Special Report: Potential of super-resolution
Super-resolution optical microscopy is a superior tool for investigating living cells, and interdisciplinary efforts are required to proof its full potential in biomedical research. Efficient functioning of the human body involves a multilevel synthesis emerging from collective brain activity to the immune system. Signalling, whether in immune, neuronal, or other cells, is usually controlled by the local redistribution and interactions of a relatively large set of proteins, both on the cell surface and interior.
The spatial scale of these organisational changes ranges from micrometres to single molecular dimensions, i.e. nanometres. In addition, these changes may occur on multiple timescales from micro/milliseconds to minutes/hours. A striking example is the re-organisation of proteins on the cell surface of immune cells, which likely starts within seconds at the molecular scale, is followed within minutes by the re-organisation of receptors into submicrometre-scale protein assemblies (micro-clusters), and after minutes to hours culminates in the formation of a micrometre-scale structure of the so-called ‘immunological synapse’, probably the most essential step during an adaptive immune response.1 Typically, these processes are accompanied or actively driven by collective re-organisations of the immune cells’ interior cytoskeleton, both occurring on macro and microscales over minutes. Similarly during neuronal communication, cellular structures such as axons and synapses have been identified to play a crucial role. They contain key molecules, whose re-organisation is required to maintain and mediate their communication mechanisms.1
Working to understand
Understanding the complex interactions of the molecular processes underlying these mechanisms is one of the main objectives of biomedical research. Disorders of these dynamic interplays can lead to disease, but their comprehension to the development of novel therapies. Considering the complexity of, for example, autoimmune diseases or cancer, to-date medical practice demands the most physiological conditions for their research – which can be best matched by studying living species, e.g. live cells and/or organisms. Covering the aforementioned large range of spatial (nanometres to larger than micrometres) and temporal (microseconds to minutes/hours) scales, this necessitates modern observation techniques.
Fig. 1: A) Super-resolution optical microscopy: Confocal (outer) and super-resolution (STED, inner part) images of immunolabelled Titin Z-disk in sarcomers in fixed myofibroblasts. Only the super-resolved image discloses certain details of molecular organisation. B) Trade-offs in optical microscopy: A toolbox of complementary fluorescence-based optical imaging techniques has been developed in order to optimise needs by biomedical applications. The ultimate goal is the development of a microscope technology that fulfils all of these properties
Scientifically, it is important that the methods do not influence the biological system during observation. The most suitable tool that can cover all of this is optical far-field fluorescence microscopy, i.e. the use of light-emitting (fluorescent) molecular labels in combination with focused light (far-field optics). Fluorescence imaging comprises several characteristics: 1) light hardly influences the system during observations; 2) the use of a lens-based system and focused light allows the observation to take place micro to millimetres away from the given optical element(s), preserving the non-invasiveness and the ability to observe deep inside living cells or organisms; and 3) the fluorescence readout, i.e. the light-induced excitation of emission from a fluorescent molecule (for example, an organic dye or fluorescent protein) labelling a specific protein, achieves the specific and highly sensitive detection of cellular constituents, and thus permits the disclosure of molecular distributions and dynamics.
Limitation
Unfortunately, far-field microscopy still has limitations when it comes to temporal and spatial resolution: 1) submillisecond recordings are challenged by the amount of light, and thus the information yield, emitted by the fluorescent labels; 2) minutes to hours-long recordings are limited due to potential phototoxic effects by the light, i.e. light-induced photo-bleaching of the labels and changes or degradation of cells; and 3) the diffraction of light prevents resolving structures at a length-scale below around 200nm.
