FEATURE
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Non-invasive innovation

William Payne on the emerging area of biophotonics, which sees lasers applied to many aspects of biology

Biophotonics is one of the fastest growing applications for laser instrumentation and measurement. According to life science market research firm Kalorama Information, the biophotonics market is growing at an annual rate of 31 per cent, and will be worth $133bn by 2016.

The main thrust of biophotonic applications is the rapid, non-invasive identification of protein and genome structures. Biophotonic applications in mass spectroscopy, cytometry and microscopy are beginning to show major advantages over conventional techniques in accuracy, speed and cost.

This flexibility and range of function is turning biophotonics into a disruptive technology that is fast becoming the basis for standard measuring applications in pharmaceutical discovery and development, life sciences research, especially in proteomics and genomics, medicine – particularly histology, cytology, oncology and virology – food sciences, agriculture and aquaculture.

Beating heart disease

It’s the world’s single biggest killer. Half of all men, and a third of women will develop it. Coronary artery disease can be difficult to diagnose, but conventional tests either take too long, or can be misleading. Now, researchers are developing new tests based on biophotonics that can provide clear, accurate answers in just minutes.

When a heart attack strikes, doctors have about 60 minutes to save a person’s life. This is the ‘golden hour’. But the symptoms of a heart attack are often confusing. They can be the classic chest pain. But just as often, patients having a heart attack complain initially of a pain in the arm, or even toothache. With such confusing symptoms, doctors are often left not knowing if the patient should be on the operating table, or sent home with a Rennie tablet.

Clinicians need an immediate way of telling whether someone is suffering a heart attack. ECG monitoring is quick. But its output can be confusing and mistakes are easily made. The definitive diagnostic test is a blood chemistry analysis. This detects proteins that leak from the heart during an attack. Unfortunately, current enzyme tests take around six hours to detect elevated levels, and a further six to show a definitive result.

A team from Massachusetts General Hospital, MIT and Harvard University, has developed a new test based on laser mass spectroscopy that can return a conclusive result just minutes after the beginning of a heart attack. The researchers coupled a robotic sampler to an Applied Biosystems triple quadrupole mass spectrometer, equipped with a 200Hz Nd:YAG laser. The system is able to analyse hundreds of metabolites in minutes. It is also able to identify accurately a range of metabolic changes that happen right at the beginning of an attack.

Eradicating heart attacks

With coronary artery disease, prevention is better than cure. If diagnosed in time, coronary artery disease can be halted in its tracks, and the danger of heart attacks averted.

In diagnosing coronary artery disease, ECG monitoring suffers many drawbacks; interpreting its waveform output is difficult. Patients often have to undergo lengthy stress echo testing. Even then, mistakes can be made.

These days, clinicians prefer alternate diagnostic tests. Analysing the myocardial fibres of the heart is the preferred approach. Understanding the threedimensional structure of the fibres tells clinicians how far coronary artery disease has progressed, and the likelihood of future heart attacks.

The two main tools used today for myocardial fibre imaging are diffusion tensor imaging (DTI) and histological sectioning. DTI is non-invasive. However, only the most advanced – and expensive – MRI scanners can achieve a sufficient resolution (<100μm) to provide clinically useful imaging. Histological sectioning provides better resolution. However, this approach is clinically invasive, time-consuming and far more complex.

Doctors at SUNY Upstate Medical University in Syracuse, New York, working with physicists at Kaunas University in Lithuania, have developed an alternative approach based on biophotonic technology, using a technique known as ‘optical clearing’.

Optical clearing makes specimens transparent by matching the intra and extracellular refractive indices. By adding a fluorescent dye, which binds to cell membranes, and optically clearing the heart, the researchers have been able to construct 3D renderings of the myocardial fibre organisation in mammal hearts.

The team carried out confocal imaging, using a Zeiss LSM 510 system with a Zeiss Axiovert 200M inverted microscope with 103 dry and 403 water immersion objectives. Fluorescence was detected using 514nm and 633nm laser excitation and 560nm long-pass and 650-710nm band-pass emission filters for the dyes di-4-ANEPPS and di-4-ANBDQBS, respectively. Depth-correction of the excitation intensity and photodetector gain (z-correction function in Zeiss software) was used to reduce signal intensity variation during deep (>500μm) scans. Repetitive scans (up to 16) were implemented to reduce the noise in the images. Whole heart scans were performed using multiple time series software, with tiled scanning and subsequent compilation into a single image.

The team claims that its fibre mapping method combines the advantages of both DTI and histological methods. It allows non-destructive myocardial fibre mapping, like DTI. And it has a spatial resolution of a few microns, as histological methods do.

Boosting global suppliers of fish

Worldwide demand for fish continues to rise at a fast pace. Western developed nations import around 33 million tonnes of fish a year. In Asia, demand is rising at a rapid pace: countries such as China have traditionally coveted fish, but had little access. Now with increasing wealth, Chinese fishing fleets are being sent out across the Pacific and into the Indian and Atlantic oceans.

The growing gap between falling wild fish stocks and rising human demand is being covered by farming fish, known as aquaculture.

However, normal fish have drawbacks when farmed. Crowd too many fish into pens and they respond by becoming stunted. Normal fish typically grow slowly. This slow growth slows even further in fish farming pens. Genetically modifying fish – often seen as key to increasing yields of farmed fish – has its drawbacks too, as farmed fish easily escape into the wild. It is seen as extremely undesirable to mix genetically modified fish genes with marine wild fish stocks.

