Microscopy Buying Guide

Microscopy and Image Analysis image

This buying guide provides an overview of the current techniques involved in microscopy, key features and considerations of both light (optical) and electron microscopy, application information, and examples of future developments.


INTRODUCTION

The main consideration for all types of microscopy is your application:

  • Do you need a microscope to suit more than one purpose, e.g. both light and fluorescence?
  • What samples will you be using? For example, living cells or fixed cells, whole tissue sections or bacterial cultures; and will these be stained or unstained?
  • What level of detail do you require?
  • Are you going to look at tissue morphology or localization of staining within a cell, or counting yeast or bacteria? If you are looking at bacteria or localization of features/staining within a cell, you will require a much higher magnification than if you are looking at overall tissue morphology.

There is a section on applications in this guide, which provides some interesting examples of the use of the recent microscope technologies for different applications.

LIGHT (OPTICAL) MICROSCOPY TECHNOLOGY AND CONSIDERATIONS

The light microscope is an instrument used for magnifying research specimens. Light microscopes are essential analytical laboratory tools that have the potential to allow scientific investigators to view objects to as much as a thousand times their original size.

In its simplest form, it is composed of a clear lens that magnifies the sample and a light source to illuminate it. However, most light microscopes are much more complex and house numerous fine-tuned lenses with tightly controlled dimensions, all within the body of the microscope itself and in components such as the objectives and eyepieces.

Basic light microscope operation begins with bringing light to the sample and ensuring that the light source is of the correct intensity, directionality and shape in order to produce the best quality image. Samples are made up from different structures, so light traveling through it is absorbed in different ways. The sample must also be magnified properly and brought into focus to view the region of interest. These variations to the light microscope provide an almost limitless range of options for visualizing specimens in the lab; from viewing of stained or unstained cells and tissues, to resolving small details of specimens, and even magnifying a region of interest during surgery to assist with complex procedures on the micron scale.

Confocal microscopy is a highly advancing type of widefield optical microscopy, which includes the ability to control depth of field, elimination or reduction of background information, and the capability to collect sections from specimens several opticals thick. The signal-to-noise ratio (S/N) is improved in comparison to widefield (traditional brightfield and darkfield optical) techniques, and it can be used with both live and fixed specimens. One of the fundamental advantages of confocal over a widefield microscope is the restricted manner in which light reaches the photomultiplier through a pinhole. In widefield fluorescence, the image of a thick biological specimen will only be in focus if its axial dimension is less than the wave-optical depth of focus specified by the objective parameters. In cases where this condition is satisfied, the in-focus image information from the specimen plane of interest is mixed with out-of-focus image information, arising from regions outside the focal plane. This reduces image contrast and increases the share of stray light detected. If multiple fluorophores are being observed, there will also be a color mix of the image information obtained from all of the channels involved. Confocal microscopy is discussed further in the ‘Applications’ section.

Principle_of_Confocal_Microscopy

Figure 1: Principle of Confocal Microscopy

(Image credit: cc SA3.0)

Fluorescence microscopy is a light microscopy technique that dramatically improves contrast. This is done by putting a dye on to the sample, which becomes attached to a specific part of the cell (e.g. the dye DAPI only stains a cell's nucleus). By adding several dyes, which stain different parts of the cell, the contrast of the sample is increased and more of the biological structures of interest are seen, and less of the background structures surrounding them.

Table 1 details some of the light (optical) microscopy and instrumentation and techniques known to us presently.

Table 1: Light (Optical) Microscopy Categories

Sub-Category

Techniques

Uses

Transmitted Light

Brightfield

Simple microscopy. Allows limited visualization of unstained samples. Most appropriate for stained samples.

Darkfield

Well suited for uses involving live and unstained biological samples.

Phase Contrast

Ideal for the research of unstained living cells in processes such as cell division.

VAREL Contrast (Variable relief contrast)

Ideal for examination of living cells in culture vessels.

