The 17th-century scientist Robert Hooke is probably best known for formulating his law of elasticity (Hooke’s Law), but his interests ranged from physics and astronomy, through to chemistry, biology, geology, naval technology, and architecture – he was instrumental in rebuilding London after the Great Fire in 1666. He is also widely thought of as ‘the father of microscopy’ for his studies of various biological structures and organisms through compound microscopes.
Hooke’s observations were set down in a book published in 1665 entitled Micrographia, the most famous of which is his microscopical study of thin slices of cork, in which he noted the structure ‘to be all perforated and porous, much like a honey-comb’. The individual pores he named ‘cells’, as they reminded him of the cells of a monastery. What he had observed were the hollowed out, dead remnants of plant cells, with the cell walls all that remained forming the honeycomb pattern.
Others have been involved in the history of microscopy, most notably Anthony van Leeuwenhoek, a Dutch scientist operating around the same time as Hooke, whose studies led to the discovery of bacteria and other microorganisms.
Using imaging techniques in science is by no means limited to microscopy, but this is one of the largest areas, one that has revolutionised science and is still a key component of scientific research today. The technology has advanced so far, that a transmission electron microscope installed at the Department of Energy’s National Center for Electron Microscopy (NCEM) at Lawrence Berkeley National Laboratory in the US, is capable of producing images at a half-ångström resolution – smaller than the diameter of a single hydrogen atom.
There are many microscopy techniques, which vary in the staining or preparatory procedures, as well as the wavelength of radiation used, among other distinguishing characteristics. Luminescence is one area where improvements have been made to microscopes used to view these events.
Luminescence microscopy
Images of luminescent events have traditionally been difficult to capture due to the low light output. Many applications use luminescence readers, which are sensitive, but have a low resolution. Olympus has developed the LV200 bioluminescence microscope, which employs a completely new optical concept with improved resolution to allow a standard camera to capture the image.
‘Luminescence readers provide a measurement of the amount of light emitted, but don’t show images from the sample,’ says Werner Kammerloher, luminescence business manager at Olympus. ‘Using a luminescence microscope, scientists can see what is happening within the sample. For example, a microscopy system used to analyse cell cultures allows the percentage of active cells emitting light to be seen, which isn’t possible using a luminescence reader.’
For in vivo studies, 99 per cent of customers use a fluorescence microscope, in which the sample is excited by high-intensity light photons to generate the fluorescent effect. However, using luminescence, the light is emitted naturally by a chemical or biological reaction within the cell, which avoids phototoxic effects observed with fluorescence.
Therefore, in a study where the expression of a particular gene is investigated over time, luminescence would provide a better optical measure than fluorescence, as there is no phototoxic damage to the cells. In addition, certain luminescent markers, such as firefly luciferase, have a very short half life and decompose quickly in the cell. ‘This is particularly advantageous for gene expression studies as both up and down regulation of the gene can be monitored,’ explains Kammerloher. A quantitative description of gene expression over time can therefore be determined using bioluminescence, which is more difficult to achieve using fluorescence, as fluorescent molecules tend to remain active in the cell.
Studies involving gene expression, for instance in a mouse embryo, are where this technology excels. A gene coding for a bioluminescence protein can be cloned and attached as a biological marker to the gene of interest. Expression of that gene during embryo development results in the marker protein being expressed, which leads to light being emitted.
Investigation of calcium signalling within the cell is also a key application of bioluminescence. Calcium is an important cellular messenger and is instrumental in processes such as muscle contraction. Imaging calcium fluxes using a luminescent protein is preferable to using a toxic fluorescent dye and light excitation, which potentially could produce artefacts in the images. Luminescence is especially beneficial if the calcium signal occurs at an unknown time point as the study can be carried out over an extended timeframe.
‘Due to the low signal produced, the microscopy system is completely light tight,’ states Kammerloher. ‘The cells are also kept warm and under a defined CO2 concentration within the microscope, ensuring they remain active over the course of the experiment.’
The LV200 microscope is designed specifically for luminescence studies. ‘In comparison to standard microscopes, the distance between the objective and camera is reduced drastically. This, along with the high numerical aperture of the tube lens and a reduction in the number of mirrors and lenses in the system, ensures as much light is captured as possible,’ he says.
