Guide to Advanced Microscopy Techniques

From whole animals to single molecules perched on the surfaces of an animal’s cells, there is a type of microscopy that can capture the image. Today’s microscopy techniques continue to push against their constraints to gain improved spatial and temporal resolution. This article reviews common advanced microscopy techniques used in imaging today, and discusses their main benefits and limitations.

Optical microscopy

Widefield or brightfield microscopy, an essential form of optical microscopy, has the advantage of speed and simplicity. Yet even widefield systems are becoming more advanced by incorporating artificial intelligence. “In many cases, user-friendly systems like the APEXVIEW APX100 can allow researchers to not only gain the information they need to solve their questions, but also image more efficiently thanks to AI technology,” says Wei Sun, Senior Product Manager at Evident (formerly Olympus Scientific Solutions). However, basic light or fluorescence microscopy can be phototoxic to the specimen, and lack the ability to image at different depths.

Confocal microscopy techniques solve this by illuminating less of the sample at once, and for less time. As such, they are slower but less phototoxic. Furthermore, they image a series of planes that are combined to reconstruct 3-dimensional images. For example, Evident’s FLUOVIEW laser scanning confocal systems “provide high spatial resolution in three-dimensions, low photo-toxicity, and deep penetration depth into your samples,” says Sun.

Many advanced fluorescence techniques are combined with confocal imaging today. For example, fluorescence lifetime imaging (FLIM) is considered the gold standard for functional imaging, and also enables multiplexing, according to Julia Roberti, Product Manager in Life Science Research at Leica Microsystems. The fluorescence lifetime of a probe can yield information about its physical environment at the molecular level, such as changes in pH, ion concentration, or lipophilicity. “FLIM can improve image quality by separating unwanted signals like autofluorescence,” says Roberti. “This technology is available with STELLARIS 8 FALCON.”

Multiphoton (also called 2-photon) microscopy is designed for deeper imaging into tissues and organs, or live animals using longer wavelengths of light. “The advantages of using a more extended excitation laser source come from two physical mechanisms, less scattering in samples, and less out-of-focus absorption,” says Roberti. “To maximize the benefit of imaging deep in tissue, researchers can combine lifetime imaging technologies to obtain valuable information from autofluorescence arising from endogenous fluorophores or tissue structures.”

Evident’s multiphoton imaging is used in many applications. “For example, our customers have imaged the brain cells of a living animal using our FVMPE-RS laser scanning multiphoton microscope, while simultaneously stimulating with light using optogenetics to decipher how the network of circuitry of neurons are connected and formed,” says Sun.

Importantly, optical microscopy methods share a disadvantage: they are constrained by the diffraction limit of light, which means that fluorescence-based techniques are unable to resolve structures smaller than about 240 nm. Super resolution microscopy techniques were developed to overcome this barrier.

Super resolution microscopy

All super resolution techniques offer the advantage of higher lateral resolution than can be obtained with optical techniques—down to 10s of nm depending on the specific technique. Several common ones include structured illumination microscopy (SIM), stimulated emission depletion (STED), and single molecule localization microscopy (SMLM). General disadvantages can include cost, and greater complexity to operate. Super resolution techniques require higher intensity light and therefore tend toward greater phototoxicity or photobleaching compared to optical methods. Because of this, two types of SMLM—stochastic optical reconstruction microscopy (STORM) and photo activated localization microscopy (PALM)—are especially challenging to use with live cells.

However, new improvements are making super resolution techniques more cell-friendly and more widely used. One of the gentlest is SIM, which is often used for live cell imaging with resolution down to about 100 nm. This is thanks to technological developments in SIM that allow imaging at still lower light levels, such as using deconvolution algorithms of lower-light data.

STED is another super resolution technique for nanoscale resolution of subcellular structures. Leica’s TauSTED augments STED further by using FLIM, which is especially useful in live cell imaging studies where the ability to image multiple colors and dynamic events is crucial. “With TauSTED technology, STELLARIS STED can reach the same resolution with a small fraction of the power needed in conventional STED,” says Roberti, which is especially helpful in preserving cell health for live cell imaging. TauSTED allows for greater STED resolution and negligible background, despite using less excitation light. “This combination is key to achieving proper temporal sampling to track fast biological processes, such as vesicle and actin dynamics, and [using] lower light doses where light sensitivity is a known limiting factor,” says Roberti. “With STELLARIS STED, for example, it is possible to capture three-dimensional, two-color STED information of highly dynamic events, e.g., reaching one cell volume in less than 10s.”

Atomic force microscopy

Atomic force microscopy (AFM) is an advanced imaging technique that produces 3D surface images and characterizes the nanomechanical properties of samples of living cells, biomolecules, and tissues. Because it uses a sensitive probe to scan the sample’s surface, AFM is free from the limitations of diffraction and light intensity, as seen with optical and super resolution techniques, respectively.

Bruker’s platform of atomic force microscopes includes BioAFMs, which are specifically adapted for the study of biological samples. BioAFMs are often combined with optical microscopy systems to gain further insights into what’s happening on the surface, and within the depths of, cells or tissues simultaneously. “Combining microscopy techniques has the advantage of co-localizing the features of an AFM measurement with the optical image of living cells,” says Florian Kumpfe, Product Specialist at Bruker BioAFM. “We have specialized software tools to overlay those images.” For example, researchers recently combined super resolution STED microscopy with AFM to study the properties and regulation of desmosomes—intercellular junctions responsible for adhesion—in living cells.

The AFM’s resolution is comparable to that of an electron microscope, and researchers apply it in a wide range of interests. “By looking at the surface, you can visualize small details, such as pores on membranes or single microvilli of living cells,” says Kumpfe. “Our customers study everything from single molecules, viruses, and bacteria, to organoids, tissues, and biopsies.” The AFM’s cantilever probe can also measure biomechanical properties, such as stiffness, viscoelasticity, and adhesion. In other recent work, researchers studying osteoarthritic tissue from a human knee joint used BioAFM to measure the progression of stiffness within the cartilage and how it correlates to disease progression.

Because scientists are continually pushing for greater results, imaging capabilities will only continue to grow—stay tuned for the next installment of the amazing achievements of advanced microscopy.