How to Buy Microscopy Equipment

Microscopy is used across most life sciences disciplines and in academic, clinical and commercial settings. Beyond life sciences, microscopy is a firmly established analysis tool in materials, formulation and information technology.

Microscopy technologies and applications are diverse and progressive. This guide provides an overview of the current techniques involved in microscopy, key features and considerations. Infrared and FTIR microscopy are, however, not included in this guide, as it is focused primarily on life sciences rather than materials science or IT manufacturing.

The main consideration for all types of microscopy is your application and what it will involve. Some general considerations may include:

• Sample type: What samples will you be using and how will you prepare them? Will you be using live cells or fixed cells, whole tissue sections or bacterial cultures? For example, cells can be harvested and then mounted on slides or viewed as a two-dimensional layer of cells across the bottom of a microplate well.
• Length of experiments: How often will you use the microscope and how long will each session be? This may affect sample photobleaching and, in turn, your choice of illumination.
• Sample number: How many samples are you going to be analyzing? Pathology labs may need a high throughput machine to handle the many samples that come in for analysis every day, whereas a research group may benefit from higher specifications and a lower throughput for detailed examination of samples.
• Image resolution: What level of detail do you require in your imaging? Are you going to look at the overall tissue morphology on a single imaging plane? Or are you measuring intracellular localization of biomolecules along multiple Z-stacks? Are you counting cells or bacteria on a lower magnification, or are you looking at protein-protein interaction within the cells, requiring a higher resolution?
• Labeling: Will your samples be unlabeled or labeled? If your samples are fluorescently labelled, does your microscopy support the wavelengths? Can you look at multiple biomolecules with multiple fluorescent channels?
• Quantitative versus qualitative: How do you want to use your images? Are you generating high resolution images as a ‘representative’ of your cohort for display and publication only? Or do you want to use them for quantitative analysis as well?

Light microscopes are essential laboratory tools that allow scientific investigators to view samples to as much as a thousand times their original size. In its simplest form, the light microscope 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. See Table 1 for a summary of the main types of light microscopy instrumentation and technology.

Stereo microscopes are typically widefield microscopes, providing the lowest magnification to yield an overall image of the sample and also used in dissections. These microscopes typically use light reflected from instead of transmitted through the sample. They are particularly useful for whole specimen observation. A good example of a stereo microscope is the Leica EZ4 HD. For higher magnification and analysis of slide-mounted specimens, a standard widefield microscope such as the Olympus SZX16 stereomicroscope is cost-efficient for more detailed sample information. Most widefield microscopes have brightfield as well as darkfield and phase contrast viewing, which offers different contrasts and views of your images.

Unlabeled cells generally provide limited contrast from the background, so good illumination is important. LEDs are now used in the latest models and offer the advantages of no sample heating, low energy consumption and long lifetime.

Confocal microscopy, in particular, using fluorescent imaging, is now a leading technology for life sciences. It is an adaptation of light microscopy that offers improved resolution over widefield microscopy. The key to this is the restricted manner in which light reaches the photomultiplier through a pinhole. This restricts the amount of unfocused light that is captured from the sample, increasing the contrast and dramatically increasing the resolution of what you are looking at. There are several different types of confocal microscopy:

• Laser scanning confocal microscopy (LSCM): enables optical sectioning i.e. imaging of thin optical slices down to 500 nanometers from thicker specimens
• Fluorescence confocal imaging: enables high resolution imaging of fluorophores
• Advanced confocal imaging: These are application-specific variants of confocal microscopy. For example, FRET and FRAP.

Confocal microscopy offers the ability to control depth of field, elimination or reduction of background information, and the capability to collect sections from specimens that are thicker. The signal-to-noise ratio is significantly improved over widefield microscopy, and it can be used with both live and fixed specimens, making it possible to produce 3D images of cellular structures.

In this video, learn how Dr. Kim Dora, University of Oxford, uses confocal imaging in her vascular research to analyze blood vessels and myocardiocytes.

Laser scanning confocal microscopy (LSCM): A confocal laser scanning microscope scans a sample sequentially point by point or multiple points at once. The pixel information is assembled into an image. This follows on from confocal microscopy to enable the user to acquire sophisticated 3D images of their sample, or individual structures within their sample. At any one focal position, a confocal laser scanning microscope can acquire multiple images over a selection of different depths and then combine these to make a 3D composite image, a technique called optical sectioning.