Polarization Contrast

View birefringent crystalline structures without staining. Ideal for plant research.

Differential Interference Contrast (DIC) (Nomarski)

As an extension of polarization contrast, this is used to enhance the contrast in unstained, transparent samples.

Confocal

Confocal Laser Scanning (CLSM)

Small slices from microscopic samples are generated. Widely used in life sciences.

True Confocal Scanning (TCS)

One diffraction limited spot is illuminated and observed. Widely used in life sciences.

Coherent Anti-Stokes Raman Spectroscopy (CARS)

Dye-free method. Images structured by rapid vibrational imaging of living cells.

Fluorescence (uses reflected light)

Super-Resolution Imaging

Fluorescent proteins (e.g. GFP) are used to fluorescence living material.

Total Internal Reflection Fluorescence (TIRFM)

Sensitive technique which allows for functional investigation in living cells.

Fluorescence-Lifetime Imaging (FLIM)

Measures the decay rate of fluorescence to give a ‘lifetime’ signal as opposed to one based on intensity. Used for receptor signaling.

Fluorescence Correlation Spectroscopy (FCS)

Used to examine the dynamics and concentration of fluorescent molecules in solution.

Fluorescence Cross-Correlation Spectroscopy (FCCS)

Two independently labeled fluorescent probes are detected by two different laser light sources.

Fluorescence Energy Transfer (FRET)

The study of the interaction of chromophores to fluorochrome.

Fluorescence after Photobleaching (FRAP)

Observing fluorescence recovery dynamics of a molecule after photobleaching.

Fluorescence & DIC Combination Microscopy

Helps to minimize the effects of photobleaching by locating a specific area of interest in a specimen using DIC.

Fluorescence & Phase Contrast Combination Microscopy

This technique limits photobleaching by locating the specific area of interest in a specimen using the technique (phase) then, without relocating the specimen, switching the microscope to fluorescence mode.


Lenses: Objectives and Eyepieces

A simple microscope or magnifying glass (lens) produces an image of the object upon which the microscope or magnifying glass is focused. There are two main types of lens in optical microscopy, the objective and the eye piece; both are important considerations when choosing a microscope.

Objective Lenses

Typically, standard optical microscopy objective lenses range between x4 and x100 in magnification. Objective lenses are available for particular applications: water immersion lenses exist for live cell imaging; color corrected and wave length optimized objectives have been designed for applications such as laser micro-dissection and fluorescence microscopy. Consider the primary use of your objective lenses, the immersion you will use, if any, and which magnifications are likely to be used the most. You may want a microscope that has easily changeable objectives to suit your application, or you may want a set that can be affordably replaced should one get damaged. Some manufacturers color- and number-code their lenses so that it is easy to look up the applications for which the lens is most suited.

Eyepieces (Oculars)

Eyepieces work in combination with microscope objectives to further magnify the intermediate image so that specimen details can be observed. Some eyepieces are specially designed for microscope users who wear glasses. Others are designed to work with specific objectives for compensation and correction purposes. Eyepieces can generally be grouped as ‘finder’, ‘wide field’ and ‘super wide field’: wide field eyepieces, as the name suggests, are designed to give a wide field of view. Eyepieces can also have scale bars and grids for measuring and counting, and moveable pointers for group work situations. Such features are normally written on the eyepiece as they are with objective lenses.

Buying Guide Tip: Manufacturers can use different terminology when describing and coding objectives and eyepieces; read around the terms if you are unsure.

Objective and Eyepiece Combinations

Most manufacturers recommend carefully choosing the objective first, and then purchasing an eyepiece that is designed to work in conjunction with the objective. Care should be taken in choosing eyepiece/objective combinations to achieve optimal magnification of specimen detail without adding unnecessary artifacts. For instance, to obtain magnification of 250x you could choose a 25x objective with a 10x eyepiece; this magnification could also be obtained with a 10x objective and a 25x eyepiece.

