The thesis aims to study the motion of individual fluorescent molecules in the gel environments, and applies to the sensitive electrophoretic and dielectrophoretic studies. Firstly, dye molecules (Rhodamine B and DiI) are dispersed, and investigate the individual fluorescent spots. Later on, we study the motion of individual B-phycoerythrin molecules under external electrical field driving. Due to the porosity, agarose gel is used to contain the liquid, and the dye molecules can freely move within the pores. Thus, one can easily observe the motion of individual dye molecules under high numerical aperture objectives. B-phycoerythrin is chosen for the high extinction coefficient, native charge, and good fluorescent properties. Our results indicate most dye molecules are attached in the rigid structure of the gel. Only very limited molecular motions are observed. Moreover, we study the dielectrophoretic interaction of the dye molecules. Nano-electrodes are fabricated by electrolysis to have sub-micron aperture silver tips. Due to the high gradient of the electric field, it is used to have strong enough attractive forces around the apex of the tip, to overcome the thermal fluctuations. It allows us further trapping and manipulating small non-charged objects, up to single dye molecules.

The purpose of this study was to assess the utility of confocal microscopy to examine nuclear organization and chromosome pairing for intact Arabidopsis male meiocytes. The efficiency of the confocal technique was evaluated by analyzing wild-type nuclei throughout meiosis. Early-mid leptotene meiocytes demonstrated the presence of several propidium iodide stained signals within the nucleolus prior to the onset of chromosome pairing in zygotene. Pachytene chromosomes were completely paired and were traced to confirm the Arabidopsis karyotype. Additionally, the confocal technique was employed on meiotic mutants, ahp2 and spo11-2-2, to characterize their meiotic defects. Leptotene ahp2 meiocytes and zygotene meiocytes in both meiotic mutants appeared normal. In contrast, pachytene meiocytes in ahp2 and spo11-2-2 mutants demonstrated a wide-spread lack of paired chromosomes. Despite this general lack of pairing, a small amount of chromosome pairing was detected on the short arms of NOR-bearing chromosomes 2 and 4 in ahp2 and spo11-2-2 mutants.

The purpose of this study was to assess the utility of confocal microscopy to examine nuclear organization and chromosome pairing for intact Arabidopsis male meiocytes. The efficiency of the confocal technique was evaluated by analyzing wild-type nuclei throughout meiosis. Early-mid leptotene meiocytes demonstrated the presence of several propidium iodide stained signals within the nucleolus prior to the onset of chromosome pairing in zygotene. Pachytene chromosomes were completely paired and were traced to confirm the Arabidopsis karyotype. Additionally, the confocal technique was employed on meiotic mutants, ahp2 and spo11-2-2, to characterize their meiotic defects. Leptotene ahp2 meiocytes and zygotene meiocytes in both meiotic mutants appeared normal. In contrast, pachytene meiocytes in ahp2 and spo11-2-2 mutants demonstrated a wide-spread lack of paired chromosomes. Despite this general lack of pairing, a small amount of chromosome pairing was detected on the short arms of NOR-bearing chromosomes 2 and 4 in ahp2 and spo11-2-2 mutants.

In this thesis the effects of single photon and multi-photon excitation on protoplasts from Arabidopsis thaliana are compared. Time-lapsed micro-spectroscopy at high spatial resolution is employed to study the response of chloroplasts within the protoplasts from Arabidopsis thaliana. We have found that the fluorescence spectra of chloroplasts exhibits dramatic changes and the protoplasts are rapidly damaged under multi-photon excitation as a result of pulsed laser illumination. In contrast, single photon excitation of chloroplasts with cw laser is relatively inert to the vitality of the protoplasts. In addition to, we have built an ultrafast laser excited cryogenic micro-spectroscopy setup to study the photoluminescence of PPV thin film. We found that the spectrum of PPV¡¦s photoluminescence should shift toward longer wavelength and the non-radiative transition should be suppressed as a result of longer electron coherence length at low temperature.

In this study we have successfully developed the techniques of time-resolved electro-luminescence (EL) and optical beam induced current (OBIC) microscopy for the mapping of photonic devices. We have applied the techniques to examine various photonic devices, including light emitting diodes (LED), organic light emitting diode (OLED), and coplanar waveguide (CPW) devices. The key development in time-resolved microscopy is the technique of modulation. By measuring the phase delay between the modulation source and the output signal, the response time of the observed devices can be extracted. In electro-luminescence mapping, the phase delay is measured between the applied sinusoidal voltage and the emitted EL, while in OBIC mapping the phase delay is measured between the modulated laser beam and the resulting photocurrent. The phase delay measurements are performed with a lock-in amplifier. In this way, large enhancement in signal-to-noise ratio can also be obtained. Additionally, the technique of varying scanning rate is also developed to synchronize the data acquisition between the LSM and the lock-in amplifier, a key enabling advancement in this thesis study.

This thesis focuses on the study of the life of a fluorescent dye. It also examines changes in its emission spectrum determined with the aid of development of fluorescence intensity over the long term sensing in several kinds if of cella. The general part of the thesis with the issues of confocal microscopy, fluorescent radiation and maks. Furthermorer, an analysis of methods for measuring fluorescence lifetime is done. In the thesis there is elaborated a description of the program, whitch was designed for the analysis of the collected output of the used confocal microscope. Sebsequently, the results are evaluated.

Yes / A confocal microscope provides a sequence of images of the corneal layers and structures at different depths from which medical clinicians can extract clinical information on the state of health of the patient’s cornea. Hybrid model based on snake and particle swarm optimisation (S-PSO) is proposed in this paper to analyse the confocal endothelium images. The proposed system is able to pre-process (quality enhancement, noise reduction), detect the cells, measure the cell density and identify abnormalities in the analysed data sets. Three normal corneal data sets acquired using confocal microscope, and two abnormal endothelium images associated with diseases have been investigated in the proposed system. Promising results are achieved and the performance of this system are compared with the performance of two morphological based approaches. The developed system can be deployed as clinical tool to underpin the expertise of ophthalmologists in analysing confocal corneal images.

Yes / A confocal microscope provides a sequence of images of the various corneal layers and structures at different depths from which medical clinicians can extract clinical information on the state of health of the patient’s cornea. Preprocessing the confocal corneal images to make them suitable for analysis is very challenging due the nature of these images and the amount of the noise present in them. This paper presents an efficient preprocessing approach for confocal corneal images consisting of three main steps including enhancement, binarisation and refinement. Improved visualisation, cell counts and measurements of cell properties have been achieved through this system and an interactive graphical user interface has been developed.

In this study, we have developed a high performance multi-photon microscopic system to perform second- harmonic (SH) imaging on a tooth and a KTP crystal . The high sensitivity of the system allows acquisition rate of 300 seconds/frame with resolution at 512¡Ñ512 pixels. The surface SH signal generated from the tooth and the KTP crystal is also carefully verified through micro-spectroscopy, polarization rotation and wavelength tuning. In this way, we can ensure the authenticity of the signal. KTP crystal and the enamel that encapsulates the dentine is known to possess highly ordered structures. The anisotropy of the structure is revealed in the microscopic SH images of the tooth and the KTP crystal samples.

In this study, we have successfully developed a high performance transmission mode Laser scanning for SHG imaging. This setup is capable of acquiring images of size 512¡Ñ512 pixels at a rate of 5.4 seconds/frame. The of samples can thus be imaged, which reflects the samples¡¦ structure and symmetry.