Experimental Results

Figure 3 shows the set-up built for proof-of-concept demonstration. It is divided into three parts. The first is the above described illumination method composed of a collimated white led, L, (6500 K) passing through a diffuser, D, close to a circular aperture, A. The diffuser is placed on the front focal plane of a lens, L1. The plane corresponding to the exit pupil of this lens is transported to the sample plane, S, by using a relay (lenses L2 and L3). The second part is a microscope consisting of an objective, MO, (Nikon 40x NA 0.60) and a doublet as tube lens, L4. This lens forms a scaled image of the microscopic sample on a plane that corresponds to the camera port of a conventional microscope. The third part of the system is the PWS whose input-plane is the previous image plane where a diaphragm, DI, is placed to control the field extension. Adjacent, a lens, L5, generates the Fourier transform on its back focal plane where a refractive pyramid is placed. Finally, the lens L6 re-images the sample on a CCD.

To test the system, we built artificial samples simulating cellular tissue using polystyrene (n_s = 1.60) microspheres of 43.3 µm Ø which were transferred to a calibrated host liquid (n_h = 1.56) inside a sealed glass chamber. As panel (a) on figure 4 shows, without the pyramid, the optical system behaves as a conventional microscope (DI fully open). Figure 4 (b) shows the image containing the sub-images I_i for a sample consisting of three microspheres in the field when the pyramid is in place and closing DI to avoid overlapping. Panels (c) and (d) show the images corresponding to S_x and S_y obtained using the image on (b). Note that information of the light absorption by the sample is not lost given that the algebraic sum of the four sub-images is the conventional microscope image that can be simultaneously displayed.

Figure 5 represents the comparison between the optical path difference (OPD) of a single sphere obtained using an algorithm for gradient data integration and the expected OPD (solid line). Figure 5 (b) shows the OPD map obtained for the images on the figure 4 (c).

Final Remarks

A new non-interferometric phase microscope that uses a pyramid sensor with dynamic range extension generated by a disc-shaped incoherent source has been presented.

The system instantaneously provides high-resolution sampling of the pahse gradient suitable for the effective numerical integration of structurally complex OPD maps. It has no moving parts, is resilient to noise and mechanical stability is not required. In conjunction with a fast integration algorithm, the microscope can provide OPD information in real time, which may be of interest for the study of structural changes in dynamic biological processes. Work is in progress to avoid the prototype's field limitation which is not intrinsic of the method and in an alternative beam splitting mode in order to use the CCD sensor area more effectible.

References
[1] Charrière F. et al.: Opt. Lett. 31, 178-180 (2006)
[2] Isikman S.O. et al.: Proc. Nat. Acad. Sci. 108, 7297-7301 (2011)
[3] Fang-Yen C. et al.: J. Biom. Opt. 16, 01100-01105 (2011)
[4] Bon P. et al.: Opt. Express 17, 13080-13094 (2009)
[5] Fu D. et al.: Opt. Lett. 35, 2370-2372 (2010)
[6] Iglesias I.: Opt. Lett. 36, 3636-3638 (2011)
[7] Ragazzoni R.: J. of Mod. Opt. 43, 289-293 (1996)

Contact
Ignacio Iglesias, PhD (corresponding author)
Universidad de Murcia
Departamento de Fisica
Murcia, Spain