Formation and analysis of widefield fluorescence microscopy images

A thesis accepted by Tallinn University of Technology for the Degree of Bachelor of Science

Maris Poroson

Tallinn University of Technology, Estonia

Martin Laasmaa, MSc

Institute of Cybernetics at Tallinn Technical University, Estonia

Pearu Peterson, PhD

Institute of Cybernetics at Tallinn Technical University, Estonia

Jaan Kalda, PhD.

Institute of Cybernetics at Tallinn Technical University, Estonia


Widefield fluorescence microscopy is used to study biological systems. This method is a significant research tool in Laboratory of Systems Biology, Institute of Cybernetics at Tallinn University of Technology. The main aim of the laboratory is to study regulation of intracellular processes and understand functional influences of intracellular interactions.

The main focus of this work is to study and analyze the image formation process in a widefield fluorescence microscope and then determine its main features. These results can then be used to develop specialized image deconvolution software to improve image quality. The studies were carried out, using fluorescent microspheres and solution for measurements and then analyzed using software developed in the laboratory.

The experiments consisted of determining the point spread function of a point light source (microspheres) and its width, the location of glass surface, the level of dark noise and finding the value of a model parameter which depends on concentration of solution, exposure time and CCD’s gain. All these parameters were then used to verify the usability of derived model to estimate intensity values at various distances from the glass surface. Comparing the results from measurements and from model equation it was found that some estimates were justified while others were totally incomparable. This means that the microscope used for carrying out measurements was not correctly calibrated and also the mathematical model need to be extended. Finding the right estimation for the parameters describing the image formation is a subject for another research that should lead to developing a deconvolution algorithm for widefield fluorescence microscopy.


The presentation of the thesis took place on June, 2012, at Tallinn University of Technology, Tallinn, Estonia.


Thesis is written in Estonian.