Diffraction phase microscopy(DPM) [1-2], which combines the benefits of high temporal sensitivity of common path interferometry and single-shot feature , has been used for quantitative phase imaging at the individual cell level. In this paper, a laser DPM system is designed and built up, and the spatial noise standard deviation of the system is measured as 5.3 nm. A standard polystyrene sphere is used as the calibration sample, and the measured phase shift shows good agreement with the expected one. The unwrapped phase image of an G.lamblia cyst is reconstructed, thereby an elliptical shape with is deduced, and the dry mass is calculated to be 54 pg. The results demonstrate that DPM can be a useful and versatile tool for the morphological and biochemical characterization of single protozoan parasite.
Schematic of experimental setup for DPM is shown in Fig.1. A He-Ne laser instead of traditional halogen lamp is used as a light source. The beam from the laser is spatially filtered, collimated and aligned to the input port of the microscope, which produces magnified image of the sample at the output port. An amplitude diffraction grating is placed at the output port, which creates multiple copies of the image at different angles. Under the 4f configuration, a spatial filter placing at the fourier plane of lens L5 allows full 0th order passing as a imaging field, and filters down 1st order as a reference field while blocking other orders. After Lens L6, the two beams from the fourier plane interfere to produce a spatially modulated interferogram at the camera plane. Before measuring the sample, the spatial noise standard deviation is characterized as 5.3 nm.
To demonstrate the accuracy of the system, individual microspheres were used as the calibration sample. Fig.2 illustrate the interferogram of a sphere and phase reconstruction steps, in which a Hilbert transform method in combination with Goldstein’s algorithm is used . The phase shift induced by a polystyrene bead is written as
Where n(x,y) and n0 are refractive index of the sample and that of the surrounding media, respectively. h(x,y) is the local thickness of the sample. As a result, the measured phase shift agrees well with the expected one .
The interferogram and unwrapped phase image of a Giardia cyst are shown in Fig.3(a) and (b), respectively. The proteins of Giardia labmlia constitute the greatest proportion of the cell solids, and the refractive index can be expressed as:
Where α and C are the refractive index increment and concentration of protein or DNA in the Giardia labmlia, respectively. Using this relationship, the dry mass of the Giardia labmlia can be calculated by integrating phase shift over the entire area of the isolated Giardia labmlia, as follows:
where α≈0.2ml/g. In this way, the dry mass of this individual Giardia labmlia cyst is approximated as 54 pg.