• INTRODUCTION
• OPTICAL SET-UP AND BASIC PRINCIPLE
• PERFORMANCES AND LIMITATIONS
• RESULTS AND APPLICATIONS
• Focus position determination
• Localisation of particles
• Biological samples
• Space applications
• REFERENCES

A conventional transmission optical microscope is characterised by a small depth of focus due to its high objective’s numerical aperture and magnification ratio. The analysis of a three-dimensional sample then requires a mechanical motion along the optical axis to scan the complete experimental volume. At the early age of holography, Gabor noticed that the information on a complete volume could be recorded in a hologram and optically reconstructed. In this configuration, the object beam relayed by the microscope lens interferes with a reference beam on a holographic plate. Nevertheless, optically refocused information is only obtained slice by slice using a mechanical scanning system along the optical axis. With the recent developments of digital holography, where the hologram information is directly recorded on a CCD camera, the optical reconstruction is replaced by a computer reconstruction. 
 

The digital holographic concept is implemented in a microscope configuration where a microscope lens magnifies the object. The interference between object and reference beams is achieved by the integration of the microscope arm in a Mach-Zehnder configuration. 

Since holographic recording requires optical coherence between object and reference beams, the use of laser sources of high coherence length is the usual approach. However, coherent laser beams are impaired by the coherent speckle noise (diffraction on scratches and digs) that severely reduces the quality of images. It results that the images obtained with laser have an optical quality that is quite below the microscopy standard. To overcome this limitation, a partially spatial coherent source (a usual LED) is used. As previously experimented in our DCCO experiment, an auto-aligned interferometer configuration allows to equalise the length of the two interferometric arms. 

The information on the complete 3-D volume is stored in only two images (intensity and phase distribution). Refocusing in any plane of the volume is obtained through numerical propagation of the optical field. The main interests of the holographic microscope with respect to the ones using mechanical translation stages are: 

• Recording in a short time of all the information related to an experimental volume
• No mechanical motion
• Important data compression for a complete 3-D knowledge of an object

The Digital Holographic Microscope (DHM) exactly refocuses like a mechanical refocus should do; at least in a given characteristic volume. Hence, typical properties (resolution, field of view, depth of focus) are the same as these of conventional microscopes, combined with a discrete visualisation system.

Focus position determination

The accuracy in the localisation of the best focus plane is derived from the intensity profile on an axis perpendicular to a target line plotted versus the refocus distance. The starting image is defocus by 80µm. After zooming on the focus zone, the resolution of focus plane localisation is measured equal to 5µm. 
 

The localisation of particles or objects in a volume is important in many research fields including fluid physics, crystal growth, protein growth, and 3-D velocimetry.  In the illustration at top of first page, glass balls of 40µm-diameter have been disposed on both sides of a thin glass plate (150µm). The distance between balls’ centres in both planes is around 190µm. Picture (a) results from a focus on an intermediate plane, inside the glass plate. Picture (b) and (c) are the computer-refocused images on glass balls on the two planes. (b) is obtained with a back propagation of 40µm and (c) with a forward propagation of 95 µm. The difference between the 135µm computed distance and the 190µm expected value is explained by considering the refractive index of the glass. This example shows that a quantitative localisation of particles or objects is possible only if particles are in a medium of known refractive index. 
 

DHM is also very promising in biological and medical analysis. Cell cultures are always realised in very thin volumes to allow microscopic visualisation of the division process. Thick volume imposes to mechanically scan the focus plane, which becomes unpractical when the division process is very fast. In essence, DHM offers the possibility to efficiently observe the complete volume. 

Tests were led on an urchin larva suspended in distilled water. The larva is about 150µm long and 50µm thick. Since the larva is mainly transparent, focusing on different planes will display its different constituents. Top left picture is the recorded image. Top right picture is the computer refocused image at 12µm. The skeletal rods, the internal cells and the border of the stomach are visible. Bottom picture is obtained after a refocus at 40µm. The larva’s end part is focused. 

Click Here to see an animation of computer reconstruction in different focus planes. 
 

In essence, DHM is a technique that produces compressed data: information on a complete volume is stored in only two images (intensity and phase distribution). This is of great interest for space environment experiments since it reduces both the volume of data storage and the download duration. 

• U. Schnars and W. Jüptner, "Direct recording of holograms by a CCD target and numeral reconstruction", Appl. Opt. 33, 179-181 (1994).
• T.M. Kreis, W.P.O. Jüptner, "Principle of digital holography", Proc. Of the 3^rd international workshop on automatic processing of fringe patterns (Fringe ’97), Bremen, 15-17 sept. (1997).
• I. Yamaguchi and T. Zhang, "Phase-shifting digital holography", Opt. Let. 22, 1268-1270 (1997).
• T. Zhang and I. Yamaguchi, "Three-dimensional microscopy with phase-shifting digital holography", Opt. Let. 23, 1221-1223 (1997).
• M. Adams, T.M. Kreis, W.P.O. Jüptner, "Particle size and position measurement with digital holography", Proc. SPIE 3098, 234-240 (1998).
• D.O. Hogenboom, C.A. Dimarzio, T.J. Gaudette, A.J. Devaney, S.C. Lindberg, "Three-dimensional images generated by quadrature interferometry", Opt. Let. 23, 783-785 (1998).
• F. Dubois, L. Joannes, J-C Legros, Improved three-dimensional imaging with a digital holography microscope with a source of partial spatial coherence, Appl. Opt., 38(34), pp.7085-7094 (1999).

Dr. Frank DUBOIS (frdubois@ulb.ac.be)