To this end, molecular dynamics and organisations can only be resolved within limited temporal and spatial regimes. A remedy to this might be the use of alternative observation technology. For example, techniques such as electron microscopy, near-field microscopy, or atomic-force microscopy can offer spatial resolutions down to molecular scales with the drawback however, of not being able to investigate the living cell, or only cell surfaces. Consequently, over centuries, a major quest has been pursued to break the limit in spatial resolution of optical far-field microscopy as given by the diffraction of light, nowadays resulting in a whole range of available super-resolution microscopy or nanoscopy techniques.1,2
Approaches such as Structured-Illumination-Microscopy (SIM), 4-Pi, and I5M push diffraction to its very limits, delivering spatial resolutions of down to around 100nm. In contrast, methods such as Stimulated-Emission-Depletion (STED) microscopy, Reversible Saturable Optical (Fluorescence) Transition (RESOLFT) microscopy, (fluorescence) Photoactivated Localisation Microscopy ((f)PALM), (direct) Stochastic Optical Reconstruction Microscopy ((d)STORM), or Ground-State-Depletion (GSD) microscopy make use of the ability to modulate the fluorescence emission, thereby ensuring that the detected fluorescence signal stems from areas much smaller than the 200nm given by the diffraction limit, i.e. they reach in principle unlimited spatial resolution.1,2 The power of such nanoscopy techniques is highlighted in Fig. 1A, which shows the organisation of cytoskeletal structures in cells, indicating that molecular arrangements can be studied inside the intact cell with so far unprecedented spatial resolution.
Unfortunately, the increased sensitivity comes at a price:1,2 1) Nanoscopy techniques are usually more sensitive to artefacts such as changes introduced by the fluorescent labels; 2) the techniques usually feature less temporal resolution than conventional methods, limiting observations to dynamics slower than seconds to minutes; 3) light-induced effects such as photo-toxicity may be increased due to the need of adding light for modulating fluorescence; and 4) the inability to investigate features deep inside cells due to deteriorations (so-called ‘optical aberrations’) when the light passes through several layers of the sample. In cases, users sacrificed spatial resolution and imaged at lower resolution using, e.g. STED or RESOLFT microscopy with lower light powers of the laser modulating the fluorescence emission (these techniques uniquely realise tuning of the resolution through adjusting that laser power), SIM, or even conventional microscopy techniques such as confocal or multiphoton microscopy.
On the other hand, the above limitations are approached by input from different disciplines, (bio)chemistry, engineering, and physics:1,2 new labels provide higher photostability and signals as well as improved ways of tagging the molecules of interest; more efficient light sources such as improved lasers realise optimised ways of exciting and modulating the fluorescence; improved optics such as adaptive optics or light-sheet and multi-spot illumination deliver the light to the sample in a more efficient way and thus minimise aberrations and phototoxicity and maximise acquisition speed; and intelligent data analysis algorithms enhance the content one can extract from an image.
Another remedy to the above limitations is the combination of nanoscopy techniques with other read-outs, which allows enhancing information contents of the recorded data as done, for example, in spectroscopic-based studies of fast millisecond molecular dynamics using STED-F(L)CS or single-particle tracking PALM (sptPALM), or when combining read-outs such as force, electrophysiology or electron with fluorescence microscopy.
Missed opportunities?
In general, the demands of biomedical research are large with applications requiring different aspects of spatial and/or temporal resolution, three-dimensional imaging deep inside the sample, and/or long acquisition times with low photo-toxicity (Fig. 1B). The fact that all microscopy approaches are complementary, whether they are diffraction-limited or with nanoscale resolution, promotes research environments with access to various kinds of microscopes and nanoscopes, depending on their suitability for the case in hand. On the other hand, research environments should be strongly interdisciplinary, allowing (bio)chemists, physicists, engineers and biomedical researchers to tightly work together to optimise technology for its use in biomedical research.
An ultimate goal would be a microscope that can combine all of the demands set by the users (Fig. 1B). Unfortunately, much advanced technology developed by engineers, physicists, or chemists has not made its way into applications, simply because of missing tight links with biomedical users. The question arises whether we have missed opportunities. Therefore, funding possibilities have to be created, promoting interdisciplinary research environments with access to a broad range of complementary state-of-the-art technology as well as with the chance to test out new approaches.
References
1 Hell, SW et al. The 2015 super-resolution microscopy roadmap. J. Phys. D: Appl. Phys. 48, 443001 (2015)
2 Eggeling, C. Willig, KI. Sahl, SJ. & Hell, SW. Lens-based fluorescence nanoscopy. Q Rev Biophys 48, 178–243 (2015).
Marco Fritzsche, PhD
Junior Principal Investigator
Christian Eggeling
Professor of Molecular Immunology
MRC Human Immunology Unit
Weatherall Institute of Molecular Medicine,
University of Oxford
+44 (0)1865 222 167
http://www.nano-immunology.org
