The development of off-axis holographic microscopy techniques

Research findings will be particularly valuable for biology and biomedicine, enabling enhanced sample imaging. The research conducted as part of the project focused especially on work at the intersection of two distinct off-axis QPI microscopy techniques: DIHM (digital in-line holographic microscopy) and TIE (transport of intensity equation). The main challenge for researchers was addressing the key issues associated with these two techniques. For DIHM, it is the difficulty of retrieving low frequencies of image phase, whereas for TIE, the key limitation is its sensitivity to noise.

“The techniques are used in biology and biomedicine for sample imaging of cells or thin tissues in a large field of vision and with sharp contrast. QPI microscopes enable imaging of the sample’s ‘thickness’, offering contrast even in transparent cells. In the long term, the research findings will be valuable for general diagnostics. Since microscopic samples will no longer need to be stained, the procedure will become accelerated, and its repeatability improved. Application work, on the other hand, was not performed strictly within the project,” emphasises Mikołaj Rogalski, PhD, from the Faculty of Mechatronics, WUT.

The project was implemented as part of the QCI Lab Group’s work on phase microscopy. Initially, the project was divided into two stages: the first focused on comparing the two techniques, and the other on integrating them. 

“In the first phase, we noticed that the DIHM method is less sensitive to noise than TIE, but it is still affected by it. We proposed a new algorithm for the DIHM method, which even more successfully minimises the impact of noise, enabling biological cell imaging at extremely low light intensities. It is crucial as it prevents stress or destruction of cells, translating into lower measurement-induced impact on the results. In the second stage, we intended to integrate both techniques; however, tests indicated that a combination of FPM and TIE methods would yield more interesting results. As a consequence, we obtained microscopic images with substantially high resolution and extended field of view (FPM) with simultaneous sensitivity to low-frequency phase changes (thanks to TIE),” explains Rogalski, PhD. 

The research work involved scientists from the Faculty of Mechatronics, WUT: Professor Michał Józwik, Mikołaj Rogalski, PhD, Piotr Arcab, MSc, and Professor Maciej Trusiak. For most of the project, Professor Józwik served as its head, and Rogalski, PhD, acted as the main contractor responsible for numerical work. Near the end of the project, he assumed the role of the project head, which was related to the completion of the doctoral thesis and regulatory requirements. Piotr Arcab was the contractor responsible for experimental work, and Professor Trusiak oversaw broad consultations, brainstorming sessions, and supervision.

As part of the project, two research papers were written: Hybrid Iterating-Averaging Low Photon Budget Gabor Holographic Microscopy and Fourier ptychographic microscopy aided with transport of intensity equation for robust full phase spectrum reconstruction.