Microscopy Buying Guide

Microscopy image

If you are looking to invest in Microscopy equipment, this essential SelectScience Buying Guide provides you with all the information you need to help you to make the right decision, based on your requirements.

Learn about key factors and application considerations when buying a new microscopy instrument, and read impartial user reviews to help you purchase with confidence.

Basic Overview

Microscopy is used across most life sciences including cell biology, developmental biology, electrophysiology, plant science, marine biology, microbiology and forensics, in academia, clinical settings and commercial research and development. Beyond life science, 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. The guide also contains links to some of the many product and supplier examples taken from SelectScience's extensive library of product reviews, product information and videos.

Infrared and FTIR microscopy are not included in this guide, which is focused primarily at life sciences as opposed to materials science and IT manufacturing.

Application Considerations

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

  • What samples will you be using and how will you prepare them? Will you be using living 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.
  • How often are you going to need to use your microscope and how long will you be using it in each session? This may affect your choice of illumination.
  • How many samples are you going to be generating? Pathology labs need a high-throughput machine to handle many samples coming in for analysis every day, whereas a research group may need lower throughput and higher specifications.
  • 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.
  • Will your samples be unlabeled or labeled? Are you planning to use fluorescent labels?
  • Image capture – how many images will you want to generate, do you want to do this digitally? How do you want to use your images – for display and publication only, or do you want to use them for quantitative analysis?

Light Microscopy

Light microscopes are essential analytical laboratory tools that allow scientific investigators to view objects 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.

Table 1: Summary of the main light microscopy instrumentation and technology.

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.


Widefield Microscopy

Stereomicroscopes provide the lowest magnification, particularly useful for whole specimen observation. A good example is the Leica EZ4 HD. For higher magnification and analysis of slide mounted specimens, a standard widefield microscope such as the Olympus CX41 is required. The Olympus CX41 is a good example of a standard brightfield microscope that is cost-efficient with excellent general performance. Most widefield microscopes will offer 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 such as the DM2500 Microscope from Leica, which was released in July 2015. LEDs offer the general advantages of no sample heating, low energy consumption, and long lifetime.

Confocal Microscopy

Confocal microscopy, in particular using fluorescent imaging, is now arguably the leading technology for life science microscopy applications. 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 un-focused 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:

  • Fluorescence Confocal Imaging– used for live cell imaging.
  • Laser Scanning Confocal Microscopy (LSCM) allows optical sectioning – imaging of thin optical slices down to 500 nanometers from thicker specimens (up to 100 micrometers).
  • Advanced confocal imaging techniques include FLIM, FRET, FRAP and FLIP, and can be used in association with TIRF imaging.

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 several opticals thick. The signal-to-noise ratio is significantly improved over widefield microscopy, and it can be used with both live and fixed specimens, allowing the possibility to produce 3D images of cellular structures.

Upright vs. Inverted Microscope?
More inverted microscopes are being developed and made available in the marketplace as applications become more advanced. Uprights are still the right choice for some applications, such as analyzing zebrafish embryos or stained tissue selection, but for most existing and emerging cellular imaging applications, the inverted microscope is favored.

The Olympus IX83 is an inverted system for long-term time-lapse imaging of living cells, as well as high-end fluorescence applications such as total internal reflection fluorescence (TIRF) microscopy. It has brightfield, phase contrast and fluorescent illumination options and includes an expanded field of view, increasing the amount of your sample you can reliably capture.

The Olympus CKX41 is another good example of an inverted fluorescence microscope for routine use; the ZEISS LSM 700 for Life Sciences can be combined with the upright microscope stand, inverted or the fixed stage set up for complete flexibility.

Fluorescence Imaging

The trade-off for improving resolution by restricting the light that is detected is that sample brightness is decreased. 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.

The use of fluorescence in microscopy enables the more specific analysis of a specimen. Cells and tissues can be stained or labeled with one or any number of dyes to allow complex visualization of structures, such a nuclei or proteins. For example, the dye DAPI, or more recently DRAQ5TM, stains a cell's nucleus only. GFP (Green Fluorescent Protein) or firefly luciferase can be expressed within cells and fused to a protein of interest, giving ready labeled specimens to view. Likewise, immunofluorescence uses labeled antibodies to bind to and highlight the structures of interest.

Draq5

Figure 1: Learn how DRAQ5TM enables multi-color analysis of complicated samples, via flow cytometry and cell based imaging

There is a wide range of dyes to use for fluorescence imaging. The key to each is what filter or activation it requires to visualize it 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; excitation and emission filter and dichroic mirrors come with the microscope to make sure you can get a good signal from your samples.

