Holography invented 1948 by Dennis Gabor is a method of recording and reconstructing amplitude and phase of a wave field providing a three-dimensional image of a sample . Digital holography introduced by Schnars and Juptner consists of recording the hologram on a digital camera in contrast to optical holography where the recording medium is a photorefractive crystal or a photographic film .The spatial resolution of a digital camera, commonly used in digital holography, is low in contrast to conventional analogue holography due to the limited pixel size. Therefore digital holographic imaging of microstructures requires a certain magnification, which is usually achieved by the use of microscope objectives. However, this lens-based magnification suffers from aberrations, which have to be compensated. Lensless digital holography is a well-established microscopic method providing the diffraction limited resolution in the order of the wavelength of the used light source [4–8]. It is based on inline holography. The distance between the source and the sample is smaller than the distance between the sample and the digital camera to achieve sufficient magnification. This leads to a small field of view. In addition a laser is focused on a small pinhole to achieve a high NA. This requires a microscope objective and mechanical alignment components. The group of Aydogan Ozcan proposed a new lensless digital holographic microscope which is capable of imaging samples with a field of view corresponding to the entire sensor active area with a resolution in the order of the pixel size of the sensor [1, 9–12].The main key concepts of this setup are the use of an LED with a pinhole of size ∽50–100 µm as point source to achieve a high throughput and the placing of the sensor chip closer to the sample.
We propose in this paper the use of a laser diode without any need of a microscope objective and a pinhole as light source for a wide-field lensless digital holo-graphic microscope. This approach allows imaging of samples with reduced number of components in the setup achieving resolution in order of the pixel size of the used sensor chip and a Wide-field of view corresponding to the active sensor area.
2 Experimental method
The experimental setup is shown in Figure 1. A divergent beam of a laser diode illuminates a sample which is placed about 5 to 15 cm away on a cover glass. The diffracted light by the sample interferes with the undiffracted part on a CMOS-sensor chip placed directly behind the sample (1–2 mm). This interference pattern called hologram is recorded by the CMOS-sensor. In order to achieve interference spatial coherence of the light source is required. In the case of LEDs, this is achieved by the use of a pinhole. In our case the spatial coherence is already achieved by the output aperture of the laser diode which is typically 1×10 µm so that a pinhole is not necessary. We operate the laser diode below lasing threshold to emit low coherence light to avoid speckle noise due to the temporal coherence of the laser source. The used laser diode has a central wavelength of 405 nm. The CMOS-sensor chip has a 2560 × 1920 pixel active area with 2.2 µm pixel size.
We use the angular spectrum method to reconstruct the hologram . The inline configuration does not allow the separation of the different parts of the hologram after the reconstruction, so that DC part, real and twin images overlap. The DC part has no disturbing effect as it consists of a constant background. The twin image in contrast provides a distortion which introduces additional noise to the reconstructed image. In many lensless microscope approaches this twin image noise effect is neglected as it appears defocused on the reconstruction plane of the real image . In other approaches effort has been made to reduce the effect of the twin image [1, 13–16]. This will be necessary for many samples such as biological samples as the inner structure may be hidden by the twin image. We use the phase retrieval approach described in  to reduce the twin image noise effect.
3 Experimental results
In order to determine the resolution of our setup, we firstly record holograms of the 1951 USAF resolution test chart.
Figure 2 shows the hologram of the test chart corresponding to the entire sensor active area. After the recon-struction the 6th element of group 7 corresponding to a resolution of 2.19 µm is resolved. This corresponds exactly to the pixel size of our CMOS sensor chip. Thus, higher resolution can be achieved with sensors having smaller pixel size.
We then follow the evaluation of our system by imaging blood smear and diatoms.
In the case of the blood smear the distortion by the twin image as discussed in the experimental methods can be seen in the Figure 3. The amplitude and phase images are better reconstructed using the phase retrieval algorithm. Blood cells can be well seen.
The upper image in the Figure 4 shows the hologram of the diatoms. We choose the red marked part for the evaluation. All the diatoms can be easily identified and counted in the amplitude and phase images after reconstruction and phase retrieval algorithm. Figure 5 highlight again the elementary diatoms identifier on a light microscope image of the same sample.
Our system is a very simple, compact and low-cost possibility of realizing a microscope capable of imaging biological samples with a Wide-field of view. The availability of the phase provides topographic information of the sample extending the application of this system to be not only for biological samples but also for general transparent microstructure. It is suitable for fault detection, shape and roughness measurements of these structures.
D. Gabor, “A new microscopic principle.” Nature, vol. 161, no. 4098, p. 777, 1948. Google Scholar
U. Schnars and W. Jueptner, Digital Holography: Digital Hologram Recording, Numerical Reconstruction, and Related Techniques, vol. 44, no. 11. Springer, 2005, p. 164. Google Scholar
J.Garcia-Sucerquia et al.”Digital in-line holographic microscopy” Applied Optics Vol.45, No.5 10 February 2006 Google Scholar
L. Repetto, R. Chittofrati, E. Piano, and C. Pontiggia, “Infrared lensless holographic microscope with a vidicon camera for inspection of metallic evaporations on silicon wafers,” Opt. Commun. 251, 44–50 (2005).Google Scholar
J. P. Ryle, U. Gopinathan, S. McDonnell, T. J. Naughton, and J. T.Sheridan, “Digital in-line holography of biological specimens,” Proc. SPIE 6311, 63110C (2006).Google Scholar
G. Pedrini and H. J. Tiziani, “Short-coherence digital microscopy by use of a lensless holographic imaging system,” Appl. Opt. 41, 4489–4496 (2002).Google Scholar
J. P. Ryle, S. McDonnell, J. T. Sheridan “Lensless multispectral digital in-line holographic microscope” Journal of Biomedical Optics 126004-1 December 2011 Vol. 16(12) Google Scholar
A. Ozcan et al. “Ultra wide-field lens-free monitoring of cells on-chip”, Lab Chip, 2008, 8, 98–106 Google Scholar
T.-W. Su et al. “Compact and Light-Weight Automated Semen Analysis Platform Using Lensfree on-Chip Microscopy” Analytical Chemistry, Vol. 82, No. 19, October 1, 2010 8307Google Scholar
S. R. Isikman “Color and monochrome lensless on-chip imaging of Caenorhabditis elegans over a Wide-field-of-view”, Lab Chip, 2010, 10, 1109–1112 Google Scholar
G. Koren, O. Thorique, U. D. P. Xi, and O. Cedex, “Iterative algorithms for twin-image elimination in in-line holography using finite-support constraints,” J. Opt. Soc. Am., vol. 10, no. 3, 1993. Google Scholar
G. Koren, D. Joyeux, and F. Polack, ”Twin-image elimination in in-line holography of finite-support complex objects,” Opt. Lett. 16, 1979-(1991)Google Scholar
L. Denis et al. “Twin-image noise reduction by phase retrieval in in-line digital holography,” presented at Wavelets XI, SPIE’s Symposium on Optical Science and Technology, San Diego, CA, USA, 2005. .CrossrefGoogle Scholar
M. K. Kim. Digital Holographic Microscopy: Principles, Techniques, and Applications. Springer, 2011. Google Scholar
About the article
Published Online: 2015-09-12
Published in Print: 2015-09-01
Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent has been obtained from all individuals included in this study. Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee.