Microscopy Buying Guide
The types of microscopy technologies and applications are diverse and progressive. This buying guide aims to offer advice for both new and existing users of microscopy equipment. The guide provides an overview of the current techniques involved in microscopy, key features and considerations, application information and examples of future developments.
TECHNOLOGY IN BRIEF
New techniques are developing all the time, as scientists push the boundaries ever forward, solutions to imaging problems are solved with new techniques. Here is a look at some of the microscopy instrumentation and techniques known to us presently.
Light (Optical) Microscopy Categories
|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||Polarized light consists of light waves which all feature the same direction of vibration, i.e. which are linearly polarized.|
|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. The sample stays intact and the slicing maybe repeated many times. Signals pass back to a detector.|
|True Confocal Scanning (TCS)||One diffraction limited spot is illuminated and observed.|
|Coherent Anti-Stokes Raman Spectroscopy (CARS)||Dye-free method. Images structures 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 photo bleaching.|
|Fluorescence & DIC Combination Microscopy||Helps to minimize the effects of photobleaching by locating a specific area of interest in a specimen using DIC.|
|Fluorescence and 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.|
Electron and Scanning Microscopy Categories
|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. Surface reactions, surface reconstructions, phase transformations and distributions.|
|Scanning Probe Microscopy (SPM)||Uses a physical probe that scans the specimen to form an image.The most common variations of SPM are below:||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:||BEEM, Ballistic Electron Emission Microscopy
CFM, Chemical Force Microscopy
C-AFM, Conductive Atomic Force Microscopy
ECSTM, Electrochemical Scanning Tunneling Microscope
EFM, Electrostatic Force Microscopy
FluidFM, Fluidic Force Microscope
FMM, Force Modulation Microscopy
FOSPM, Feature-Oriented Scanning Probe Microscopy
KPFM, Kelvin Probe Force Microscopy
MFM, Magnetic Force Microscopy
MRFM, Magnetic Resonance Force Microscopy
NSOM, Near-field Scanning Optical Microscopy (or SNOM, Scanning Near-field Optical Microscopy)
PFM, Piezoresponse Force Microscopy
PTMS, Photothermal Microspectroscopy/Microscopy
SCM, Scanning Capacitance Microscopy
SECM, Scanning Electrochemical Microscopy
SGM, Scanning Gate Microscopy
SHPM, Scanning Hall Probe Microscopy
SICM, Scanning Ion-Conductance Microscopy
SPSM, Spin Polarized Scanning Tunneling Microscopy
SSRM, Scanning Spreading Resistance Microscopy
SThM, Scanning Thermal Microscopy
STP, Scanning Tunneling Potentiometry
SVM, Scanning Voltage Microscopy
SXSTM, Synchrotron X-ray Scanning Tunneling Microscopy
Of these techniques AFM and STM are the most commonly used for roughness measurements.
KEY FEATURES OF LIGHT (OPTICAL) TECHNOLOGY TO CONSIDER
The main consideration is your application. Do you need a microscope to suit more than one purpose i.e. both light and fluorescence? What samples are you going to be using? For example, whole tissue sections or bacterial cultures, and will these be stained or unstained? What level of detail do you require? For instance, are you going to be looking 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 technology for certain uses.
Buying guide tip: We recommend that you read up on the basic principles of refraction and reflection in order to understand the magnifying process.
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 2 main types of lens in optical microscopy, the objective and the eye piece; both are important considerations when choosing a microscope.
Typically, standard optical microscopy objective lenses range between x4 and x100 in magnification. Objective lenses are available for particular applications: water emersion 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 where the objectives are easily changeable to suit your application or you may want a set that are 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 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. Eye pieces can generally be grouped as ‘finder’, ‘wide field’ and ‘super wide field’: wide field eye pieces, 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.
Objective and Eyepiece Combinations
Buying guide tip: Manufacturers can use different terminology when describing and coding objectives and eyepieces; read around the terms if you are unsure.
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 1000 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 however 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.
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.
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.
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; 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.
All microscopes are designed to include a stage where the specimen (usually mounted onto 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.
Other Light (Optical) Microscope Considerations
These are terms that you may come across when reading about light microscope specifications and some terms, such as resolution, are also used in electron microscopy. These considerations below are more process than structural orientated.
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 it 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 (SNR)
Signal to Noise Ratio (S/N) is the ratio between a given signal and the noise associated with that signal. The Signal to Noise Ratio 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 signal-to-noise ratio, while still maintaining adequate signal-to-background ratio for good image contrast.
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.
KEY FEATURES OF ELECTRON TECHNOLOGY TO CONSIDER
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.
As outlined in the EM technology table, 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. SEM produces 3D images that give information on morphological and topographical details. Some electron microscopes can encompass both SEM and TEM technology in one. 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.
Sample Stage and Chamber Size
Ensure the sample stage is the correct size in which 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 changed.
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.
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 have depth of field (DOF) and resolution at the higher magnifications. The lens types will determine the magnification so check both requirements for your applications. Obviously high magnification without high resolution is redundant.
A Low Kv Performance
By this we mean how does the microscopes 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.
The room space and atmosphere of the room is of great consideration in electron microscopy, especially for a TEM. A vacuum pump system is required. The room needs to be kept completely dust and debris free for optimum function. A special area/room will be required for sample preparation.
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. 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 in every laboratory it is an important consideration. Do negotiate on cost, extra features/equipment/software or after sales service/training. Also do approach more than one manufacturer for quotes.
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 enabling 3D imaging of structures from obtained images. The SNR is improved in comparison to widefield (traditional brightfield and dark field optical) techniques, and it can be used with both live and fixed specimens. One of the fundamental advantages of the confocal versus a widefield microscope is due to 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.
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.
Total Internal Reflection Fluorescence (TIRFM) is a highly sensitive technique to perform functional investigations in living cells. The high signal to noise ratio 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, mean that the diagnosis of illness is becoming much more exact. Fluorescence imaging is now combined with confocal 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.
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 discussion 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, 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.
Each application area is unique; however equipment can be used in different permutations to achieve individual results.
Live Cell Imaging
Cell culture and maintenance
Observation of prepared slide
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. These new techniques and ideas are portrayed by the abundance of news articles available.
The electrical engineer and bioengineer Aydogan Ozcan of University California, Los Angeles, (UCLA) is leading a group that want to directly image single virus particles using holographic microscopy. His team has demonstrated the ability to capture optical images of single viruses and nanoparticles over a comparatively large field of view (about the size of a postage stamp) using nanolenses that self-assemble around the virus particles like little magnifying glasses. A different group, led by electrical engineer Holger Schmidt of the University of California, Santa Cruz (UCSC), is detecting single particles tagged with fluorescent labels on a microfluidic chip. Both teams expect to use their work to develop commercial instruments useful for on-site diagnosis and monitoring with rapid results and fast turnaround. (The Optical Society, May 30, 2013: “New single virus detection techniques for faster disease diagnosis.”)
Dr Claudio Canale has a team at the Istituto Italiano di Tecnologia (IIT) in Genova focussing on the application of scanning probe techniques in the study of bio-materials and bio-mechanisms. One of his main projects has been to couple STED (stimulated emission depletion microscopy) and atomic force microscopy (AFM). This work has recently been published in the Journal of Optical Nanoscopy (Harke B., Optical Nanoscopy 2012, 1:3). The group’s next target is to characterize biological processes having simultaneous access to morphological and mechanical properties and coupling both of them with chemical recognition capability directly provided by STED with a resolution in the order of tens of nanometers. Working on model membranes, Dr Canale says "we can recognize target molecules or particular membrane components by STED and we can look at the fine structural and mechanical changes by AFM." (SelectScience, 16 April 2013: JPK Reports on the Research Activities of the Nanophysics Group at the Istituto Italiano di Tecnologia Based in Genova.)
The Carl Zeiss Lightsheet Z.1 is another example of a new technology. It is a fluorescent microscope 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.
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 which 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.
"This microscope and attached camera is quite versatile. Clearly seen nucleus and cell membranes. Excellent for keeping track of infections and cell health."
Stephania Widger, Novartis