How to Buy Microscopy Equipment

Microscopy is used across most life science 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. Also, does your imager allow for uninterrupted imaging at night or over the weekend, so you don’t have to always come in to capture data? For example, the IncuCyte by Essen BioScience enables longitudinal live-cell imaging so you’re always getting data even when you’re away.
• 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 labeled, 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?
• High-througphut and automation: Will your lab process multiple samples at a time, risking human error? If yes, you might need to consider whether your microscope is compatible with automation systems. When performing live cell imaging, consider an in-built or connected incubator to keep cells healthy while processing multiple samples.

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 with numerous fine-tuned lenses and 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.

For whole specimen observation, for example, during dissections, stereo microscopes, typically widefield, can be used. They provide the lowest magnifications to yield an overall image of the sample. Such stereo microscopes tend to use light reflected by the sample instead of transmitted through it.

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. Halogen illumination has been the gold standard for light microscopy, however, LED lights sources are becoming increasingly common and offer the advantage of no sample heating, low energy consumption and long lifetime with the caveat of altered colors of a stained sample. This application note outlines how to overcome the caveat with Olympus’ True Color LED.

Modern imagers are programmed to offer better contrast to yield more accurate cell counts. Inquire about the software’s ability to produce crisp intensity profiles to yield better counts. For example, this application note describes a protocol for counting unlabeled cells using high-contrast brightfield imaging.

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’re 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. This application note by Olympus discusses the signal-to-noise ratio and superresolution offered by confocal imaging.

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, superresolution 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.

Modern cell imagers enable users to toggle between brightfield, phase-contrast and fluorescence imaging. With multiple fluorescence channels for image overlay, you can perform 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..

This downloadable poster by Essen BioScience details real-time image-based quantification of phagocytosis in living cells using the IncuCyte by employing pH-sensitive bioparticles that fluoresce in an acidic environment.

In this application note, learn how the InCellis can automatically calculate cell culture confluency. In a recent SelectScience editorial interview, Dr. Bruce Morgan, University of Kaiserslautern, Germany, explains how he uses genetically-encoded fluorescent sensors to study reactive oxygen species using the CLARIOstar Multimode Microplate Reader.

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.

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. (Figure 1).

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.

In conventional cell culture experiments, protocols involve taking measurements on a microscope at specific time points. This method doesn’t offer the full picture of cell behavior that is otherwise dynamic and changing. Adding in a ‘live’ element to your microscopy can substantially elevate the data you obtain from your experiments and provide you with cell insights that are not seen through conventional methods.

• Note: Advantages of live cell imaging:
   
• It’s live: Enables real-time tracking of subtle cellular processes and in vitro changes
• It’s detailed: Offers a time-lapse, detailed understanding of cell behavior
• It’s automatic: Experiments go on and data are collected even when you’re away
• It’s minimalistic: You can get multiple data points from one batch of cell culture, thereby you can make the most out of sensitive, low-yielding primary cells
• It’s contamination-free: Eliminates handling of cell culture plates between readouts

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.

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 garnered over 90 reviews from scientists around the world on SelectScience, and recently received a SelectScience Silver Seal of Quality as a result of the consistently positive feedback. Below, we highlight case studies using the IncuCyte where scientists have used live-cell imaging in diverse range of applications, expanding the quantity of data obtained from experiments and leveraging the benefits of capturing cells ‘live’ in action. 

Case studies

     1.   Catching killer T cells live in action

Dr. Adam Case, Assistant Professor, University of Nebraska Medical Center, studies the immune system and monitors behaviors of immune cells in real time using live-cell imaging. 

To examine the immune system, Case makes a unique choice of studying cardiovascular and psychiatric diseases. The Case lab hypothesizes that in a state of ‘stress’ during a psychiatric illness, the brain triggers the immune system, which, in turn, modulates cardiovascular responses. 

His lab isolates T cells from a mouse model of post-traumatic stress disorder, and measures immune responses using live-cell imaging. “A lot of the tests measuring the immune system function involve culturing immune cells and measuring the cells’ ability to, say, fight an infection or even proliferate and expand,” says Case.

The video below (Figure 2) captured using the IncuCyte shows mouse T cells (appearing as small black circles) killing pathogenic species, in this case, human cancer cells (appearing ovoid) in real time. The bright green fluorescent marker indicates cell death.
 

     2.     Capture protein expression changes over time in living cells
Immunocytochemistry (ICC) forms a significant part of microscopy, capturing protein expression. However, a big caveat with traditional ICC is that it’s unable to capture protein expression changes over time. Live-cell ICC, however, offers a way to capture cellular dynamics of live cells, while still keeping the benefits of traditional ICC.

Tim Dale, Director of Biology, Europe, Essen BioScience, explains how live-cell ICC works in a SelectScience interview. “Live-cell ICC allows surface expression dynamics to be monitored. More importantly, these protein expression changes can be coupled to morphology and function by using this alongside many of IncuCyte’s functional applications,” says Tim (example shown below in Figure 3). Read the whole article here.
 

     3.     Live-cell analysis of cell subsets and heterogeneity 
Add another dimension to cell culture microscopy by using live-cell analysis to identify and quantify cell subsets in heterogeneous cell populations. This method illustrates how you can use live-cell imaging to identify and quantify subsets using mixed cultures of Jurkat and Ramos B cells in cell culture media. Specific antibodies to the leucocyte common antigen, CD45 and the B-lymphocyte specific antigen, CD20, were labeled with FabFluor-488. The data in Figure 4 below demonstrates the identification of Jurkat- and Ramos-positive cells in the mixed culture. Get the entire method here.

Due to the visual element of live-cell imaging, as compared to flow cytometry, you can perform subset classification based on morphology, in addition to using antibodies/cell markers. For example, in the same method, the research team at Essen BioScience has demonstrated cell-by-cell analysis of activation states of T cells using morphological changes and enlargement.

      4.    Analyze 3D spheroids for cancer biology using in real time 
3D cell models help recapitulate the tumor microenvironment. Live-cell analysis can be used to study diverse 3D tumor models – single spheroid assays, co-cultures as well as multi-spheroid assays. 

In this on-demand webinar, get protocols to quantify tumor growth, morphology and viability using label-free or fluorescent cell health markers. Learn how you can automatically acquire and analyze images of spheroids over time using brightfield imaging and the ‘spheroid module’ on your live-cell imagers.

In another example, cancer epigeneticist, Dr. Sonal Gupta, Assistant Professor, MD Anderson Cancer Center, Texas, examines 2D and 3D behaviors of mouse-derived pancreatic cancer cells. In a SelectScience interview, Gupta says, “It is a well-accepted notion now that cultured cancer cells change their physical characteristics based on the stage of cell cycle and environmental factors. For people studying epigenetic regulation of cancer cells, it's very useful to be able to track changes in cellular behavior with cell culture environment changes.”

Dr. Sammy Ferri-Borgogno, postdoctoral researcher in the Gupta lab, explains how live cell imaging enables her to learn more about pancreatic cancer cells both two- and three-dimensionally, while saving time and money. “We can track the spheroids over a period of time, to see how they grow, to check if they are shrinking,” says Ferri-Borgogno. “This live imaging is good because it helps us understand our cells three-dimensionally, and to confirm if the model we use is reliable.” Read the full interview here.

Figure 5. Read this article to learn how Dr. Sonal Gupta and Dr. Sammy Ferri-Borgogno of the MD Anderson Institute, Texas, USA, use the live-cell imaging for their 2D and 3D cell culture research on epigenetics of pancreatic cancer.