A flexible low-cost quantitative phase imaging microscopy system for label-free imaging of multi-cellular biological samples

In this thesis, a flexible low-cost quantitative phase imaging microscopy (LQPIM) system for imaging both thin and thick biological phase objects in a non-contact, non-invasive, and label-free manner is reported. LQPIM optics was developed based on classical Zernike’s phase contrast approach and an additional phase shifting module to introduce user-defined phase modulations by utilising standard optical components. The phase shifting was performed using twin concentric mirrors or laser cut apertures in the arms of a Michelson interferometer where the reference mirror can be moved in / n steps (n - number of steps) with a piezoelectric transducer. Hence, the optical phase shifting modules are 10 - 15% (approximately) of the cost compared to the more widely reported modules based on spatial light modulator. In the microscope implementation reported in this thesis, a total magnification of 25x was achieved utilising relay lenses in LQPIM optics together with a standard 10x objective lens. The imaging system was simulated in MATLAB, where two-beam interference equation with varying bandwidth (1 – 250 nm), centre wavelength (450 – 650 nm) of the illumination sources and a range of previously reported phase shift algorithms (PSA) were used. The simulation results confirm that the optimum phase resolution is achievable if a broadband source of bandwidth 30 - 50 nm is used for illuminating thin (i.e. ≤ 250 nm) and thick (i.e. ≥ 1250 nm) biological samples. The four frames at 90 PSA and six plus one frames at 60 PSA offer different compromises between image acquisition time, phase resolution and out-perform other PSAs. A phase resolution of 0.382 nm and 0.317 nm was achieved using four frames at 90 and six plus one frames at 60 PSAs, respectively for the broadband illumination from a green LED. A coherent, single longitudinal mode laser source with a rotating diffuser for speckle averaging, gave 0.667 nm and 0.512 nm phase resolution using the same algorithms mentioned above. The parasitic fringes resulted in reduced resolution; hence, incoherent LED illumination was preferred. Measurements are presented over a longer optical path difference (≥ 1250 nm) than hitherto reported for a similar microscope. The given exemplar data demonstrates an ability of LQPIM system to quantify cellular and sub-cellular structures at the nanoscale in epidermis cells of Allium cepa. Key words: Quantitative phase imaging, low-cost, optical microscopy, phase imaging and phase shift imaging