Read this SelectScience editorial article to learn how advanced confocal imaging in Dr. Ben Prosser’s lab, University of Pennsylvania, revealed a key role of the cytoskeleton in the pathology of heart disease.

The LSM 880 with Airyscan from ZEISS enables fast, sensitive, super resolution confocal imaging of live or fixed samples, to enable imaging of the smallest structures, the weakest signals or tracking of fast processes. The Airyscan module allows more photons to be captured by imaging on to a specially shaped array of 32 optimally arranged single detector elements, from which the signals are reassembled, creating an overall image with increased signal-to-noise ratio and resolution.

Other excellent examples of LSCM units are the Leica TCS SP8, the Nikon A1R MP+ Multiphoton Confocal Microscope and the new Olympus FV3000 LSCM.

Fluorescence microscopes take cell imaging one step further than brightfield or phase-contrast. With the use of fluorophores, applied topically, expressed genetically or by immunofluorescence, fluorescence microscopy helps capture the localization of target molecules involved in cell signaling, apoptosis, cell viability or other cell biology pathways.

InCellis^® from Bertin Instruments is a cell imager that enables users to toggle between brightfield, phase-contrast and fluorescence imaging. The InCellis offers up to four fluorescence channels for image overlay, providing options for multi-labeling and co-localization when studying cells either in vitro or fixed on a slide. It can also be employed for everyday cell culture activities like measuring cell confluency or ensuring transfection efficiency.

In this application note, learn how the InCellis can automatically calculate cell culture confluency. Download this method to discover how the InCellis ​enables the easy documentation of a scratch assay to study cell migration.

Fluorescent labels and dyes: The use of fluorescence in microscopy enables more specific analysis of a specimen. Cells and tissues can be stained or labeled with one or more dyes, to allow complex visualization of structures, such as nuclei or proteins. For example, DRAQ5™ is a selective nuclear stain, while DRAQ7™ selectively stains dead, dying and apoptotic cells. GFP (green fluorescent protein) or firefly luciferase can be expressed within cells and fused to a protein of interest, giving ready labeled specimens for imaging. Likewise, immunofluorescence uses fluorescently labeled antibodies to target structures of interest.

A wide range of dyes are available for fluorescence imaging. The key to each is what filter or activation is required to visualize the dye, and how many filters and channels are available on your microscope. Excitation and emission spectra of the fluorescent label must be matched with the light source; the correct excitation and emission filters and dichroic mirrors are required to get a good signal from your samples.

Fluorescent labels vary in signal strength and in the amount of background fluorescence. If a sample is to be viewed for a long time, e.g. for live imaging and tracking of cellular events, photobleaching of the fluorophore needs to be addressed; this may affect your label choice. Consider your application too: for live cell assays, fluorescent probes need to be cell membrane permeable to assess structure and function within the cell.

Multiple labels can be used to label the same sample, often co-localizing on the same part of the sample. You need to make sure you select a microscope that enables you to visualize your palette of labels. You need to consider how many labels you are going to want to use and make sure the microscope can accommodate any current dyes you are using and any you plan to make use of in the future.

In a SelectScience editorial article, Anne Marie Quinn, founder of Montana Molecular, shares how it’s possible to measure multiple signaling pathways inside a living cell using fluorescent biosensors. In another interview, Dr. J. Quincy Brown, Tulane University, explains how optical methods can examine fresh tumor biopsies to determine the tumor margins during surgery. Read on to learn how he developed an alternative to H&E staining using fluorescent dyes.

Fluorescence combined with confocal microscopy: In confocal systems, the trade-off from improved image resolution by restricting the light detection is a reduction in sample brightness. Therefore, with confocal microscopy, one or more fluorescent labels are typically employed to highlight structures of interest and improve contrast within a specimen. Even greater contrast and differentiation can be achieved when multiple fluorescent labels are used for different structures.

Active Illumination (AL) solutions can be used alongside particular microscopy techniques, such as fluorescence microscopy. Spinning disk and swept field confocal (SFC) systems are ideal for the imaging of high-speed intracellular events such as calcium ion dynamics.