One solution to increasing farmed fish yields and preventing cross contamination with marine wild fish stocks is triploidy. This involves genetically modifying the fish so that instead of carrying the normal double – or diploid – set of chromosomes, the fish instead carries three.

Triploid fish grow much faster, live longer, put on much more weight, don’t mind overcrowding, and do not become stunted. They also bring greater return on investment, as they have a much better food to weight conversion ratio. Triploid fish are also infertile.

Inducing triploidy in fish is cheap and simple – yet another benefit. Fish eggs are shocked into triploidy by sudden changes in either water pressure or temperature. The drawback of this technique is that it is not 100 per cent effective. Some diploid eggs will remain. Fish farmers therefore need a method to separate triploid eggs from the remaining diploid.

There are a number of ways of achieving this separation. But one of the fastest and most accurate methods to emerge uses laser scanning cytometry.

Cells are stained with a fluorescent dye, and then stream past a sensor. A laser fluoresces each cell as it passes. Triploid cells have roughly 1.5 times the amount of nuclear material as diploid cells. So by using a DNA-specific stain, triploid cells can be made to fluoresce at 1.5 times greater intensity than their diploid cousins.

Triploid identification by laser scanning cytometry is typically done with argon-ion lasers. But laser manufacturer Coherent believes that laser scanning cytometry is a natural fit for its low cost optically pumped semiconductor laser technology.

Beating food contamination

Biophotonics is making major inroads into the food sciences and testing for food safety. Recent worldwide food scares over milk and chocolate underline both the risk to health and the economic damage that poor food safety can pose, especially in a globalised food market.

Food analysts need to be able to detect a wider range of potential contaminants in less time, at higher volume, and with more accuracy. As a result, biophotonic testing is making inroads, with mass and near-infrared spectroscopy increasingly being adopted.

A number of vendors have produced laser-based food testing systems recently, including Bruker, Applied Biosystems, Agilent, Ocean Optics, Andor and Jobin Yvon Horiba. Detecting bacteria in chocolate is a major challenge. Cadbury is not alone in having to recall chocolate recently – almost all the major manufacturers have had to. Yet with conventional testing tools, detecting bacteria in chocolate is difficult. It can take a week using conventional methods to run the tests to detect contamination. Bruker have developed a benchtop system employing a 337nm nitrogen laser, called the Biotyper, that is designed specifically for this type of application.

Unlike conventional testing, Bruker’s BioTyper does not require any elaborate preparation. A sample is simply placed straight in the system, which provides an immediate analysis of what proteins are present within the food. By moving from conventional chemical analysis to a biophotonic application, manufacturers can test food during production and gain immediate feedback to its safety.

Biophotonics is creating breakthrough applications in a wide range of sciences. With protein and genome-based sciences extending their reach across medicine, pharmaceuticals, the life sciences, forensics, agriculture and the food sciences, biophotonics looks like continuing its rapid growth into a hundred-billion dollar industry by the end of the decade.

True-CW ultraviolet lasers

Optically pumped semiconductor laser (OPSL) technology is now a dominant technology in bioinstrumentation applications, because both the output power and wavelength are freely scalable throughout the visible. But, in the areas of flow cytometry and confocal microscopy, it is the OPSL’s ability to produce true-CW ultraviolet output in a low-noise yet costeffective platform that is now proving to be important.

An example of this type of laser is the recently-launched Genesis 355 series from Coherent, developed and tested in close-cooperation with bioinstrumentation suppliers. Coherent’s Matthias Schulze, the company’s director of marketing for OEM components and instrumentation, explains: ‘A growing number of bioinstrumentation applications require fluorescence excitation by an ultraviolet laser source. Standout examples are flow cytometry for embryo selection and cell sorting, as well as newer applications in confocal microscopy. Some of these applications have used mode-locked solid state lasers with output at 355nm. However, these quasi-CW lasers are not always an optimum solution for bioinstrumentation applications, because of their high peak power; for example, a 100mW laser with a pulse duration of 10ps can generate peak powers of more than 100W. Focusing this high power into live cells can potentially damage the DNA. In addition, it can cause non-linear optical effects and damage in the fibre optic delivery systems. To avoid this, we have developed a true-CW ultraviolet laser, based on its OPSL technology that produces tens of milliwatts at 355nm.’

Why not use the tried and tested frequency-tripled DPSS laser to produce 355nm for this market? Schulze explains: ‘Because it depends on peak power, frequency-tripling is much more efficient and easier to implement with pulsed lasers rather than CW designs. That’s why modelocked DPSS lasers have been used for bioinstrumentation in spite of their obvious peak power drawbacks. A CW DPSS laser can operate at 355nm using intra-cavity frequency tripling, but you have to address the notorious “green noise” problem. This is noise caused by rapid power fluctuations between different cavity modes, which affects the coherent frequency mixing/doubling processes. While green noise can be eliminated by using a stabilised single-mode cavity, OEM bioinstrumentation applications demand a lower-cost, simpler solution. Fortunately, because an OPSL essentially has a zero upper state lifetime, there is no stored gain and the laser modestructure is therefore completely stable. This eliminates green noise without resorting to a stabilised single-mode design.’