Life through a lens

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Greg Blackman on the importance of optics for those working in the field of life sciences, from microscopy to measuring fluorescence with ultrafast spectroscopy

Modern microscopes are highly advanced pieces of equipment allowing scientists to probe deeper into tissue and resolve ever finer structures. Super resolution microscopy techniques, such as photoactivated localisation microscopy (PALM) or stimulated emission depletion microscopy (STED), are the current state-of-the-art, providing higher resolution than standard confocal microscopes. Confocal microscopes are typically diffraction limited to around half the wavelength of the illumination source.

While the technology surrounding microscopy continues to be refined at the cutting-edge, the challenges facing those building lower-spec microscopes with a limited budget remain considerable. Researchers at the Universidad Peruana Cayetano Heredia (UPCH) and the Universidad Nacional de Ingeniería in Peru, led by Dr Mirko Zimic, have developed a low-cost inverted microscope from stock optical components. The team hope to improve the diagnosis of endemic diseases in poverty-stricken areas with the device. Dr Zimic and his team are currently trialling one of the prototype systems at a health centre in Trujillo in the north of Peru for diagnosing tuberculosis.

The researchers built the system from optical components from Edmund Optics (EO); the work won first place in Edmund Optics’ 2011 higher education grants, with the team receiving $10,000 worth of EO products.

‘The project began with tuberculosis, although it can be expanded to other diseases,’ explains Dr Zimic. ‘This is not a state-of-the-art system, but a simple microscope comprised of affordable standard optical elements. It doesn’t have tremendous capacity, but has enough functionality for TB diagnosis. We hope the microscope will reduce diagnosis of multi-drug-resistant TB in Peru from 10 months to seven days.’

Tuberculosis is a major public health problem in Peru and most developing countries. Early diagnosis of the disease is not common in Peru and multi-drug-resistant strains take even longer to diagnose. In 2000, Dr Gilman’s tuberculosis laboratory at UPCH developed a method to culture TB in a liquid rather than a solid medium (microscopic observation drug susceptibility). This allows TB to be diagnosed in seven days directly from a sputum sample plus the determination of multi-drug resistance. However, the diagnostic test requires an inverted microscope, which is not a cheap piece of equipment and not many laboratories can afford it, especially not Peru’s ministry of health. The MODS reading also requires skilled technicians to interpret the patterns and correctly classify TB. Both of these reasons have meant it’s not been feasible to implement this equipment to any large degree in Peru, although this method is now being used in other countries including Singapore, Thailand, South Africa, Bangladesh, Ecuador and Bolivia.

‘We wanted to find a way of replacing a costly microscope as well as the skilled technician to make the diagnosis,’ states Dr Zimic. In Peru, most newly-diagnosed patients are automatically put on a 10-month regime of first-line empirical drugs. If the patient doesn’t respond to the treatment, they are declared to have a multi-drug-resistant strain and put on a course of second-line drugs. During this time the patient remains ill and can transmit the disease.

Dr Zimic’s team took a two-pronged approach to the problem: firstly, building an inexpensive digital microscope and secondly, developing a mathematical algorithm to automatically identify the Mycobacterium tuberculosis in a digital microscope image. The prototype microscope used optical components from Edmund Optics, including stages, lenses and a 45° mirror to deflect the light beam at 90°. The system was designed to be used for both manual readings and to capture digital images of the slides, which can be sent for automated analysis at the UPCH-Bioinformatics laboratory servers with the algorithm. The algorithm takes 15 seconds to run on a standard PC and is 99.4 per cent sensitive and 99.7 per cent specific, according to Dr Zimic.

‘We proved that there was no significant difference between results from our system and a $10,000 Nikon inverted microscope,’ Dr Zimic comments. After the seven-day culture period, the plate can be read under the microscope, the image from which is processed by the automated algorithm at the University servers to make the analysis. ‘The physician will know in seven days, with high accuracy, if the patient has TB and simultaneously if that patient is multi-drug resistant,’ he continues. ‘This test cuts out the 10-month empirical treatment of TB sufferers with first-line drugs by determining multi-drug-resistant strains initially, which means treatment can immediately commence with second-line drugs.’

The microscope can be built for as little as $400-500 using a simple dichroic lamp as the illumination source. Nikon or Olympus inverted microscopes cost from $8,000 upwards.

The components from Edmund Optics were used to demonstrate the proof-of-concept system. ‘We bought different sets of lenses and stages from Edmund to design a microscope fit for this purpose, using the most efficient and cost-effective combination of optical components. We could economise on the design further by removing the 45° mirror, but we wanted to have the dual ability to look through the eyepiece as well as capturing digital images of the slide,’ Dr Zimic says.

‘The Peruvian health system pays for TB treatment and all the indirect costs associated. If we can reduce the time of TB diagnosis and determination of multi-drug resistance from 10 months to one week, we will have a very important impact on the prevalence of the disease,’ he concludes.

Although this project began with TB, Dr Zimic explains that the same principle can be applied to any disease that could be diagnosed with pattern recognition on digital images. ‘We’ve developed other image recognition algorithms for intestinal parasites, malaria, and are currently working on cervical cancer to create automatic Papanicolau-smear slides,’ he says.

Cervical cancer is a common form of cancer in women and, especially in rural areas in developing countries, diagnostic equipment is not available. Therefore, slides have to be shipped to designated laboratories, which takes time. Women might have to wait 4-5 months after a sample was taken before receiving the diagnostic results.

‘We want to do exactly the same thing [with cervical cancer] as we’re doing with TB,’ states Dr Zimic, ‘to bring the diagnostics of smears directly to the health centres. We’re currently developing the optical system and algorithm, in collaboration with the University of Washington in Seattle, for screening samples to reduce the number sent to pathology labs. This would help reduce the time taken for a complete diagnosis.’

