Fourth Finnish Medical Physics and Medical Engineering Day
Abstracts of the Awarded Master's Graduate Theses

Sanna Malinen, Helsinki University of Technology
Cardiac-Triggered fMRI: Application to Study Somatotopic Organization in the Human Brain

Functional magnetic resonance imaging (fMRI) enables the studies of both the human cortical and subcortical brain areas. However, accurate functional imaging of thalamus may be impaired in deep brain structures because of inadequate spatial resolution of functional MR images or cardiac-cycle related movement of the brain tissue.

The aim of this study was to find somatotopic organization in the human thalamus and in the secondary somatosensory cortex. To eliminate the effect of cardiac-related pulsations in the deep brain areas, cardiac-triggered fMRI was utilized so that the functional images were collected always at the same phase of the cardiac cycle. The drawback of heart-rate synchronized image acquisition is that it can lead to varying levels of blood-oxygen-level-dependent signal saturation. Therefore, images were corrected with two different methods using voxel-based approach.

Ten healthy subjects underwent an fMRI experiment, where tactile stimuli were delivered to their lower lip, fingers and toes. The heart-rate synchronization was performed using pulse-oximeter. The fMRI data were preprocessed and analysed using SPM2 software and Matlab version 6.1. The statistical significance of activations was determined by Student's t-test. The results from cardiac-triggered image acquisition were compared with results from conventional fMRI.

Group analysis (n = 10, threshold p < 0.01 and extent threshold of four voxels) of the cardiac-triggered data with post-acquisition signal correction indicated somatotopic organization in the human thalamus. With conventional imaging only response for finger stimulation was seen. In single subject analysis, more subjects showed thalamic activations with cardiac-triggered imaging than with non-triggered. The reference somatotopic organization was found in SII (threshold p < 0.001 and extent of 20 voxels), with activation pattern consistent with previous fMRI studies.

To evaluate the effect of cardiac-triggering and the related signal corrections, the pre-processed signals of cardiac-triggered series (seven subjects showing both cortical and thalamic responses) were compared with conventional imaging. The regions of interest (ROIs) were spheres of 5-mm radius, chosen from finger representation sites. The sizes of the thalamic BOLD responses (measured as percentage of the signal change) did not differ between cardiac-triggered vs. non-triggered conditions, but the variability of the signal (measured as standard deviation) decreased both in thalamus and SII during cardiac triggering. This decrease explains the better detection of activations in the cardiac-triggered series.

Julia Mednichihina, University of Oulu
Physical Characterization of New Bio-Optical Materials

Current methods for recognizing biological molecules have limitations regarding speed, accuracy and sensitivity of the measurements. Methods with good sensitivity are often slow and complicated to perform and require strict laboratory conditions and skilled personnel. Label-free methods that are in use today are faster than the most conventional recognition techniques but are generally expensive and require bulky and high-priced equipment.

To be able to detect small biological signals, high resolution of measurement set-up as well as good optical and physical properties of sensitive element of the biosensor are crucial. Solgel materials are widely used in biosensor technology as an excellent platform for the biological interactions. Important physical properties of these hybrid materials are their thermal stability, good electromagnetic properties in the visible wavelength area, scratch and bend resistance.

Aim of the work was to characterize the use of the porous solgel material (HybridGlass™) as biologically selective surface in biologically friendly temperatures. Sensitive element was created using porous HybridGlass™ materials and spin-coating technique. Stabilization series were performed with three material versions before biological experiments in order to observe changes in thin-film properties during long-term thermal processing. Biological experiments were performed on stabilized thin-films using streptavidin-biotin–bridge to constrain a biological recognition complex. Human Leptin and IGFBP-3 were used as target molecules. Specific and non-specific binding of the target proteins to the sensitive layer of the biosensor were tested using Metricon prism coupler in clean-room laboratories to measure refractive index and thickness of the thin-films.

In heat stabilization series complete stability phase was reached after 24 hours of 50°C incubation. Average total 5mRIU refractive index shift over this period was observed. In biological experiment series, samples with streptavidin immobilization through surface application gave the best ratio of the specific and non-specific binding of the protein. Samples with streptavidin had positive effect on antibody binding. Plain water did not substantially affect the thin-film properties.

Antti Nikkanen, Tampere University of Tecnology
A New Method for Determination of Fluorescence Lifetime

The goal of this thesis was to design and realize a measurement system for determination of fluorescence lifetime. The measurement system was realized with programmable logic by using only one programmable FPGA chip. High resolution was obtained by multiplying and phase shifting the clock frequency. Short time interval measurement system realized in this way has not been published earlier in scientific literature.

Functioning of the measurement system is based on the measurement of the short time interval between two pulses, start and stop. Measurement starts with an excitation pulse which excites the sample and activates the time counter. Counter stops when a photon emitted from the sample is detected. Excitation is done by a laser and a photomultiplier tube is used for detection of emission. Measurement can also be done vice versa in the way that emission pulse is used to activate the counter and time is measured until the next laser pulse. When measurement is reproduced several times a probability histogram of photon counts versus time is built up. From this graph one can determine the lifetime of fluorescence.

