Multi-spot live-image autofocusing for high-throughput microscopy of fluorescently stained bacteria

Screening by automated high-throughput microscopy has become a valuable research tool. An essential component of such systems is the autonomous acquisition of focused images. Here we describe the implementation of a high-precision autofocus routine for imaging of fluorescently stained bacteria on a commercially available microscope. We integrate various concepts and strategies that together substantially enhance the performance of autonomous image acquisition. These are (i) nested focusing in brightfield and fluorescence illumination, (ii) autofocusing by continuous life-image acquisition during movement in z-direction rather than at distinct z-positions, (iii) assessment of the quality and topology of a field of view (FOV) by multi-spot focus measurements and (iv) acquisition of z-stacks and application of an extended depth of field algorithm to compensate for FOV unevenness. The freely provided program and documented source code allow ready adaptation of the here presented approach to various platforms and scientific questions

Development of a handheld fiber-optic probe-based raman imaging instrumentation: raman chemlighter

Raman systems based on handheld fiber-optic probes offer advantages in terms of smaller sizes and easier access to the measurement sites, which are favorable for biomedical and clinical applications in the complex environment. However, there are several common drawbacks of applying probes for many applications: (1) The fixed working distance requires the user to maintain a certain working distance to acquire higher Raman signals; (2) The single-point-measurement ability restricts realizing a mapping or scanning procedure; (3) Lack of real-time data processing and a straightforward co-registering method to link the Raman information with the respective measurement position. The thesis proposed and experimentally demonstrated various approaches to overcome these drawbacks. A handheld fiber-optic Raman probe with an autofocus unit was presented to overcome the problem arising from using fixed-focus lenses, by using a liquid lens as the objective lens, which allows dynamical adjustment of the focal length of the probe. An implementation of a computer vision-based positional tracking to co-register the regular Raman spectroscopic measurements with the spatial location enables fast recording of a Raman image from a large tissue sample by combining positional tracking of the laser spot through brightfield images. The visualization of the Raman image has been extended to augmented and mixed reality and combined with a 3D reconstruction method and projector-based visualization to offer an intuitive and easily understandable way of presenting the Raman image. All these advances are substantial and highly beneficial to further drive the clinical translation of Raman spectroscopy as potential image-guided instrumentation

Automatic Recognition of Light Microscope Pollen Images

This paper is a progress report on a project aimed at the realization of a low-cost, automatic, trainable system "AutoStage" for recognition and counting of pollen. Previous work on image feature selection and classification has been extended by design and integration of an XY stage to allow slides to be scanned, an auto focus system, and segmentation software. The results of a series of classification tests are reported, and verified by comparison with classification performance by expert palynologists. A number of technical issues are addressed, including pollen slide preparation and slide sampling protocols

Automated microscopic analysis of optical fibre transmission surfaces

Outlined in this thesis is the design of a prototype device for the inspection of optical fibre endfaces. The device designed uses lenses with different magnification’s to acquire scaled microscopic images of the endfaces for analysis purposes. The design specifications for the device are established based on the optical transmission requirements of the fibres and the impact of defects on transmission losses in various regions of the optical fibre endface. The specifications of this device are as follows: • Optical System o 3 lens automated changeover • Imaging System: o Minimum Resolvable Object Size of 2.43j.im o Maximum Field of View of 0.9mm o Resolution of 740 X 560 pixels • Autofocus System with Focus Resolution of 1.25pm • Coaxial Illumination System • 12Mbits/sec USB video acquisition hardware The device designed realises all the mechanical, optical and electronic functionality required to automate the inspection process of optical fibres. The hardware and software challenges involved in designing and building the prototype are discussed in detail in the thesis. A complete evaluation of the design is also carried out, difficulties and problems that occurred with the project are analysed, and recommendations for the improvement of the design are made