Ultrafast spectroscopy

Leaving the topic of microscopy for the moment and moving onto spectroscopy, researchers at NTT Photonics in Japan, which conducts photonics R&D mainly for telecommunications, have developed an optical beam scanner that’s used in a high-speed spectrometer. The spectrometer’s rapid scanning properties make it ideal for observing ultrafast luminous phenomena in bioanalytical science, among other potential applications.

The scanner-based spectrometer, fabricated from potassium tantalate niobate (KTN), can measure a wide optical spectrum in microsecond-order timescales. AMS Technologies, based in Martinsried, Germany, will supply the KTN spectrometer, the technology surrounding which, as Dr Torsten Ledig, sales manager at AMS, explains is still under development.

NTT Photonics based its scanner on a KTN crystal, a nonlinear crystal that can be used to deflect light beams from point to point at high frequency (megahertz) modulation. ‘The scanner operates at faster scanning frequencies compared to commercially available spectrometers,’ states Dr Ledig. ‘Solid-state spectrometers using CCD lines cannot make measurements over these timescales. Likewise, spectrometers using a slit and rotating the prism mechanically would also be much slower.’

With a standard spectrometer, the incident beam travels through a prism or diffraction grating to split the light into different wavelengths. The dispersed light then passes through a slit and the wavelength-dependent signal is measured, typically either with a CCD line or a photomultiplier detector. This, however, is not very fast. NTT looked to develop an optic that would scan the beam in front of the dispersion element to achieve a different angle of incidence on the prism over time. ‘The slit and detector are in the same position and by scanning the incident light in front of the prism, the result is a wavelength-dependent signal detected over time,’ explains Dr Ledig.

The system developed by NTT is only a proof-of-principle device at the moment, according to Dr Ledig. So far, laser beams at specific wavelengths have been used as a light source to test the device, whereas, in reality, the spectrometer would be used to measure a fluorescence signal.

Fluorescence studies

In the life science arena, fluorescence is one of the major tools scientists have at their disposal. Fluorescence provides a means of viewing proteins and cellular components; the expression of a fluorescently labelled protein, for instance, can be seen and tracked over time.

When excited by one wavelength of light, fluorescent proteins, such as GFP, emit photons at a longer wavelength. ‘Fluorescence microscopes have to be able to separate the very intense excitation light from the very faint emitted light,’ explains Nicolas George, director of product marketing at US optics manufacturer Semrock. In modern microscopes, the excitation and emission light pass through part of the same optical system and are separated by a dichroic mirror. The mirror reflects the shorter excitation wavelengths coming back through the system and transmits only the longer emission signal.

Semrock, the sister company of CVI Melles Griot, both of which are owned by Idex Corporation, supplies excitation filters, emission filters and dichroic mirrors used in fluorescence microscopes. The filters have very high blocking (optical density of six or seven) in all regions of the spectrum apart from a narrow band (approx ±10nm) around the wavelength of interest. The dichroic mirror has to have high reflectivity in the shorter wavelengths and then switch to high transmission in the longer wavelengths – the transmission profile is like a step function.

Semrock supplies hard-coated filters for fluorescence microscopes. These are fabricated using ion-beam sputtering to deposit metal oxides on the glass. The technique produces a very dense and uniform coating. ‘Soft-coated filters are less expensive but have to be replaced over time,’ comments George. ‘The hard-coated filters can be cleaned with acetone and are much more robust to changes in humidity.’

Fluorescence is relatively inefficient; the protein will fluoresce in all directions but only that light passing through the eyepiece will be detected. Also, microscope optics will have some losses. ‘Because the process is relatively inefficient, a powerful laser light source is used to illuminate the sample,’ explains George. ‘Therefore, there’s a lot of excitation light contributing to the background noise that needs to be attenuated by the optics. Hard-coated filters are particularly advantageous as they are robust enough to handle the intense laser light sources.’

The latest technology surrounding microscopy mean the optics have to work even harder. Super resolution microscopy techniques like PALM and STORM take advantage of photoactivation at different wavelengths to activate and deactivate different subsets of labelled proteins. ‘Traditionally, the dichroic mirror would only have to contend with a single laser wavelength,’ explains Jim Passalugo, product manager at Semrock, ‘whereas, with these techniques, the dichroic beam splitter has to have wider transmission and reflection bands to accommodate the various different excitation and emission wavelengths. This can be challenging when you want to maintain all the other properties of the mirror.’

Another big market for photonics in life sciences, according to Dr Matthias Schulze, director of marketing at Coherent, is flow cytometry, used in clinical applications such as testing blood samples of HIV sufferers. Flow cytometry requires a non-circular beam overlap to make the counts and beam shaping optics, such as a Powell lens, are often used to translate a Gaussian beam profile into a line or custom profile for flow cytometry equipment. Coherent provides beam shaping optics for flow cytometry applications.

‘The market is asking for a laser with integrated beam shaping optics,’ Schulze says. ‘By incorporating the beam shaping capabilities into the optics for the laser, instrument builders no longer have to do this themselves. The life science instrument community always wants to simplify complex subsystems from an engineering perspective.’

Coherent has also released the OBIS family of lasers, combining laser diode and OPSL solutions in the same package. OBIS lasers are suitable for flow cytometry, confocal microscopy, and DNA sequencing, among other applications.

Schulze notes that DNA sequencing for patient care and personalised medicine is another important area for photonics within life sciences. ‘Currently, the growth rate in DNA sequencing has been slow due to a variety of considerations surrounding the technology, such as data handling, but this could be very important market for photonics in the future,’ he says. ‘If disease treatment could be improved simply by sequencing a patient’s DNA, that will open the door to massive growth.’