Fluorescence lifetime is rarely used as a sole measurement parameter in fluorescence measurements. Most often measurement of the lifetime is used as an extra parameter to produce certain accuracy or extra information to measurements. Real time measurements often reveal information which can not be obtained from steady state measurements. Lifetime is effected by several factors as binding reactions between molecules, change in orientations and relative locations and different kind of changes in molecule and its immediate surroundings. Thus defining the lifetime and observing the changes in it gives us important information about the sample and its surroundings of interest.

On the basis of the measurements we can find out that it is possible to specify the lifetime of fluorescence by designed measurement system and the results are totally comparable with expensive commercial measurement system. In addition to the specification of the fluorescence lifetime the measurement system can also be used for other short time interval measurement demanding applications.

Mikko Hakulinen, University of Kuopio
Ability of Ultrasound to Predict Mechanical and Structural Properties of Trabecular Bone

Osteoporosis is major public health problem and it causes significant burden to the society. The only widely accepted clinical technique for assessment of fracture risk is DXA (Dual energe X-ray Absorbtimetry) –method. During last decade, quantitative ultrasound (QUS) techniques are introduced as an alternative method for osteoporosis diagnostics. Ultrasound devices are portable, relatively cheap and they do not involve ionizing radiation. At the moment two QUS parameters, i.e. broadband ultrasound attenuation (BUA) and speed of sound (SOS) are commonly used in clinic. In this study, ability of ultrasound backscattering (Broadband Ultrasound Backscatter, BUB) and reflection (Integrated Reflection Coefficient, IRC) to predict density and mechanical properties of bovine trabecular bone (n = 41) was investigated. In addition, relationship between commonly determined QUS parameters (BUA,SOS) and scattering parameters (BUB,IRC) was determined. Mechanical properties (ultimate strength, Young’s modulus, resilience) were determined by applying destructive test. Bone mineral densities (BMD) of the samples were measured with DXA- technique.

Reproducibilities (standardized CV%) of BUB (3.5%) and IRC (1.5%) were comparable to those of nBUA (2.3%) and SOS (0.5%). BUB showed moderate correlation with the volumetric BMD (r = 0.61, p < 0.01), Young’s modulus (r = 0.40, p < 0.01) and ultimate strength (r = 0.40, p < 0.01). Moreover, IRC demonstrated strong correlation with volumetric BMD (r = 0.92, p < 0.01) and with the most of the mechanical parameters (0.81 < r < 0.85). Strong correlations were also found between mechanical parameters and SOS (0.87 < r < 0.90). No significant correlation was found between attenuation (nBUA) and either volumetric BMD or mechanical parameters. To conclude, BUB and IRC are promising parameters for the assessment of density and mechanical properties of trabecular bone. Advantageously, BUB and IRC can be determined easily with a single transducer, hypothetically enabling measurements at many clinically relevant osteoporotic fracture sites.

Mikko Lukkari, Tampere University of Technology
Electrical Detection of a Contact between a Microinjection Pipette and Living Adherent Cells

Current state-of-the-art cell injection micromanipulators are typically manual or semi-automatic and joystick controlled. Therefore they are slow and require experienced persons to operate. Automation of the injection system is important for making a faster, repeatable and more reliable research instrument. The bottle-neck in making a fully automatic injection micromanipulator is the detection of the contact between a micromanipulator pipette and cells. Presently, there are no reliable methods for detecting the contact. In this work, a device for measuring the contact between a microinjection pipette and a cell has been developed for automation of the intracellular injection process of living adherent cells. Also the breakage and clogging of the pipette can be detected with the developed device and from the same measured signal. This work is a part of the AIM (Integration of Automatic Intracellular Microinjection and Bioelectrical Recordings) project, funded by the Academy of Finland. The device is designed for use together with the injection micromanipulator developed by the Micro- and Nanosystem Research (MST) Group at Automation and Control Institute (ACI) at Tampere University of Technology (TUT). The implementation of the contact detection device in the micromanipulator was taken into account in design, which required the simplification and miniaturization of the device.

The operation of the developed contact detection device is based on measuring the resistance of the pipette in a similar way as in a patch clamp current/voltage recording device, but using injection pipettes, which have considerably sharper tip compared to patch clamp pipettes. This M.Sc. thesis work first included preliminary tests with the patch clamp recording equipment (available in the laboratory of Docent Tuula Jalonen) and FemtotipII injection pipettes. The work then included system and electronic design of the contact detection device and implementation of the device. The design had to be carefully made due to high demands in the measurement (low signal levels). Finally, the testing of the contact detection device was done in the same environment as the tests with the patch clamp device.

The test measurements with the developed contact detection device were successful and all the desired changes, the contact, breakage and clogging of the pipette, were detected with the device. With the designed contact detection device and the FemtotipII injection pipettes we were able to measure easily up to 15 contacts using one pipette, which is also an important result from the microinjection point of view.

The contact detection device has now been successfully integrated and tested with the injection micromanipulator. It is now used as a part of an expert system providing the user additional information on the injection – information not available in any other system today. Furthermore, the designed contact detection device makes it possible to develop even an entirely automatic microinjection system for intracellular injection of single adherent cells.