So which is most appropriate? Numerical aperture (NA) of the objective helps to decide this. The NA defines the resolution for a microscope; the minimum resolution is 500 multiplied by NA and the maximum is 1,000 multiplied by NA. These are arbitrary but commonly used formula.

Example: a 10x objective has a NA of 0.25. The minimum and maximum useful magnifications are therefore 125x and 250x.

Combining a 10x objective with a 25x eyepiece is within this range, but at the upper limit.

A 25x objective has a NA of 0.4. The minimum and maximum useful magnifications are therefore 200x and 400x.

Combining a 25x objective with a 10x eyepiece is within the middle of these ranges and is therefore considered a more appropriate combination to achieve a 250x magnification.

Eyepiece and objective combinations that fall outside the objective NA formula ranges will result in empty magnification or poor quality images.

Once you understand the terminology and principles in objective and eyepiece combinations you are on your way to choosing the most appropriate equipment for your needs.

Buying Guide Tip: Do check the NA of the objective; if a 60x objective and a 100x objective have the same NA number, the 100x lens will not provide any extra detail.

Microscope Illumination

Quite often, sophisticated and well-equipped microscopes fail to yield excellent images due to incorrect use of the light source, which usually leads to inadequate sample illumination. When optimized, illumination of the specimen should be bright, glare-free, and evenly dispersed in the field of view. There are numerous light sources available to illuminate microscopes, both for routine observation and critical photomicrography. Tungsten lamps are common sources of illumination due to their cost and long-life. Some lamps have minimum and maximum recommended times of use, so consider how long you are likely to be using the microscope in one session: if you require long periods of use, perhaps in an industry setting, a heavy-duty lamp may be more appropriate for your needs. Another consideration is whether you want your specimens lit from above or below. Inverted microscopes provide light from above and are useful for viewing Petri dishes and other cell culture vessels. Most confocal systems use lasers for illumination, so safety is an important feature when choosing such a microscope.

Microscope Staging

All microscopes are designed to include a stage where the specimen (usually mounted on to a glass slide) is placed for observation. Stages are often equipped with a mechanical device that holds the specimen slide in place and can smoothly translate the slide back and forth as well as from side to side. A stage can be classified according to design and functionality. Many microscopes have a simple stage with an attachable mechanical stage which can be easily manipulated; some stages are robotic to allow precise and reproducible imaging to take place. There are many other types of stages: specialized, inverted, universal and precision, to name a few. Some stages are designed to include a heated/humidified chamber for live cell imaging. Your application needs define the most appropriate microscope stage. The flexibility of an open frame modular microscope system and how this allows flexibility to combine several applications, such as live cell imaging, can be seen in this video from Olympus.

Flexible_Modular_Microscopy

Figure 2: Flexible Modular Microscopy

Other Light (Optical) Microscope Considerations

These are terms that you may come across when reading about light microscope specifications; some terms, such as resolution, are also used in electron microscopy. The considerations below are more process than structurally orientated.

Resolution

Resolution is a measure of how fine a detail can be detected, in terms of either space (spatial resolution), time (temporal resolution), or intensity. It is the shortest distance between two separate points in a microscope’s field of view that can still be distinguished as distinct entities. The greater the resolution of the image, the more information can be determined. In fluorescence applications using low light and/or thicker specimens, confocal imaging can greatly improve resolution.

In light (optical) microscopy, resolution is affected by several factors including numerical aperture, light diffraction and the light wavelength.

As described above, a microscope may offer high magnification, but if the lenses are of poor quality the resulting poor resolution will disrupt the image quality.

Super-resolution describes a series of methods used to improve the resolution of light microscopy by surpassing the diffraction limit (see below) to further improve the output image. Applications are broad, from dynamic vesicle movements in the sub-100 nm range to fluorescence images of sub-cellular structures, enabling researchers to see fine details only previously possible with electron microscopy techniques.

Signal-to-Noise Ratio (S/N or SNR)

Signal-to-noise ratio (S/N) is the ratio between a given signal and the noise associated with that signal. The S/N usually increases (improves) as the signal increases and is directly associated with the image quality. The S/N is affected by sample fluorophores, sample autofluorescence and even the optical components themselves, such as the lens cement. An optimum aperture size will exist that maximizes the S/N, while still maintaining adequate signal-to-background ratio for good image contrast.

Diffraction Limit

There is a fundamental maximum to the resolution of the light (optical) microscope, which is caused by the diffraction of light. Super-resolution microscopy uses methods to push this boundary and so increase resolution.

ELECTRON MICROSCOPY TECHNOLOGY AND CONSIDERATIONS

There are a number of key features to consider when choosing an electron microscope. Some of the features detailed in optical microscopy are also applicable when choosing an electron microscope, such as staging and resolution.

As outlined in Table 2 below, Transmission Electron Microscopy (TEM) produces high quality and detailed images by passing electrons though a thin section of sample; for instance it can be used to view details of mitochondria in cells. TEM can also provide information on element and compound structure. Figure 3 Shows the structure and make-up of a TEM. Scanning Electron Microscopy (SEM) produces 3D images that give information on morphological and topographical details, such as basic surface characterization. A focused ion beam system (FIB) is a tool that has a high degree of accuracy and can be used to reveal artifacts below the surface in materials and devices. DualBeam (FIB/SEM) systems are the preferred solution for 3D microscopy. Some electron microscopes can encompass both SEM and TEM technology in one, such as the Quanta™ Scanning Electron Microscope, from FEI Company.

Consider sample preparation; with most types of electron microscopy, sample preparation can be quite time consuming with risks of introducing artifacts. CryoSEM avoids complex preparation and can be useful when studying samples containing water/moisture, such as botanicals and food stuffs. However, it has limitations such as sample shrinkage. Consider the different brands, as each one has a different mechanism, varying accessories (add-ons) and optics. Also consider the microscope’s capability to be automatically controlled using included software, rather than by manual control of functionality such as zooming, often requiring a specialist.

Structure_of_a_Transmission

Figure 3: Structure of a Transmission Electron Microscope (TEM)

Table 2: Electron and Scanning Microscopy Categories

Category

Sub-Category

Description

Transmission Electron Microscopy (TEM)

 

Passes energetic electrons through the sample. Electron beam passes through thin slice of specimen. Resolution limit approx 0.05 nanometers. Able to distinguish surface features, shape, size and structure.

Scanning Electron Microscopy (SEM)

 

Investigates the surface of bulk objects by scanning the surface with a fine electron beam. A 3D view is obtained giving surface detail of specimens. Resolution limit is approx 0.4 nanometers. The preparation of samples can result in the production of artifacts.

Reflection Electron Microscope (REM)

 

A combination of imaging, diffraction, and spectroscopy techniques. Applicable to metal, semiconductor, and ceramic surfaces.

Scanning Probe Microscopy (SPM)

 

Scans several images of interactions simultaneously using various probes. The resolution varies depending upon the probe and technique used. Some high resolution techniques have resolution to a precise atomic level.

Atomic Force Microscopy (AFM)

High resolution type of SPM. Resolution is in the nanometer range. Ideal for imaging, measuring and manipulation at the nanoscale.
There are many types:
- Contact AFM
- Non-contact AFM
- Dynamic contact AFM
- Tapping AFM

Scanning Tunneling Microscopy (STM)

Images surfaces at the atomic scale. Can be used in ultra-high vacuum, air, water, and other liquid or gas ambient states.

Ultrasonic Force Microscopy (UFM)

Gives detail and image contrast of flat areas of interest.

Photonic Force Microscopy (PFM)

High precision technique measuring scattered light and orientation of a particle.

