Travel Grant for Adaptive Optics in CARS Microscopy

Optical microscopy has provided scientists, and clinicians, with the ability to image living tissue with sub-cellular resolution for around four hundred years. This ability has lead to a number of scientific breakthroughs both in the life and physical sciences. Significant advances in general optics have frequently been achieved by the desire to image samples with increasing resolution so that the finest structures can be resolved. Indeed just over 130 years ago Abbe advanced the study of optics with his work on improved optical microscopy with the introduction of diffraction theory and the study of optical aberrations. However, crucial to all forms of microscopy is the ability to have contrast between the different structures present within the sample. For most of the last fifty years this has been provided by the addition of fluorescent labels to the sample culminating in the use of genetically modified samples so that they naturally express a fluorescent label. Even in this case the sample is not in its natural state and thus the method is not practical for a number of samples, and makes the application of microscopy to living tissue difficult, and impossible for many clinical applications.Coherent Anti-Stokes Raman Scattering (CARS) microscopy is a method by which optical microscopy can be undertaken using the spectral fingerprint of a specific molecule as the contrast agent. By tuning two laser sources to the correct wavelengths specific molecular spectral features can be excited enabling highly specific microscopy. The process is non-linear and hence the imaging volume is restricted in three dimensions offering the ability of the microscope to naturally generate high resolution optical slices which can be reconstructed to produce three dimensional images.However, as one wishes to image more deeply into a sample the laser light has to pass through the sample and is aberrated leading to a loss of signal and spatial resolution at depths much beyond 100microns. This problem is similar to that faced by ground based astronomers examining space through the atmosphere. Through the use of active optical elements (adaptive optics) they have demonstrated that the atmospheric aberrations can be removed, or at least minimised. Previously we have adopted the same technology to multiphoton and confocal microscopy with considerable success; however, both of these imaging modalities require the addition of fluorescent compounds to the sample in some form thus perturbing the natural state of the tissue. The aim of this project is to explore the use of adaptive optics (AO) in CARS microscopy by linking, for two weeks, world leading expertise in both the application of adaptive optics to microscopy and CARS imaging. By fitting the Institute of Photonics AO system to the Harvard CARS microscope we will rapidly be able to determine if AO has a role to play in CARS microscopy as is strongly suspected. This proposal thus offers a rapid, low cost route to the evaluation of AO for in-depth CARS microscopy.