Digital Holography

A patented technology

The Digital Holographic Microscopy (DHM®) as a patented technology makes use of a video (CCD) camera to record a hologram produced by the interference between a reference wave and a wave emanating from the specimen. The captured image is transmitted to a computer where numerical procedures are applied to reconstruct a 3D image of the specimen. This process is called “image reconstruction”. The innovation of DHM® is the possibility to retrieve optical topography information from a single image grab, without any scanning. Moreover, it offers the intervention of digital processing at a level that had not been reached before in microscopy.

Many application cases have established that this new concept enables fast 3D imaging of microscopic specimen with a high resolution (nanometer scale in the vertical direction), with  easy to use, flexible and cost effective systems. Complete description of the method is given in the reviewed paper by E. Cuche et. al.: “Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms

Measurement principle

The DHM® technology generates, in true real-time, high resolution 3D digital images of a sample using the principle of holography. Holograms are generated by combining a coherent reference wave with the wave received from a specimen. They are recorded by a video camera and transmitted to a computer for real-time numerical reconstruction. A single hologram is acquired and computed in a few microseconds. The DHM® proprietary software computes the complete wavefront emanating from an object and provides:

  • Intensity images providing the same contrast as with classical optical microscopy
  • Phase images providing quantitative data, defined at a sub-wavelength scale, used for accurate and stable 3D measurements

In reflection, the phase image reveals directly the surface topography with a sub-nanometric vertical resolution. In transmission, the phase image reveals the phase shift induced by a transparent specimen, which can be interpreted in terms of many underlying biological processes. This digital approach to holography allows the application of computer based procedures at a level unreached in video-microscopy.

In particular the strength of DHM® principle allows unique software compensations. Optical aberrations, digital image focusing, and numerical compensation for sample tilt and environmental disturbances can be performed.

Principle of Digital Holography

Introduction to digital holography

Holography is a well-established imaging technique. Since its discovery by Denis Gabor in 1948 [GAB48, GAB49, GAB51, GAB66] who was awarded the Nobel price in physics in 1971, large developments have been carried out. A description of the different forms and applications of classical holography is already developed in details in several books [GOO68, HAR96, COLL71, HAN79, STR69, SMI69, FRA87]. Furthermore, an exhaustive list of historical papers and an interesting overview of the developments of holography from its discovery to the present can be found in E.N. Leith’s overview [LEITH97].

It is however important to mention that even if digital holography took a great importance in the last years, holography progresses in non digital or more classical holography cannot be reduced to 3D spectacular “art images”. Research launched in the 50s and 60s by several famous personalities as D. Gabor, E.N. Leith, A. Lohmann, R.J. Collier, J. Upatnieks, G. Stroke, N. Hartman, Yu. N. Denisyuk, S. Benton, R.F. Vanligte, J.W. Goodman, R. Dändliker and N. Abramson, emerge onto a lot of different applications such as holographic data storage [SHEL97, ORT03], photorefractive crystals hologram capture [ROO03], light-in-flight for ultrafast phenomena recording [YAMA05], metrology applications, TV holography [POO05] and so on. Furthermore, all aspects of holography is a source of inspiration for the development of digital holography. For example, the configuration for DHM presented by E. Cuche [CUC99b] and used in this work was presented for the first time in 1966 by R.F. VanLighten and H. Oserberg [VAN66].

Concerning digital holography, the first computer reconstructions of holograms go back to the late sixties, some twenty years after the publication of Gabor’s landmark papers. The idea was proposed for the first time in 1967 by J.W. Goodman and R.W. Laurence [GOO67]. Numerical hologram reconstructions were initiated in the early 1970s by M.A. Kronrod and L.P. Yaroslavsky [KRO72a, KRO72b]. Nevertheless, the holograms were still recorded on a photographic plate, developed, optically enlarged and finally sampled before being numerically reconstructed.



Hologram recording on photographic plateHologram recording on photographic plate
Hologram reconstructionHologram reconstruction

A complete digital holographic set-up in sense of digital recording and reconstruction was achieved first by Coquoz et al. [COQ92, COQ93a, COQ93b] followed by U. Schnars and W. Jüptner in 1994 [SCHN94a]. The introduction of a coupled charged device (CCD) camera to record Fresnel holograms suppresses the recording on media such as photographic plate, photopolymers and photorefractives. It allows faster acquisition and reconstruction rates, as well as larger flexibility.

Important steps in the evolution of the technique and algorithms have been: the acquisition through endoscopic device [COQ95, SCHE99, SCHE01, KOLE03, PED03], the use of high wavelength [ALL03], and short-coherence laser source [CUC97, IND00, PED01a, PED02, MAS05, MAL05]. A very important step was the retrieval of the phase information in addition to the amplitude. Different techniques exist to reconstruct the phase. In-line techniques require phase-shifting procedures performed either by several successive hologram acquisitions [YAM97, ZHA98, LAI00, GUO02, YAM03, AWA04, MILGA05] or by simultaneous acquisitions [KOLI92, MILL01, WYA03]. In off-axis configurations, U. Schnars [SCHN94b] has demonstrated the possibility of measuring specimen deformations by evaluating the phase difference between two states of the specimen. However, this double exposure technique does not give the absolute phase of the specimen. A solution for absolute phase measurements has been proposed by E. Cuche in off-axis geometry [CUC99a] and delivered patent [CUCPat]. A flat reference surface is taken on a flat part of the specimen and a procedure is performed to compensate for the phase deformation. The complete wavefront is thus reconstructed out of a single hologram, which retrieves the initial aim of holography. E. Cuche also showed [CUC99b] that the phase compensation technique could be applied in holographic microscopic methods. Later on other techniques were developed to perform the phase reconstruction out of a single hologram [LIE03, LIE04]. P. Ferraro [FERR03a] performs a method in which a reference hologram is recorded and then subtracted to the hologram of interest to compensate for the phase deformations. G. Indebetouw developed a method, called Spatiotemporal digital holograph, that avoids the need for high spatial bandwidth detectors and high spatial coherence, leading to speckle noise [IND99, IND01]. Nowadays, digital holographic microscopy has become a wide used method [ZHA98, TAK99, TIS01, XU01, YAM01, DUB02, CARL04, COP04].
Other developments include color digital holography [KAT02, YAM02, ALM04, JAV05b], polarization digital holography [LOH65, COLO02b, COLO04, COLO05], synthetic wavelength digital holography [ONO98, WAG00, GAS03], tomography [KIM99, KIM00, DAK03, MONT06], optical diffraction tomography [CHAR06a, CHAR06b] and several aberration compensation techniques [CUC99b, STA00, PED01b, DEN02, FERR03b, FERR03a, IND01, MAL05, YON05, COLO06a, COLO06b, COLO06c]. The development of DHM allows truly non-invasive examination of biological specimens and therefore it becomes an increasing powerful technique for biomedical applications [COLO02a, TIS05, MAS05, MAL05, MARQ05, JEO05, JAV05a, RAP05].