Other, less used, types of SPM include:

Ballistic Electron Emission Microscopy (BEEM), Chemical Force Microscopy (CFM), Conductive Atomic Force Microscopy (C-AFM), Electrochemical Scanning Tunneling Microscope (ECSTM), Electrostatic Force Microscopy (EFM), Fluidic Force Microscope (FluidFM), Force Modulation Microscopy (FMM), Feature-Oriented Scanning Probe Microscopy (FOSPM), Kelvin Probe Force Microscopy (KPFM), Magnetic Force Microscopy (MFM), Magnetic Resonance Force Microscopy (MRFM), Near-field Scanning Optical Microscopy (NSOM or Scanning Near-field Optical Microscopy, SNOM) Piezoresponse Force Microscopy (PFM), Photothermal Microspectroscopy/Microscopy (PTMS) Scanning Capacitance Microscopy (SCM), Scanning Electrochemical Microscopy (SECM), Scanning Gate Microscopy (SGM), Scanning Hall Probe Microscopy (SHPM), Scanning Ion-Conductance Microscopy (SICM), Spin Polarized Scanning Tunneling Microscopy (SPSM), Scanning Spreading Resistance Microscopy (SSRM), Scanning Thermal Microscopy (SThM), Scanning Tunneling Potentiometry (STP), Scanning Voltage Microscopy (SVM), Synchrotron X-ray Scanning Tunneling Microscopy (SXSTM).

Sample Stage and Chamber Size

Ensure the sample stage is the correct size to fit your samples. Most stages have a travel range and a set capacity. The longer the travel ranges of the stage, the larger the samples you will be able to analyze. The chamber size is often selected upon purchase, or the microscope is provided with different chamber sizes that can be interchanged.

Electron Detectors

The capabilities of an electron microscope are dependent upon which detectors it accommodates. Most electron microscopes can house a variety of detectors. For instance, SEM detectors are designed to detect secondary electrons that are emitted from the sample surface as a result of excitation from the primary beam. If you want a multi-purpose electron microscope, ensure the detector is suitable for multiple applications or easily changeable as required.

Magnification

Generally, a TEM has a much higher magnification than a SEM. However, consider the sample preparation required for the use of a TEM before purchase. Ensure you still havedepth of field (DOF) and resolution at the higher magnifications. The lens types will determine the magnification, so check both requirements for your applications. High magnification without high resolution is redundant.

A Low kV Performance

How does the microscope’s electron optical performance operate at low voltages? Some electron microscopes have a high resolution at as little as 1kV. Consider the applications of the microscope, as for some applications, high resolution at low voltages is not so important. Surface sensitive imaging with high material contrast is better guaranteed with lower voltages of acceleration. The lower voltage prevents beam penetration into the sample.

Microscope Surroundings

The room space and environment of the room is of great consideration in electron microscopy, especially for a TEM. Room considerations and requirements are:

  • Vibration dampening
  • Dimming Lights
  • High voltage access
  • Electromagnetic shielding
  • Air movement control and cleanliness, using aircon and vacuum pumps
  • Separate sample preparation room

Often a company engineer will make a site visit to assess a room’s suitability.

OTHER KEY FEATURES TO CONSIDER

Microscope analysis software is available from many manufacturers and again will vary depending on the application. Many problems are caused by software being incompatible with your technique and type/brand of microscope.

Cameras for microscopy CCD and EMCCD are available. These enable the user to take a still photograph or video of the substance or structure being analyzed. For example, the integrated high definition digital camera included in the Leica EZ4 HD stereo microscope provides fast, live images direct to a storage device. Consider where you will be publishing your photomicrographs, if at all: will they be used in high quality publications or posters? If not, does your laboratory actually require the large file size and image detail of some high performance cameras? The number of megapixels on a camera generally equates to how big you can enlarge the image without losing quality; if you will not be using large images then perhaps the quality and size of the camera sensor may be of higher importance to you.

Miscellaneous Features: consider what else you may require with your instrument; for example will more than one person be using it at once? If this is the case, you may require a multi-headed microscope.