Fluorescent labels vary in their strength of signal and also 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, possible photobleaching of the fluorophore needs to be addressed; this may affect which labels you choose. 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 allows 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.

The key for microscope selection here, is making sure your instrument carries the necessary filters and number of channels to detect whichever dyes you plan to use. Some microscopes are limited to one or two detection channels, more and more are now offering five, ten or more.

The ZEISS LSM 710 boasts complete freedom to use any dye in your fluorescence microscopy. You can work with up to 10 dyes and use ‘continuous spectral detection across the complete wavelength range’. One reviewer states: “The thing I love the most about 710 is the availability of UV laser, which gives me the capability to use DAPI, CO2 and temp control, which is great for live imaging and the external fine focus and field adjustor.”

LSM170

Figure 2: Fluorescence imaging and confocal microscopy combines in the LSM170, providing enhanced contrast characteristics and noise reduction.

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. Spinning disk and Sweptfield confocal systems are ideal for the imaging of high-speed intracellular events such as calcium ion dynamics.

Confocal Laser Scanning Microscopy

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 follow on from confocal microscopy allows 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.

The ZEISS LSM 880 is an example of a stand-out confocal microscopy product and won a SelectScience Scientists' Choice Award®. LSM 880 provides outstanding resolution, beyond that of other standard confocal microscopes. The LSM 880, as with the 800 model, uses a linear scanning laser scanning confocal microscope with the aim of providing a constant signal-to-noise level and uniform exposure throughout the scanned area. The 880 and 800 microscope is designed particularly for live cell imaging. Images from the 800 are 512×512 pixels with a capture rate of up to 13 frames per second; importantly for live cell imaging applications, it also has incorporates low laser power to avoid photobleaching and phototoxicity.

As with other ZEISS confocal microscopes, there is the option of adding the ZEISS ‘Airyscan’ module. This is effectively an upgrade to standard confocal detection systems. With Airyscan it is possible to capture more of the photons available by imaging on to a specially shaped array of 32 optimally arranged single detector elements, from which the signals are reassembled, overall creating an image with increased signal-to-noise ratio and resolution.

LSM_880

Figure 3: A short video of mouse retina imaging using with the ZEISS LSM 880

Other excellent examples of LSCM units are the Leica TCS SP5 II and the Nikon Eclipse C1 Plus. The Eclipse C1 Plus is a modular confocal system that gives a very high quality digital imaging while being compact and lightweight.

Live Cell Imaging

Live cell imaging allows for some groundbreaking studies to be carried out, producing images and real-time tracking and imaging of even the most subtle of cellular process and changes. Microscopy with live cell imaging is now a commonplace technology for cell biology such as cell signaling studies, cell culture maintenance, developmental biology and other life science applications; many fluorescence and confocal microscopes now offer live cell imaging capabilities.

In live cell imaging, the primary considerations are signal-to-noise, image acquisition speed and specimen viability. You’ve got to provide the right environmental conditions, such as temperature and CO2 / O2 for your samples so they remain viable for the length of time you wish to observe.

Exposures for long periods will negatively affect the viability of live samples (phototoxicity) and can also cause photobleaching. For live cell imaging applications, a low power illumination is desirable.The BioTek CytationTM 5 is a big step forward for live cell imaging. As well as a standard system for live cell imaging (CO2/O2 control, incubation to 65 °C and shaking for cell-based and other assays), the Cytation5 combines high resolution digital fluorescence microscopy with a plate reader in a single instrument, which permits the user to obtain high-quality cellular imaging and information with well-based quantitative data at the same time.

With up to 60x magnification, the microscopy part allows high-quality cellular and sub-cellular imaging in fluorescence, brightfield, color brightfield and phase contrast. The microplate reader incorporates variable bandwidth monochromator optics (9 nm to 50 nm in 1 nm increments), as well as filter-based detection optics for high-sensitivity detection. For imaging, it offers image stitching and stacking for 3D digital pictures, plus image analysis software – it has an enormous range of capabilities.

Caleb_Foster

Figure 4: Overview of the BioTek CytationTM 5 from Caleb Foster, Product manager in the BioTek Development Group.

Another good example is the FluoView FV1200 Confocal Laser Scanning Microscope from Olympus, which has five simultaneous fluorescent detection channels. 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.The UltraVIEW® VoX 3D Live Cell Imaging System from PerkinElmer allows users to acquire high-speed, high-resolution, multi-dimensional images of live biological samples including cultured cells, tissues and embryos.