Total internal reflection fluorescence microscopy (TIRFM)
TIRFM is a highly sensitive technique that enables you to perform functional investigations in living cells using fluorescence. It only images structures within a very thin layer as it uses total internal reflection to illuminate cells contacting a surface and only produces a very narrow excitation depth. Because the illumination is so focused, TIRFM enables very high resolution 2D images, down to less than 200 nm. This is not a high-throughput technique but one for analyzing very specific, small cellular events at the surface.

TIRFM is the method of choice to visualize single molecules in living cells, in particular fluorescent molecules located at cell adhesion sites, cell membranes and membrane proximal cytoplasmic organelles. TIRF microscopy systems are now commercially available from many microscope suppliers, including ZEISS, Leica and Olympus.

Live cell imaging enables real-time tracking of even the subtlest of cellular processes and in vitro changes, helping you obtain a more detailed understanding of cell behavior and get more information on your cells without ever removing them from the incubator. Such a live cell imaging approach, coupled with fluorescence or confocal microscopy, can produce images, videos and data that could otherwise go unnoticed in conventional approaches. Microscopy with live cell imaging is now getting a lot of traction in many life sciences applications, including wound healing, cell-cell communication, signaling studies, 3D cell cultures, developmental and vascular biology, among others.

In live cell imaging, the primary considerations are signal-to-noise ratio, image acquisition rate, and sample viability. To capture cellular processes, it’s important to ascertain that your cells have the right environmental conditions, such as temperature and CO²/O², so they remain viable throughout the entire the time period of imaging. In setting up your live imaging protocol, care should be taken to space out the imaging to avoid long periods of light exposure which can be phototoxic to cells. For live cell imaging applications, a low power illumination is desirable.

The IncuCyte S3^® Live-Cell Analysis System by Essen BioScience automatically acquires and analyzes images around the clock – without ever removing your cells from the incubator. Its walkaway feature helps you capture physiologically relevant processes as you spend your valuable time performing other experiments. Without displacing cells or moving cell culture plates, you can collect images throughout the course of your experiment, thereby offering you a ‘snapshot’ into true physiological behavior. Plus, the image analysis software helps you analyze kinetics, bringing additional insights into your captured images.

The IncuCyte has gained over 60 reviews from scientists around the world on SelectScience, and recently became one of the very first products to receive a SelectScience Bronze Seal of Quality as a result of the consistently positive feedback. In this application note, learn how to perform quantitative live cell analysis for optimization of culture conditions for induced pluripotent stem cells (iPSCs), using the IncuCyte S3. Also, learn how to use live cell imaging to model angiogenesis in vitro.

In this SelectScience editorial article, cancer epigeneticist, Dr. Sammy Ferri-Borgogno, of the MD Anderson Cancer Center, Texas, explains how live cell imaging reveals more information about pancreatic cancer cells both 2- and 3-dimensionally, while saving time and money.

Watch this video of Dr. Kim Wicklund, Director of Product Management, Essen BioScience, describing how the instrument can be used for many different in vitro assays in cancer research, from measuring cell proliferation to assessing immune cell killing, all from within the incubator.

Another live cell imaging system is the BioTek Cytation™ 5, the winner of the Scientists’ Choice Award® for Best New Life Sciences Product of 2015, that combines high-resolution digital fluorescence microscopy with a plate reader in a single instrument. Other live cell imaging platforms include the new CellASIC^® ONIX2 Microfluidic System from MilliporeSigma and the UltraVIEW^® VoX 3D Live Cell Imaging System by PerkinElmer.

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 the details of mitochondria in cells. TEM can also provide information on element and compound structure.

Electron microscopes can be coupled with standard 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 nanotechnology 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 that limits the resolution of what we can see in the electron microscope, rather than the microscope itself.

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 stuff. 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, which often requires a specialist. The Titan™ - Transmission Electron Microscope by FEI is a good example of a versatile scanning/transmission electron microscope (S/TEM).

SEM produces 3D images that give information on morphological and topographical details and basic surface characterization, such as surface study of plant pollen, stem and root systems. 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.

Nikon’s JCM-6000 Neoscope™ Scanning Electron Microscope is an affordable benchtop SEM, ideal for advanced and versatile imaging.  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. 3D 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 ZEISS EVO 18 SEM.