Buying Guide Tip: Cost has not been covered in this guide. However, it is of course an important consideration in every laboratory.

WHICH TECHNOLOGY SUITS YOUR APPLICATION?

Many microscopy users utilize one or two combinations of different techniques in order to optimize results for specific applications. Some examples:

Confocal microscopy, for example, is an optical imaging technique that enables 3D imaging of structures from obtained images. Carl Zeiss demonstrates some scientists’ own applications of confocal microscopy in neurons of animal models and drosophila.

In Laser Scanning Confocal Microscopy (LSCM), for example, it is possible to exclusively image a thin optical slice out of a thick specimen (ranging in physical section thickness up to 100 micrometers), a technique known as optical sectioning. Under suitable conditions, the thickness (z-dimension) of such an optical slice can be less than 500 nanometers.

Fluorescence Confocal Imaging is used for live cell imaging. Spinning disk and Sweptfield confocal systems are ideal for the imaging of high-speed intracellular events such as calcium ion dynamics. Confocal imaging is fundamental to advanced imaging techniques such as FLIM, FRET, FRAP and FLIP, and can be used in association with TIRF imaging. The revolutionary laser light stimulation scanner (SIM Scanner) in the Olympus FluoView FV1200 achieves simultaneous stimulation and imaging for real-time visualization of rapid cell responses.

Total Internal Reflection Fluorescence (TIRFM) is a highly sensitive technique to perform functional investigations in living cells. The high S/N and resolution allows the visualization and analysis of vesicles in transport and signaling events, as well as kinetic studies and single molecules detection.

Coherent Anti-Stokes Raman Spectroscopy (CARS), for example, can be used in biological, pharmaceutical and dermatological research, biomedical imaging, food processing, and materials science. Its potential has been demonstrated for various biomedical applications, such as the imaging of lipid transport, protein concentrations, DNA, RNA, tissue in a living organism, and order in liquid crystals. By integrating CARS technology into modern optical scanning microscope systems, the researcher has the latest technology in hand combined with an easy-to-use confocal system.

The use of fluorescence in microscopy enables the more specific analysis of a specimen. Permanent coupling of fluorescence molecules (e.g. Green Fluorescent Protein, GFP) with biological substances, e.g. antibodies, means that the diagnosis of illness is becoming much more exact. Fluorescence imaging is now combined with confocal microscopy; for example, in the LSM170 (Figure 4) from Carl Zeiss, and optical methods to enhance techniques for image analysis. Active Illumination (AL) solutions can be used alongside particular microscopy techniques, such as fluorescence microscopy. They are used in photoactivation (e.g. with fluorescent proteins), FRAP and marking, among other uses.

Fluorescence_imaging

Figure 4: Fluorescence imaging and confocal microscopy combines in the LSM170, providing enhanced contrast characteristics and noise reduction.

Lightsheet Fluorescence

The Carl Zeiss Lightsheet Z.1 is a new technology advance in fluorescent microscopy that uses lightsheet fluorescence to illuminate samples from the side. This minimizes photobleaching and increases speed so that live cells or organisms, such as zebrafish, can be imaged in real-time. This type of technology is expected to grow, as more researchers need the ability to translate in vitro research to in vivo applications.

Digital microscopy is a variation on the traditional optical or confocal microscopes that can be used in conjunction with an image analysis system for specific applications. For example in live cell imaging, the primary considerations are signal-to-noise, image acquisition speed and specimen viability. Fluorescence microscopy such as TIRF, FLIM, FRAP, and laser scanning, as an example, would be combined with a CCD or an electron magnifying CCD (EMCCD) for image acquisition. Some new techniques involve high resolution digital fluorescence microscopy with conventional microplate detection. The combination of these techniques provides detailed phenotypic cellular information with high quality quantitative data.

Buying Guide Tip: We recommend you look at the individual requirements for live-cell imaging applications and determine the best equipment to use following discussions with key manufacturers.

