski, M. Schorb, et al. The Journal of Cell Biology. 2011. DOI: 10.1083/jcb.201009037.
  6. “Super-resolution Microscopy” – University of San Francisco
  7. “An Introduction of Electron Microscopy – Practical Applications of Electron- and Ion Beam Microscopy” – FEI part of Thermo Fisher Scientific
  8. “Transmission electron microscope (TEM)” – Encyclopedia Britannica
  9. “Scanning Electron Microscope” – Purdue University
  10. “Scanning Electron Microscope” – Microscope Master
  11. “An Introduction to Electron Microscopy – Focused Ion Beam Systems and DualBeam Systems” – FEI part of Thermo Fisher Scientific
  12. “ZEISS Crossbeam 550 sets new standards in 3D analytics and sample preparation” – Zeiss
  13. “Correlative Microscopy” – ZEISS
  14. “ZEISS ZEN Correlative Array Tomography” – ZEISS
  15. “Correlative light and electron microscopy (CLEM) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets.” L.E., Reddick, N.M. Alto. Journal of Visualized Experiments. (2012). DOI: 10.3791/3650.
  16. “How an AFM Works” – nanoScience Instruments
  17. “AFM Principle – Basic Training” – Park AFM
  18. “The Cypher Atomic Force Microscope” – Asylum Research
  19. “Atomic Force Microscopes” – Bruker
  20. “Peak Force Tapping – How AFM Should Be” – Bruker
  21. “Introductory Confocal Concepts” – Nikon
  22. “3D X-ray Microscopes (XRM) for Scientific and Industrial Research” – ZEISS
  23. “Phenom ProX” – Phenom World
  24. “U9320B 8500B Field Emission Scanning Electron Microscope (FE-SEM)” – Keysight Technologies
  25. “Tabletop SEM (Scanning Electron Microscopes)” – NanoImages

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