Optical phase imaging has many applications, it allows to image transparent objects like cells without staining, which is a huge benefit in biology, and also allows to measure the phase from reflecting objects to determine their topography. Furthermore, there are many applications in life science, material science, healthcare and industry for optical phase imaging.

There exist different methods to image phases, phase contrast microscopy converts the phase information into amplitude contrast, but is difficult to interpret quantitatively, quantitative phase microscopy (QPM) on the other hand tries to determine the phase information quantitatively. Several full-field QPM methods exist, they can be divided in off-axis approaches and inline approaches. Off-axis approaches need a special microscope due to the reference beam and suffer from the high coherence of the illumination resulting in phase noise. Inline approaches could be divided in phase-shifting methods, common-path methods, digital inline holography, ptychography, Shack–Hartmann wave front sensors and focal series reconstruction, e.g. from the transport of intensity equation (TIE). Some of these approaches need special equipment, like laser illumination, gratings or spatial light modulators, which makes them expensive or introduces artefacts from the experimental setup or from the reconstruction algorithm.

Focal series reconstruction has the advantage, that it only needs a standard optical microscope plus a computer and no additional equipment, this makes quantitative phase imaging from focal series reconstruction both an easy and a low-cost method.

Figs. 1 & 2 present results obtained by our current approach to optical quantitative phase imaging, where we use focal series reconstruction with partially coherent illumination (a green LED source). The images were aquired using a Zeiss Axiovert 200M microscope and a pco edge sCMOS camera by varying the object height (z = 0µm is the focal plane). We apply our flux-preserving iterative reconstruction algorithm [1] and combine it with a TIE-like approach [2]. It aligns the images during reconstruction and applies gradient-flipping regularization [3]. This full-resolution inline holography (FRIH) algorithm [2] was originally developed for electron microscopy, but here we apply it to optical microscopy. We will discuss the validity of the reconstruction, especially with respect to artefacts and present the algorithm and the results in more detail.

[1] C.T. Koch, A flux-preserving non-linear inline holography reconstruction algorithm for partially coherent electrons. Ultramicroscopy 108 (2008),141–150. DOI: 10.1016/j.ultramic.2007.03.007

[2] C.T. Koch, Towards full-resolution inline electron holography. Micron 63 (2014) 69-75. DOI: 10.1016/j.micron.2013.10.009

[3] A. Parvizi, W. Van den Broek, C.T. Koch, Recovering low spatial frequencies in wave front sensing based on intensity measurements. Advanced Structural and Chemical Imaging (2016) in press

Figures:

Fig. 1: a) Phase image of HeLa cells reconstructed from a focal series recorded using a 10x objective (z-range = -20µm .. +20µm in 2µm steps). b) Phase image of human osteosarcoma (bone cancer) cells reconstructed from a focal series recorded using a 40x (NA = 1.35) objective (z-range = 0µm .. 20µm in 1µm steps). Both of these phase maps contain more than 2000 x 2000 pixels. c) Close-up of the sub-area indicated in b).

Fig. 2: a) .. c) Images of human osteosarcoma (bone cancer) cells recorded at 40x (NA = 1.35) at different object height (z-stack). d) and e) Amplitude and phase reconstructed from a series of 20 images spanning the same range of object height shown in a) .. c), i.e. z = 0 .. 20µm, but recorded in 1µm steps.

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EMC Abstracts - https://emc-proceedings.com/abstract/optical-quantitative-phase-imaging-by-focal-series-reconstruction-with-partially-coherent-illumination/