Advances in electron microscopy mean that these instruments can now also be coupled with imaging equipment such as CCD and EMCCD. Software correction of spherical aberration in TEM has allowed the production of images with sufficient resolution to show carbon atoms in diamond separated by only 0.089 nm, and atoms in silicon at 0.078 nm at magnifications of 50 million times. The ability to determine the positions of atoms within materials has made the TEM an indispensable tool for nano-technologies research and development in many fields (see the section ‘The future of microscopy’, including heterogeneous catalysis and the development of semiconductor devices for electronics and photonics. Within life sciences, it is still mainly the specimen preparation which limits the resolution of what we can see in the electron microscope, rather than the microscope itself.

The non-destructive nature of X-ray microscopy (XRM) allows for multi-length scale or multi-modal imaging of the same sample for vital analysis of hierarchical structures. Three-dimensional imaging can be achieved through high contrast and submicron resolution imaging, even for relatively large samples. The capability of excited X-ray fluorescence can be integrated into an existing SEM system such as in the Carl Zeiss EVO 18 SEM (Figure 5 below).

Analytical_Scanning

Figure 5: Analytical Scanning Electron Microscopy with X-ray Geometry

In a recent publication in Science , scientists captured the first images of one of the most important physical interactions in the world, the hydrogen bond, which holds DNA together and gives water its unique properties. These photos are an encouraging advancement in atomic force microscopy (AFM), is a method of scanning that can see details at the fraction of a nanometer level.

In another example using a combination of two high-power analytical techniques, synchrotron radiation microscopy and AFM, scientists in Italy have been able to map vital elements in a single cancer cell. This multimodal approach provides molar concentration, cell density, mass and volume of carbon, nitrogen, oxygen, sodium and magnesium.

Each application area is unique; however, equipment can be used in different permutations to achieve individual results. Table 3 below demonstrates many of these applications.

Table 3: Typical Microscopy Applications

Live Cell Imaging

 

 

Developmental biology

Cell signaling

Cell culture and maintenance

Research

 

 

 

 

 

 

Genetics

Neurobiology

Electrophysiology

Molecular pathology

Plant science

Marine biology

Regenerative studies

Clinical

 

 

 

 

 

 

 

 

 

 

Cytology

Cytogenetics

Embryology/IVF

Hematology

Histology

Immunology

Microbiology

Pathology

Genitourinary

Veterinary

EQA

Education

 

 

Virtual slide

Observation of prepared slide

Gross observations

Pharma/Drug Discovery

 

 

 

Formulation science

ADME-Tox

Target identification

Cell culture

Other

 

 

 

 

 

 

Biophysics

Chemical biology

Lab-on-a-chip

Forensic science

Paleontology

Environmental science

Food science

Materials applications


This list is not exhaustive but gives an indication of how diverse and widespread microscopy is. Consider whether your lab uses a combination of these topic areas. A microscope system that can interface with a number of these techniques may be preferable.

THE FUTURE OF MICROSCOPY

There are regular developments in this field as scientists seek to push the boundaries and develop new techniques. But what of the future of microscopy; what are the trends in technology, the new applications and the new combinations of microscope on the horizon?

Quantum Entanglement

Shigeki Takeuchi and his team in Japan have created a microscope that uses quantum entanglement to increase its sensitivity. In their paper in Nature Communications, they describe how they generated ‘entangled’ photons by converting a laser beam and special nonlinear crystals to achieve the superposition of the photons’ polarization states. The polarization states in this case were horizontal and vertical and were considered as ‘entangled’, and an action on one of them should affect the other, regardless of the distance between them. This new research is especially important for applications in the investigation of transparent samples such as biological tissues and, in particular, living cells.

