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The off-axis DHM microscope was set up with an inverted microscope with a continuous laser wave source as detailed in He et al. (2016) and shown below in Figure 5.1 (A) and (B). The post-sampling region recognition algorithm and program (Figure 5.1 C) was validated through the measurement of air-dried erythrocytes. The inverted microscope was set up with a laser beam light source, which is split into a sample beam ko and reference beam kr (Figure. 5.1 A).

The sample beam ko is transmitted through the sample and then recombined with the

reference beam kr at an angle (). The off-axis hologram obtained is then processed through

the iterative algorithm to obtain three-dimensional phase (spatial) images of cells (Figure 5.1 C). The iterative algorithm uses Fast Fourier Transform (FFT), which deconstructs a signal into discrete frequency signals and then differentiates background noise frequency from sample frequency.

Figure 5-1 Digital Holographic microscope set up and region recognition methodology (He et al.

2016). A) A schematic of the optical light path for DHM from laser source to sample. M indicates mirrors, MO1, MO2, MO3 are microscope objectives, F-S is the optical fibre splitter that splits the beam into two, CO is the optical collimator that fixes the beams to be parallel and is used before L3 for the sample beam ko and before L1 for reference beam kr. L1-L4 are optical lenses that either condense or expand the beam, BS is beam splitter that recombines the two split beams and CCD stands for the charged couple device camera that captures raw images. B) The physical setup of the DHM microscope showing the sample stage set up for slide samples. C) Post image sampling space-recognition iterative methodology used to filter sample from background and process hologram images into phase images.

Measurement of erythrocytes with off-axis DHM

The resolution of the DHM setup and capability of the FFT method to recognise erythrocytes were both validated by visualising air-dried uninfected and infected erythrocytes. Air-drying fixes the erythrocyte to the slide and allows the measurement of static cells without

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Figure 5-2 Phase maps of infected and uninfected erythrocytes. Images from (He et al. 2016). A)

Processing of hologram of cell visualised under 100x. First image is a hologram of cell with interference phase fringe at 15 pixels per fringe. Red arrow indicates an interference phase fringe. Second image is the Fourier transform of the hologram. The red circle in the image indicates sample region filtered by region recognition method. Third image is the processed phase image of cell. The coloured bar indicates height of the cell in microns, maximum height h1 = 2.9 m. B) 3-dimensional phase maps of an infected and a uninfected erythrocyte visualised under a 100x objective. The coloured bar indicates height of the cell in microns, maximum height h2 = 2.77 m.

The conversion of a hologram of an uninfected erythrocyte to its corresponding phase image is shown in Figure 5.2 (A). Here the post-sampling FFT method identifies the sample interference signal (red circle) from the background interference signal and then converts the interference data to the corresponding phase data (third image in Figure 5.2 A). Subsequently uninfected and infected erythrocytes were compared to test whether the method could recognise changes in the morphology and height of the cell (Figure 5.2 B). The phase image, reconstructed from the hologram, depicts the spatial position of several phase values in the sample and can be used to infer the height and topology of the cell. The uninfected erythrocyte shows a biconcave shape, which is consistent with known external morphology of erythrocytes. The infected erythrocyte shows an irregular height topology with the peak in height of the cell biased towards one side, which is a result of the parasite position within the

host cell. This is consistent with known external morphology of infected erythrocytes as seen in scanning electron microscopy images (Gruenberg et al. 1983).

Figure 5-3 Height distribution of uninfected and infected erythrocytes. Images from (He et al. 2016. A) Phase distribution of uninfected cell (shown in insert with 2m scale bar) on a 2-dimensional plane. Black dots indicated normalised phase values, the red line is the Gaussian fitted curve, the red shaded area indicates the FWHM value (= 0.303). B) Phase distribution of infected cell (shown in insert with 2m scale bar) on a 2-dimensional plane. Black dots indicate normalised phase values, the blue line is the Gaussian fitted curve, the blue shaded area indicates the FWHM (value = 0.222).

C) Distribution of FWHM for twelve infected and twelve uninfected cells each to indicate the

distribution of height in both cell types. The blue line indicates distribution of FWHM of infected cells and the red line indicates FWHM of uninfected cells.

The shape and height of uninfected and infected cells were also quantified and compared by measuring the distribution of height. The phase values on the cell surface were plotted for an uninfected cell (Figure 5.3 A) and infected cell (Figure 5.3 B) to observe the height distribution. The distribution was quantified by calculating the full width at half-maximum (FWHM) of the fitted curve. FWHM is a function of the histogram distribution and is the difference between the two most extreme phase values at which height was half its maximal value. A higher FWHM as seen for the uninfected cell (Figure 5.3 A) indicates a larger variance in height, which is a result of the biconcave shape of the cell. Comparatively, the infected cell has a lower

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uninfected and infected cells (Figure 5.3 C) by comparing the distribution of FWHM values. The larger FWHM value and the wider distribution of FWHM values between uninfected cells indicate that there is larger height variability within a cell and between cells whereas height variation within an infected cell and between infected cells was more consistent. In infected cells the variation in height would be caused by the internal parasite and thus the decreased variation in height between cells would suggest the size and shape of the parasites are similar, which is to be expected as they were synchronized to the mid-trophozoite stage (30-40 h.p.i). Additionally, DHM was able to resolve the height of the cells (2.7-2.9 m) in the 100nm range. Thus, these initial measurements validated the use of DHM and the FFT method to obtain high resolution height quantification for uninfected and infected erythrocytes.