AFM is a method of scanning that can see details at the fraction of a nanometer level. Using a combination of AFM and synchrotron radiation microscopy, 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.

Electron detectors

The capabilities of an electron microscope are dependent upon the 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.

A low voltage performance

How does the microscope’s electron optical performance operate at low voltages? Some electron microscopes have a high resolution at as little as 1 kV. However, high resolution at low voltages may not be 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.

Electron microscope room considerations

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

• Vibration dampening bench
• Dimming lights
• High voltage access
• Electromagnetic shielding
• Air movement control and cleanliness, using air conditioning and vacuum pumps
• Separate sample preparation room

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

Recently, there has been a drive to combine the best parts of light and electron microscopy; to obtain details on a broad, molecular level and combine them with the intricate, intracellular details to examine the entire scope of the underlying biology. Such a combination has become known as correlative microscopy.

Combining AFM and Raman Imaging, the alpha300 RA from WITEC enables the acquisition of Raman images from chemical investigations to be linked to the AFM topographic information from the same sample area. Download this application note to learn about this correlative method in studying blood vessels.

The ZEISS ZEN Correlative Array Tomography can automatically image hundreds of sections across length scales and can combine them into one single correlative volume data  set for light and electron microscopy. This application note outlines how to it can connect your light and electron microscopes to reconstruct your sample in 3D.

Watch this presentation to hear Dr. Peter O’Toole, Director of the Bioscience Technology Facility, Head of the Imaging and Cytometry Laboratory at the University of York , explore the benefits of microscopy for biologists (Figure 9). Dr. O’Toole discusses innovations in and applications of superresolution microscopy, 3D imaging and correlative microscopy.

Also, in another SelectScience interview, Dr. Louise Hughes, researcher and microscopist at Oxford Brookes University, explains how she uses correlative microscopy to study plant parasites, viruses and general cell ultrastructure. “It’s important for us to get to the point where you take one sample all the way through – from live imaging looking at the fluorescent markers through to the EM level looking at three dimensions with electron microscopy,” she says. Learn more in this video.

Sample prep for correlative microscopy may need a different approach. To learn the details on sample prep for successful imaging, download this exhaustive protocol compendium.

Digital image capture and analysis has now largely overtaken earlier imaging methods. Digital cameras and imagers enable the user to take images or videos of the substance or structure being analyzed direct from the microscope. Cameras for microscopy, namely CCD and EMCCD and CMOS, are available. There is a wide range of such cameras available and further details can be found in the SelectScience Microscopy Camera section, with independent user reviews to help you compare and select the best camera for your needs.

For digital microscopy, it is important to consider what data you want to get from your sample. Whether you require only qualitative images for display or more detailed images for quantitation will determine the imaging and software specifications that you need. Digital microscopy and sophisticated imaging software are available that enable image stitching and 3D image stacking, which can be used to make montages and composite images.

Microscope analysis software is available from many manufacturers and again will vary depending on the application. Check that any software you obtain is compatible with the instrument(s) you are using. You can find a great deal of information about software products in the SelectScience Microscopy Software directory, again featuring independent reviews to guide you.

Consider where you will be publishing your images, 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.

Note: 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. We also recommend approaching more than one manufacturer for quotes.

The development in the field of microscopy often relies on advances in optics or detectors, with improvement in resolution, speed and sensitivity. More recently, an aspect of imaging that is being considered is the accessibility of technology. Key pain points in microscopy include the need for unique expertise to acquire the images, and later, another set of skills to manage and analyze all the images generated. More companies are now adapting ease-of-use as their next step in microscopy advancement – where a lack of expertise will not limit researchers from using a microscope or troubleshooting their experiment.

Listed below are a few recent conceptual advancements in microscopy:

There are many different types of microscopy instruments on the market. Finding the correct one for your application may seem daunting but by applying a few simple considerations, selecting the right microscopy tool for your research can be made an easier task.

Microscopy equipment will continue to develop and new technologies will emerge. Visit the SelectScience product directory to view multiple microscopy instruments all at once. Read reviews from your peers to help make better decisions. Additionally, you can use the SelectScience application note library to keep up-to-date with the latest methods in imaging and sample prep.