How acousto-optical tunable filters enhance the versatility of confocal microscopy

Confocal microscopy, also known as confocal laser scanning microscopy (CLSM), has been applied across life sciences for decades. From ophthalmology to neuroscience, confocal microscopy supports life-saving diagnostics, treatments and research.

Today, biomedical applications for confocal microscopy increasingly rely on acousto-optical tunable filters (AOTFs). There are good reasons for this. The precise control, agility and speed created by AOTF technology enhances the versatility of confocal microscopy, which, in turn, enables further scientific innovation. As the demand for greater image clarity and flexibility increases, AOTF solutions are likely to become more complex, requiring particular integrated expertise and capabilities.

Uses and benefits of AOTF in confocal microscopy

Acousto-optic (AO) components have many uses in a confocal microscope. Find them in modulation, power control, laser switching, laser coupling and beamsplitting. Within a single scan head, such as Leica Microsystems’s STELLARIS 8, AO is used extensively.
In particular, AOTFs are a star performer, delivering multiple benefits, including:

Sharper images

One challenge in examining living tissue is acquiring multispectral data fast enough without specimen movement or molecular change or 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).

Pixel-by-pixel wavelength and power control

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 and laser. One use of an AOTF is to select the excitation wavelength and set the power of white light lasers.
Advanced laser multiplexing schedules
An AOTF controls the wavelength and intensity by selecting the laser source in the system and controlling its intensity. The acousto-optic allows for rapid and precise control of transmission and wavelength selection. Then used as an excitation filter, the AOTF, can be adjusted ‘on the fly’. This is in contrast to traditional dielectric band pass filter where any adjustments mean that a new filter needs to be purchased. Also, there is always a limitation on how many filers that can be mounted in the microscope.

Environmental stability

AOTFs in confocal systems eliminate any potential frequency drift caused by temperature or humidity changes by enabling agile, rapid electronic tuning and intensity control of multiple laser lines. This is much harder to achieve with mechanical tuning scheme using filter turrets/wheels.

Technology and specifications

In an AOTF, a RF drive frequency is applied to a piezoelectric transducer, typically lithium niobate, which generates an acoustic wave. This 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 un-diffracted beam. The crystal geometry is critical to obtain the needed performance.

Most high-end AO devices are made to specification and G&H is a leading specialist offering a wide range of AO tunable filters covering wavelengths from the UV through mid-IR, with bandwidth of less than 1 nm. G&H’S acousto-optic tunable systems includes an electronic control, configurable drivers for improving operator flexibility and feedback stabilizing systems to maintain wavelength stability whatever the environmental conditions. G&H has also implemented a patented side lobe suppression technology to improve the spectral purity.

Crucially, G&H is the only optical systems developer to grow its own superior quality tellurium dioxide (TeO2) crystals. This helps to maintain the consistency and reliability, resulting in more consistent and repeatable AOTF products. This ability to grow, polish and fabricate crystals at one of our US facilities (with ITAR compliance) ensures industry-leading standards.

Next generation drivers

For OEM customers and end users, feedback on the direction of travel for confocal microscopy indicates that versatility is a key requirement. Demand is growing for a wider range of tunable wavelengths from within a single system, offering flexibility, as well as better value. G&H already provides a single source to cover the range 400-2400 nm (rather than the three filters usually needed) and is implementing further advances in driver flexibility.

Management of temperature effects is another area for further innovation. AOTFs are very sensitive to temperature changes. To overcome this, G&H driver designs are based on a chip that maintains the temperature and then adjusts the output to keep it constant using a feedback system, a process called wavelength locking. This integrated system also contains accessible information about crystal structure, serial number and so on.

To maximize the potential of an AOTF systems and their benefits to users, G&H collaborates with microscopy system manufacturers at the very start of the process of designing their own next generation microscopes. This collaborative approach enables manufacturers to improve the performance of their microscopes and supercontinuum sources.

To summarize, experience has demonstrated the advantages of adopting an evolutionary rather than revolutionary approach to superior AOTF and driver system manufacture and integration. G&H works in tandem with the microscopy industry to improve AOTF technology to keep pace with the fast-moving advancements in life science and biophotonics application.

Lars Sandström is Vice President, Life Sciences at G&H.