Kammerloher believes that: ‘Luminescence will not overtake fluorescence in its use as an imaging technique. However, the improved optics of Olympus’ microscope opens up more possibilities and new channels of research where luminescence can be used. It also allows studies, which were previously carried out using fluorescence techniques, to be revisited using luminescence imaging, and any detrimental effects associated with fluorescence due to dyes or phototoxicity can be established.’
Multiphoton excitation microscopy
The use of fluorescence to image cellular components is a well-used technique in microscopy. Multiphoton excitation (MPE) microscopy is a fluorescence microscopy technique, but works on the principle of multiple (usually two) photons of lowenergy radiation arriving simultaneously at a fluorophore to elicit the fluorescent response, rather than a single, high-energy photon. A fluorophore is a fluorescent molecule that, in absorbing photons of a specific wavelength, is excited to emit light. Fluorophores are used as fluorescent stains or contrast agents in techniques utilising fluorescence to visualise cellular components in biological samples.
As opposed to confocal fluorescence microscopy, where a continuous wave laser illuminates the specimen at a specific wavelength causing it to fluoresce, MPE microscopy uses a pulsed laser operating at a longer wavelength, typically in the infrared region, to elicit a response. As multiple photons reach the fluorophore within femtoseconds of each other, the effect is the same as illuminating the specimen with a single, shorter wavelength beam. As an example, a two-photon excitation at 680-1,080nm is equivalent to a one-photon excitation at half the wavelength, (340-540nm).
Marco Arrigoni, director of marketing in the scientific segment at Coherent, explains that: ‘Using an infrared laser light source increases the depth of visualisation and allows living tissues to be examined with minimal damage. The technique allows imaging of tissues at a depth of 0.5-1mm and is currently used mainly in research laboratories.’ German company JenLab has been using the technique for in vivo studies into the diffusion and accumulation of topically applied cosmetic and pharmaceutical ointments in the upper layer of the skin. Coherent provides the laser systems used in MPE microscopes.
Coherent’s Chameleon Ultra and Chameleon Vision laser systems can be tuned to 1,080nm wavelength, making them suitable for MPE microscopy. ‘Different fluorophores absorb at different wavelengths and often in staining a sample a combination of these markers are used,’ explains Arrigoni. ‘This requires excitation at different wavelengths and so a typical confocal system will have three or more lasers integrated into it.’ Confocal illumination sources are often solid state lasers operating at 532nm and 490nm or red diodes at 635nm.
‘In MPE microscopy, by contrast, the key laser source is tuneable, so a Ti:sapphire laser, like Chameleon Vision, can be tuned between 680nm and 1,080nm to cover most of the contrast agents commonly used. Therefore, a tuneable laser used in MPE microscopy can replace the suite of lasers required for confocal microscopy, which simplifies the overall system design,’ says Arrigoni. However, he goes on to say that the cost of an MPE laser system is in the region of $170-230K, which is still much more expensive than a suite of lasers used for confocal microscopy – a group of five lasers costing no more than $20-50K.
In vivo studies in organisms such as mice, which can be genetically engineered to express non-toxic, naturally occurring fluorescent proteins such as GFP (Green Fluorescent Protein), DSred and mCherry, have become a widespread imaging protocol used both in confocal and MPE microscopy. ‘The combination of the low cellular damage associated with MPE and the lack of toxicity of these fluorescent proteins, has produced spectacular results, such as in-depth imaging in the brain of live mice, which survive and behave normally for many months,’ says Arrigoni.
‘However, if MPE is to become a diagnostic tool for humans, it needs to work with contrast agents directly produced by our bodies, without external manipulations,’ Arrigoni notes. He goes on to explain that some cellular components are naturally fluorescent, such as NADH (the reduced form of nicotinamide adenine dinucleotide), albeit weakly, while others, like collagen, when excited by two infrared photons, produce second harmonic generation (SHG) light at exactly half the absorbed wavelength. ‘Native fluorescence and SHG microscopy both show promise for use as a diagnostic tool in humans, as a stain is not needed.’