Mobile Retinal Imaging

Daniel Fletcher, a professor of Bioengineering at UC Berkeley has designed a mobile phone microscope. Using mobile phone technology, Fletcher has redesigned the otoscope – the instrument doctors used to visualize your ear canals. This updated version, nicknamed Oto, attaches on to a mobile phone, generating an easy-to-analyze image of the ear for easy diagnosis.

Linking Light and Electron Microscopy

Recently there has been a drive to combine the best parts of light and electron microscopy into one piece of equipment, known as Correlative Light and Electron Microscopy (CLEM). The resulting technology provides some excellent new application benefits, such as improved image acquisition, sample preparation and structural analysis.

The recent correlative microscopy combination of Raman and SEM or AFM can be seen in the WITec RISE Microscopy Raman-SEM (Raman imaging and SEM) product and the capability to add on chemical analysis in this Renishaw AFM-Raman System.

The combination of fluorescence and TEM can be seen in FEI’s i-Corr correlative microscope. The integration of hardware and software merges EM imaging with optical microscopy, both in reflection mode, as well as for fluorescence imaging. This capability improves the visualization of ultra-structural details and multiple points of interest without transfer of the sample between instruments.

The combination of focused ion beam system (FIB) and a mass spectrometer (secondary ion mass spectrometer) has recently been developed. This allows the chemistry of surfaces to be analyzed as the FIB removes material.

Confocal fluorescence microscopy has also been combined with an inverted SEM column in this example published in Methods Cell Biology. An inverted SEM column allows electron images of wet samples to be obtained in ambient conditions in a biological culture dish, via a silicon nitride (SiN) film window in the base. This allows the SEM to view the same features as the light microscope.

Finally, a collaboration between JEOL and Gatan is to produce a correlative system that creates perfectly aligned image stacks of thousands of sequentially-imaged slices of the freshly cut, resin embedded block face sample. 3D structures of biological and materials samples at ultrahigh resolution can be viewed using the JEOL JSM-7100F Field Emission Scanning Electron Microscope with an integrated Gatan 3View® Serial Block Face Imaging System.

There are many other recent examples of new technologies being developed by scientists and manufacturers. Our advice is to investigate the limitations and possibilities of individual equipment. Learn how to combine equipment, and how you can customize it to suit your requirements. Manufacturers produce equipment that can be upgraded, developed and enabled to accept additional components. Think about choosing equipment which is best suited to your core requirements and develop it further from there.

Editor's picks

Michelle Maxwell

Associate Editor

ZEISS LSM 880 (Zeiss Microscopy)

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5 out of 5

“A fantastic advancement from Zeiss. Microtubules were resolved to a level that I hadn't seen before. The ease to which the images were obtained and the quality of the images makes this a must-have confocal microscope.”
Paul Pryor, University of York

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Leica EZ4 HD (Leica Microsystems)

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5 out of 5

“This high-end product is very nice, easy to use, and gives great depth.”
Jess Boyer, Waynesburg University







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FluoView FV1200 Confocal Laser Scanning Microscope (Olympus)

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5 out of 5

“Fluoview 1200 has entirely changed my live tissue imaging. I can label the tissues with antibodies against the extracellular epitope and visualize them live to track the labeling of antibodies within a specific tissue. It gives very low or no background noise.”
Ramandeep Takhter, Mayo Clinic

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IX83 (Olympus)

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4 out of 5

“The instrument is simple and easy to use. It has very good after-sales services and it is value for money. I would recommend to other colleagues for their various applications.”
Shuchi Agarwal, Ngee Ann Poly



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EVO 18 SEM (Zeiss Microscopy)

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4 out of 5

“The scanning electron microscope is better for the surface study of the plant root, stem and pollen. We studied the different types of pollen present on different varieties of the same species. This will be useful for the classification of plants.”
Manoj Parakhia, Junagadh Agricultural University

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Quanta™ Scanning Electron Microscope (FEI Company)

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4 out of 5

"Easy to use, versatile and very good equipment. It is a very good product for first time users. I recommend it, good value."
Pedro Diaz, Western Refining Co.


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