Total Internal Reflection Fluorescence Microscopy (TIRFM)

This highly sensitive technique allows you to perform functional investigations in living cells. 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 allows very high resolution 2D images, down to less than 200nm. This is therefore 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 the cell adhesion sites, and in cell membrane and membrane proximal cytoplasmic organelles. TIRF microscopy systems are now commercially available from many microscope suppliers, including ZEISS and Olympus.

Electron Microscopy

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).

Transmission Electron Microscopy (TEM)

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 5 shows the structure and make-up of a TEM.

Transmission Electron Microscope

Figure 5: Structure of a Transmission Electron Microscope (TEM)

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 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.

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, which often requires a specialist.

The Analytical TableTop Microscope TM3000 from Hitachi was designed to cover the widest range of industrial and academic applications and research, making it a good entry level machine. It allows high-quality resolution images with no special sample preparation, such as coating with metal films for hydrated, oily or non-conducting samples. It has a simple imaging system similar to a digital camera.

Since its introduction in 2005, the superior design of Titan - Transmission Electron Microscope by FEI, and its proven ability to deliver ground-breaking results have made it the preferred scanning/transmission electron microscope (S/TEM) of leading researchers around the world.

Scanning Electron Microscopy (SEM)

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.

JEOL produces a range of SEMs such as the JSM-6490LV Scanning Electron Microscope, which has a resolution of 3.0nm. Some electron microscopes can encompass both SEM and TEM technology in one, such as the Quanta Scanning Electron Microscope, from FEI Company.

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 (Figure 6).

EVO 18 SEM

Figure 6: Analytical Scanning Electron Microscopy with X-ray Geometry

Atomic Force Microscopy (AFM)

AFM is a method of scanning that can see details at the fraction of a nanometer level. Using a combination of AFM with synchrotron radiation microscopy and standard 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. The MFP-3D-BIOTM from Asylum Research boasts the highest sensitivity and most accurate images and measurements possible on an inverted optical platform. According to the manufacturers “The NPS™ closed loop nanopositioning sensors on all three axes ensure distortion-free images on samples as small as proteins and as large as cells – in both air and liquid.”

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.

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. 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 Placement

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.

Correlative Light and Electron Microscopy

Recently there has been a drive to combine the best parts of light and electron microscopy. Here are some examples of these technologies combined into one piece of equipment:

  • Raman and SEM or AFM is combined in the WITec RISE Microscopy Raman-SEM (Raman imaging and SEM) product with the capability to add on chemical analysis in this Renishaw AFM-Raman System.
  • Fluorescence and TEM can be seen in FEI’s Tecnai i-Corr correlative microscope. This improves the visualization of ultra-structural details and multiple points of interest without transfer of the sample between instruments.
  • A combination of focused ion beam system (FIB) and a mass spectrometer (secondary ion mass spectrometer) has recently been developed by TESCAN. This allows the chemistry of surfaces to be analyzed as the FIB removes material.
  • Confocal fluorescence microscopy has 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.

Imaging and Analysis

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. Many are presented and reviewed on product pages. Some examples of CCD camera include the CoolSNAPTM HQ2 Interline CCD Camera from Photometric, the iXon Ultra 897 EMCCD Camera from Andor Technology, the EvolveTM 128 EMCCD Camera from Photometrics.

leicahd Microscope

Figure 7: An introduction to Leica’s HD Imaging Systems for microscopic inspection in Industry and Life Science applications

For digital microscopy, what data do you want to get from your sample? Qualitative images only for display or could you benefit from using more advanced imaging and software to record quantities data on your sample? Digital microscopy and increasingly sophisticated imaging software allows image stitching and 3D image stacking 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 Microscopy analysis software section in our product pages.

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.Live cell imaging systems are available, for example the IncuCyte ZOOM by Essen Bioscience, which allow you to program when and where the images are taken over time.

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. Also, do approach more than one manufacturer for quotes.

The Future of Microscopy

Light Sheet Fluorescence Microscopy

Ten years of development in light sheet microscopy have led to spectacular demonstrations of its capabilities. The technology is ready for mainstream use to help biologists in tackling scientific problems. Its key features are low phototoxicity and high-speed imaging, allowing gentle imaging of biological samples with high resolution in 3D and over long periods of time. Instead of illuminating or scanning the whole sample through the imaging objective, as in widefield or confocal microscopy, one illuminates the sample from the side with a thin (practically 2D) plane or sheet of light. The emitted fluorescence is then detected from above or below the sample, along an axis perpendicular to the light sheet. Light sheet fluorescence microscopy has recently been used to image living hearts and functioning brains and to track moving cells within developing embryos. The ZEISS Lightsheet Z.1 brings light sheet fluorescent technology to the marketplace.