Currently, an MPE microscope costs around $600,000. The equipment is also cumbersome and for these reasons the technology is restricted to research laboratories. Arrigoni feels that to implement this technology in hospitals and medical centres, there must be a reduction in cost to below $100,000 and techniques that don’t use fluorescent markers need to be developed.
Microspectroscopy
The technology behind microspectroscopy was driven by scientists using microscopes who wanted additional information on what they were seeing. The technique combines the visual benefits of microscopy with the advantage of being able to obtain a spectroscopic analysis of a sample area. The sample area is located through the objective and then spectral information is obtained (mainly through laser-stimulation) and captured by a standard spectrometer-detector detection system.
‘The technology allows scientists to carry out Raman mapping, for example. Using this technique, spectra are gathered at set points to give a “map” of the chemical makeup of a sample,’ explains Antoine Varagnat, market development manager for spectroscopy at Andor Technology.
The technique can be used in the pharmaceutical industry, looking for contamination in drug samples, as well as for medical research in the validation of disease diagnosis.
In one instance, a scan of a slice of the retina has been used to identify the location affinities of infections in the eye structure through a Raman technique. Varagnat says: ‘If the presence of a particular disease is suspected and the chemical structure of this is known to produce a specific peak on a Raman spectrum, then microspectroscopy systems can be used as a quick and easy diagnostic tool. It’s an easy technique to implement, as there is virtually no sample preparation. The area under analysis is simply found, and the spectrum produced and correlated to a physical location on the sample.’
At present, the technique is still being developed as an ex vivo tool for human disease characterisation and behaviour understanding, such as retinal infections, skin cancer, and artery ageing. ‘Validation of the technology to ensure there are no adverse effects to the laser is still ongoing,’ notes Varagnat. However, using an Electron Multiplying Charge Coupled Device (EMCCD) to amplify the Raman signal allows a safe level of irradiation to be used, which ensures a rapid diagnosis.
Imaging shockwaves
VISAR (Velocity Interferometer System for Any Reflector) is a technique used to measure the basic properties of a material. The technique uses a laser, which reflects off the surface of the material creating laser fringes, or bands, produced by the interference of light waves. The material is then impacted by shockwaves and by looking at the rate of fringe motion, the velocity of the reflecting surface can be directly measured. The spacing between the fringes can indicate induced stresses in the material, and changes in reflectivity can be used to infer material properties such as density and conductivity.
Measurement of the movement of fringes is carried out using a streak camera, which can record the picosecond timeframes involved in this type of shockwave experimentation. Sydor Instruments (represented by Armstrong Optical in the UK) has provided its Ross streak cameras for use in VISAR studies conducted by the Laboratory for Laser Energetics (LLE) at the University of Rochester in the US.
The LLE is involved in studies investigating inertial confinement fusion (ICF) – a process where nuclear fusion reactions are initiated by heating and compressing a fuel target. ‘In laser fusion experiments, there is much study and effort to produce uniform compression of the spherical targets,’ explains Yoram Fisher, senior systems engineer at Sydor Instruments. ‘The efficiency of fusion energy out to the energy put in is directly proportional to how dense the fuel in the target can be compressed.’
ICF uses a combination of long laser pulses to slowly compress the spherical target into a smaller, higher density sphere, while simultaneously ‘kicking’ the target with short, higher intensity pulses. The shorter pulses produce shockwaves of very high density and initiate the fusion burn. ‘Understanding how the shockwave propagates through the target, as well as the speed, and density of the shockwave, is fundamental to the fusion process,’ says Fisher. VISAR is used to measure the shock physics of ICF.
Fisher feels that from these studies the ICF process will be refined to provide better compression of targets, and thus yield higher amounts of fusion energy. ‘Work is continuing at the LLE to get higher yield shots. It has recently installed a new high power laser that will produce stronger shockwaves, and therefore stronger target compression, yielding more fusion energy.’
The LLE, along with the Lawrence Livermore National Laboratory (LLNL) in California and a collection of other laboratories, is also using VISAR to study shockwaves in helium. The experiments are designed to gain a better understanding of the evolution and internal structure of Jupiter, Saturn and other extrasolar giant planets where the main constituent is helium.
