Confocal Microscopy Evolves with AO Tunable Filters

Mark Stevens, Director of Business Development Life Sciences, 13 September 2016
Mark Stevens G&H Director of Business Development Life and Health Sciences
Mark Stevens, Director of Business Development Life and Health Sciences

Biomedical applications for confocal microscopy such as in vivo inspection increasingly rely on acousto-optical tunable filter (AOTF) technology. AOTFs increasingly replacing conventional and cumbersome mechanical filtering techniques. The advantages of the acousto-optic tunable systems, include: electronic control, configurable drivers improving operator flexibility, feedback stabilizing systems to maintain wavelength stability whatever the environmental conditions, and our custom-grown tellurium dioxide (TeO2) crystals to ensure optimal performance.

Acousto-Optic Tunable Filters in a Confocal MicroscopeAn AOTF controls both the wavelength and intensity of multiple lasers electronically and gives the user precise control of transmission by making extremely rapid adjustments to the parameters of the diffracted beam. In this way, microscopists can maintain a high scan rate and, at the same time, adjust the image on a pixel-by-pixel basis. The balancing of different signal levels is achieved through assigning different intensities to each wavelength.

Conventional mechanical tuning is invariably subject to environmental factors such as heat and humidity, which change device dynamics over time. Traditional confocal systems utilize interference filter turrets and wheels; these filters have many limiting characteristics. Each filter has a fixed central wavelength and passband. Several filters are needed to provide monochromatic imaging and the filter wheels are subject to vibration upon rotation and relatively slow switching speeds. Utilizing AOTFs in confocal systems eliminates any potential frequency drift caused by temperature or humidity changes by enabling agile, rapid electronic tuning and intensity control of multiple laser lines..

In an AOTF, a RF drive frequency is applied to a piezoelectric platelet, typically lithium niobate, which generates an acoustic wave, which is coupled into an acousto-optic material such as tellurium dioxide (TeO2). This creates a diffraction grating in which the refractive index of the crystal varies with drive frequency. As a coherent optical beam passes through the crystal, only a narrow band of frequencies will meet the phase-matching condition and exit the crystal at an angle that differs from the undiffracted beam.

Sharper Images with Acousto-Optical Tunable Filters

One challenge in examining living tissue is acquiring multispectral data fast enough without specimen movement or molecular change (damage). The versatility of AOTFs allows living cells to be analyzed. This means that scientists can monitor dynamic cellular process thanks to the rapid intensity and wavelength switching capabilities allowed by an AOTF-based system.

Researchers can now accurately monitor the complete dynamic cellular process due to rapid intensity and wavelength switching capabilities. There has been great progress with techniques such as fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and small user defined specimen areas (ROI regions of interest). The precise control, agility and speed created by AOTFs further advances confocal microscopy and provides the necessary tools for scientific advancement.

G&H offers a wide range of AO tunable filters covering wavelengths from the UV through mid-IR, with resolutions of less than 1 nm. Our acousto-optic tunable filters are manufactured using high quality TeOcrystals grown in-house, polished and fabricated to rigorous standards. As we are the only optical systems developer to grow our own TeO crystals, this helps us to maintain the consistency and reliability of our AOTFs. By this means, our product quality tends to be more consistent and repeatable; we hear from our customers that we have the edge in terms of repeatability of performance and device consistency.

Next-Generation Drivers

What is clear from the feedback we get from industry is that customers want greater value for their money and they want a wider range of tunable wavelengths from a single system. We are constantly working to meet these needs, such providing a single source to cover the range 400-2400 nm (rather than the three filters that range currently needs). Furthermore, we are also working on another advantageous approach: increased driver flexibility.

To maximize the potential of an AOTF systems and their benefits to users, it is important for the supplier to collaborate with the microscopy system manufacturer while they are in the process of designing their own next–generation microscopes. We’ve seen the advantage this collaborative approach brings to product development; improving our range of drivers and AOTF integration while simultaneously helping manufacturers to improve the performance of their microscopes and supercontinuum sources.

Another area where our approach to refining our AOTFs is boosting performance is in the management of temperature effects. AOTFs are very sensitive to temperature changes. The designs we are using with our new drivers are based on a chip that maintains the temperature and then adjusts the output to keep it constant using a feedback system, a process we call wavelength locking. This integrated system also contains accessible information about crystal structure, serial number and so on.

To summarize, experience has demonstrated the advantages of adopting an evolutionary rather than revolutionary approach to superior AOTF and driver system manufacture and integration. We work in tandem with our clients to improve the performance of both our components and their end products.

G&H and Acousto-Optics

G&H has been manufacturing acousto-optic devices and drivers for nearly 40 years with more than 100 engineers on staff. G&H has invested in vertical integration of the design and manufacturing of our entire acousto-optic product line, including crystal growth of lithium niobate and tellurium dioxide, crystal orientation, cutting and shaping, polishing, anti-reflective coating, transducer bonding, and packaging. Our AOTFs are manufactured internationally, with plants in Fremont, California and Ilminster, UK.

If you’d like further information on our AOTF technology, please contact Mark Stevens.