S. Poland

Adaptive optics for enhanced signal in CARS microscopy.

We report the use of adaptive optics with coherent anti-Stokes Raman scattering (CARS) microscopy for label-free deep tissue imaging based on molecular vibrational spectroscopy. The setup employs a deformable membrane mirror and a random search optimization algorithm to improve signal intensity and image quality at large sample depths. We demonstrate the ability to correct for both system and sample-induced aberrations in test samples as well as in muscle tissue in order to enhance the CARS signal. The combined system and sample-induced aberration correction increased the signal by an average factor of approximately 3x for the test samples at a depth of 700 microm and approximately 6x for muscle tissue at a depth of 260 microm. The enhanced signal and higher penetration depth offered by adaptive optics will augment CARS microscopy as an in vivo and in situ biomedical imaging modality.

Adaptive optics for deeper imaging of biological samples.

Optical microscopy has been a cornerstone of life science investigations since its first practical application around 400 years ago with the goal being subcellular resolution, three-dimensional images, at depth, in living samples. Nonlinear microscopy brought this dream a step closer, but as one images more deeply the material through which you image can greatly distort the view. By using optical devices, originally developed for astronomy, whose optical properties can be changed in real time, active compensation for sample-induced aberrations is possible. Submicron resolution images are now routinely recorded from depths over 1mm into tissue. Such active optical elements can also be used to keep conventional microscopes, both confocal and widefield, in optimal alignment.

Impact of wavefront distortion and scattering on 2-photon microscopy in mammalian brain tissue.

Two-photon (2P) microscopy is widely used in neuroscience, but the optical properties of brain tissue are poorly understood. We have investigated the effect of brain tissue on the 2P point spread function (PSF2P) by imaging fluorescent beads through living cortical slices. By combining this with measurements of the mean free path of the excitation light, adaptive optics and vector-based modeling that includes phase modulation and scattering, we show that tissue-induced wavefront distortions are the main determinant of enlargement and distortion of the PSF2P at intermediate imaging depths. Furthermore, they generate surrounding lobes that contain more than half of the 2P excitation. These effects reduce the resolution of fine structures and contrast and they, together with scattering, limit 2P excitation. Our results disentangle the contributions of scattering and wavefront distortion in shaping the cortical PSF2P, thereby providing a basis for improved 2P microscopy.

Evaluation of fitness parameters used in an iterative approach to aberration correction in optical sectioning microscopy

A major problem when imaging at depth within a biological sample in confocal or nonlinear microscopy is the introduction of sample induced aberrations. Adaptive optical systems can provide a technique to compensate for these sample aberrations and often iterative optimizations are used to improve on a particular parameter of the image (known as the fitness parameter). In this investigation, using a deformable membrane mirror as the adaptive optic element, we examine the effectiveness of a number of fitness parameters, when used with a genetic algorithm, at determining the optimal mirror shape required to compensate for sample induced aberrations. These fitness parameters are compared in terms of the number of mirror changes required to achieve optimization and the final axial resolution of the optical system. The effect that optimizing each fitness parameter has on the lateral and axial point-spread function is also examined.

Dynamic closed-loop system for focus tracking using a spatial light modulator and a deformable membrane mirror

A dynamic closed-loop method for focus tracking using a spatial light modulator and a deformable membrane mirror within a confocal microscope is described. We report that it is possible to track defocus over a distance of up to 80 microm with an RMS precision of 57 nm. For demonstration purposes we concentrate on defocus, although in principle the method applies to any wavefront shape or aberration that can be successfully reproduced by the deformable membrane mirror and spatial light modulator, for example, spherical aberration.

Search-based active optic systems for aberration correction in time-independent applications

We describe a protocol for the use of a control feedback loop incorporating an iterative optimization routine for a range of time-independent adaptive optics applications. These applications are characterized by the quasi steady state of the aberrative effects (>0.1 s) and contrast, for instance, to astronomical applications where the aberrations constantly vary at frequencies above 10 Hz. For optimal performance in such time-independent applications, the control systems typically require specialized tailoring. A typical example of two different types of time-independent adaptive optics applications--an adaptive optic microscope and an adaptive optic laser platform--are detailed and compared. It is shown that implementing a number of minor, but crucial, application-specific modifications to the control system results in an improved efficiency of an already extremely successful technique for aberration compensation. We present a description of the crucial parameters to consider in a search-based adaptive optics system.

Fast wavelength multiplexing of a white-light supercontinuum using a digital micromirror device for improved three-dimensional fluorescence microscopy

We report on the use of a computer-controlled digital micromirror device (DMD) to select discrete wavelength ranges from a white-light supercontinuum and the application of the light source to confocal laser scanning microscopy (CLSM). The fast switching rate of the DMD enabled video-rate wavelength tuning, while the high resolution allowed precise control over the bandwidth selection. The DMD system was used in conjunction with a CLSM system to characterize the wavelength switching capability, prior to performing three-dimensional CLSM of a variety of multiple-labeled samples in order to demonstrate the efficacy and practical application of the system.

Mask-less ultraviolet photolithography based on CMOS-driven micro-pixel light emitting diodes

We report on an approach to ultraviolet (UV) photolithography and direct writing where both the exposure pattern and dose are determined by a complementary metal oxide semiconductor (CMOS) controlled micro-pixellated light emitting diode array. The 370 nm UV light from a demonstrator 8 x 8 gallium nitride micro-pixel LED is projected onto photoresist covered substrates using two back-to-back microscope objectives, allowing controlled demagnification. In the present setup, the system is capable of delivering up to 8.8 W/cm2 per imaged pixel in circular spots of diameter approximately 8 microm. We show example structures written in positive as well as in negative photoresist.

Development of fibre-optic confocal microscopy for detection and diagnosis of dental caries

We report on the development of a fibre-optics-based confocal imaging system for the detection and potential diagnosis of early dental caries. A novel optical instrument, capable of recording axial profiles through caries lesions using single-mode optical fibres, has been developed. The practical study illustrates that miniature confocal devices based around single-mode optical fibres may provide additional diagnostic information for the general dental practitioner.

5B-2 3D Imaging of Teeth Using High Frequency Ultrasound

It was shown in the late 1960s that the internal structures of teeth could be investigated using ultrasonic pulse echo techniques with 4 MHz contact probes. However, the low frequency limited the resolution of the system and therefore the thickness of dental structures which could be observed. More recent reports have increased frequencies into the region of 10 to 20 MHz. With such frequencies, the resolution in enamel is improved to 0.5 mm. However, the average thickness of enamel in human teeth is around 1.5 mm, implying that even the improved resolution is still inadequate for detailed images and diagnosis. As well as the considerations about the resolution of the system, it has been shown that the attenuation and losses due to acoustic boundaries in tooth structures are detrimental to image reconstruction, with potentially useful information lost or degraded. Therefore, it is essential to have maximum energy transfer into, and back out from, the tooth. The work presented here introduces a novel high frequency focused ultrasound transducer operating at 35 MHz. In order to avoid the natural complexities of the human tooth in the experiment, human incisors were prepared so that only one layer of enamel and dentine were present. The sample was then immersed in distilled water on a translation stage and an x-y raster ultrasound scan was performed. A number of signal processing algorithms were applied to the raw data including correction of distortion and position via correlation and high and low bandpass filtering. The image processing application IMAGEJ was then used to reconstruct a 3D representation and rotation of the processed dataset. The individual A-scans which in turn create the B-scans and 3D images are of a much higher resolution in both the temporal and spatial domain than previously published. The 35 MHz operating frequency gives a resolution of 0.19 mm in the enamel layer, which is at a useful level for the detection of dental caries and more specifically acid erosion. The high frequency also produces a spotsize of 110 mum which allows for accurate localisation in the individual A-scans. The results are believed to be the first known 3D high resolution ultrasound images of the enamel-dentine junction.