Dr. Annette Bergter

Figure 8: Watch a video about long term imaging of samples with light sheet technology from Annette Bergter, ZEISS

Advanced Imaging and Analysis Software

The evolution of more powerful imaging and analysis software will bring the biggest advances in microscopic research in the short term. Microscopes now have the capability to produce thousands of images of incredible spatio-temporal resolution and complexity, with a massive increase in the quality and amount of data that can be detected and elucidated; the challenge is to interpret as much out of it as possible. Software to handle all the images and tracking are also now needed to detect patterns and sequences from huge numbers of images. Organizing and making sense of spatio-temporal images and data is the next biggest challenge, as researchers push to track and observe activity and development not just within cells but multicellular structures and model organisms, such as zebrafish and nematodes.

Extending the Spectrum of Fluorescent Labeling

Brainbow is a genetic cell-labeling technique where hundreds of different hues can be generated by stochastic and combinatorial expression of a few spectrally distinct fluorescent proteins. Unique color profiles can be used as cellular identification tags for multiple applications such as tracing axons through the nervous system, following individual cells during development, or analyzing cell lineage. In recent years, Brainbow and other combinatorial expression strategies have expanded from the mouse nervous system to other model organisms and a wide variety of tissues.

In Vivo Imaging of Tumors

This allows researchers to understand the subtleties of aberrant cell division using quantitative intravital microscopy. Currently this is being achieved by researchers by manual annotation of select image events. It is now possible to combine image analysis with machine learning methods for automated 3D segmentation and cell cycle monitoring of individual cell nuclei within complex tumor environments.

Optical Projection Tomography at the UCL Centre for Advanced Biomedical Imaging (CABI)

Optical Projection Tomography (OPT) is a new technique for 3D imaging large biological samples (of the order of 1 cubic centimeter). OPT is a novel and exciting technology and represents the next generation of optical microscopy. It is particularly suited to study fundamental biological processes using light emitted from inside the organ, via optical fluorescence. The main advantage of this new imaging modality is that it avoids the need to physically section the sample. Furthermore, OPT is able to take advantage of fluorescent dyes, and three different wavelength channels can be used. This allows the observation of the autofluorescence of the tissue (to inform on tissue structure), alongside the mapping of gene and protein expression. The principle aim of the group is to use the OPT scanner installed in CABI for cancer research, using particular fluorophores to stain tumors and study their vasculature.

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.

Summary

There are many different types of microscopy instruments on the market and 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 find out about the latest Microscopy instruments from leading manufacturers and read user reviews. Use the SelectScience application note library to keep up-to-date with the latest methods.

Editor's Picks

Kerry Parker Kerry Parker
Editor-in-Chief

ZEISS LSM 880 with Airyscan (ZEISS Microscopy)

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4 out of 5

"The very latest and highest performance confocal microscope on the market. With the optional upgrade, the increased sensitivity makes it second to none..."
Peter O'Toole, University of York

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CKX41 Inverted Microscope (Olympus Europa SE & CO. KG)

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5 out of 5

"Compared to our standard upright microscope, you get a lot more and better microscope for less money with this system..."
Mikael Evander,, Lund University, Department of Biomedical Engineering

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Quanta™ Scanning Electron Microscope (FEI Company)

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5 out of 5

"Easy to use, with excellent customer support. I have been using the instrument for about a year, and support took care of the regular maintenance and training..."
Ajay Zalavadia, Advanced Chemical Imaging Facility

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Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc.)

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5 out of 5

"This is so easy to use. The Cytation 5 integrates user-friendly Gen5 software that incorporates autofocus, auto LED intensity, and auto exposure, and allows for easy sample positioning..."
Douglas Hughes, Thermo Fisher Scientific

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Leica DM1000 LED (Leica Microsystems)

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4 out of 5

"The microscope is easy-to-use for students, and has great value when compared with other brands."
Rene Basso, University of Baja California


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IX83 - Fully-Motorized and Automated Inverted Microscope System (Olympus Europa SE & CO. KG)

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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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Cytation 5 Imaging Multi-Mode Reader


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