Problems of Bad Contrast in Conventional Microscopy Solution and Speckle Elimination with a Laser Fourier Holographic Microscope

The problem of bad contrast in conventional microscopy is well-known and was solved in part by colouring the samples. It is shown theoretically that a laser Fourier holographic microscope produces images undisturbed by speckle-noise. A laser holographic microscope (LHM) is investigated experimentally. The instrument uses visible radiation of λ = 0.514 μm, Mach – Zehnder scheme optical setup, and CCD detector of the hologram. Images are reconstructed digitally. The standard slide of Parascaris Univalens Iarva (ascaris) is studied without any drying as for electron microscope. Comparison of the pictures of the same ascaris cell, observed by the LHM and high-quality Nikon conventional optical microscope with immersion oil and green filter indicates dramatically different contrast. The ultrahigh contrast of the LHM gives much more micromorphological information.


Introduction
A conventional optical microscope (COM) [1] is a popular human instrument, which will hardly fall into oblivion. Not considering the advantages, it is reasonable to be reminded of its disadvantages, limiting the obtainable information. Thus, the problem of bad contrast is well-known and was solved in part by colouring the samples [2].
Light emitted from a lamp is a carrier of information in a COM. Physically it is incoherent visible radiation with the central frequency 0

The Problem of Speckle-Noise
Considerable distortions arise from the speckle-noise [5,6] if the image is formed with high coherent laser radiation. Moreover, the speckle-noise disturbs images reconstructed from the holograms [7].
The speckle-noise is produced both by reflecting from rough surfaces and transmitting phase nonuniform optical elements. The speckle-noise contributors are placed before the sample, inside and after it. In this way it is reasonable to divide the problem into three steps. In the first step, a plane wave or Gaussian beam, which do not possess speckle-structures [8], have to be used for sample illumination.
In the second step, the speckle-noise produced by the sample, for certain conditions, does not disturb the image. Our optical scheme ( Figure 1) is similar to that of a COM [2]. Here a monochromatic plane wave of wavelength λ  Then [1,9] is the radius-vector in the plane of the lens; is the value of wave vector; The lens is supposed to be ideal i.e. greater than the sample and image. If the "focusing condition" is valid, then (1) reduces to [9] ( ) amplitude and phase. After substituting (3) into (2), we obtain: The phase factor of (3) is not involved with (4). A real lens is described with numerical aperture NA and focal depth 0 Z ∆ [1,8]: In such a case, the plane detector "sees" focused not only the sample plane, but also the volume of the length approaching 0 Z ∆ . If the typical transverse size of the phase roughness is 0 d (Figure 2), then the distance 1 Z ∆ of transformation of the phase distortions into amplitude ones, i.e. the depth of speckle-noise formation [1] is: For the third step of the speckle-noise removing it is necessary to put away all sources of the parasitic scattering and reflection placed between the sample and detector. The construction of a COM [2] does not allow it. Indeed, the larger the numerical aperture the smaller its objective lens.
Here F is known operator of propagation. The sample wave carries the intensity: A conventional detector (retina, photo film, CCD, etc.) is responsible to the intensity (7), but losses the phase. That leads to impossibility of the image reconstruction with (6). We need a detector based on holography [1]. The principles of an LHM were explained in [9,10]. Results of the study of a real biological sample, specifically, a standard slide of Parascaris Univalense Iarva (ascaris) with an LHM are presented. An experimental setup, based on the Fourier holography [9, 11 -13], is shown in Figure 3. Here, a cw Ar -ion laser 1 provides a continuous, linearly polarized, single transverse and longitudinal mode beam of wavelength 0.514 m λ µ = . A shutter 2 creates a pulse with controlled duration. A beamsplitter 3 divides the beam into two parts, specifically, reference (transmitted) and sample (reflected). Intensities of both beams are controlled. The reference beam after reflection from a plane mirror 4 is focused by an objective 5. The waist W 1 can be considered like a point source of a spherical wave, which after reflection from a beamsplitter 6 reaches the CCD detector 7. The sample beam is reflected by a mirror 8 and then focused by an objective 9. A sample 10, which is a standard slide with a thin section of ascaris, is placed in a focal waist W 2 . A scattered wave is a result of interaction between the sample and sample wave. A transmitted unscattered beam is blocked by an absorbing blocker 11. The scattered light transmitted the beamsplitter 6 incidents the CCD detector 7. The scattered field interference pattern with the reference wave (a Fourier hologram) is captured by the detector. The hologram is recorded, digitized, and stored by a personal computer 12. The holographic data are then transferred to a Stardent GS 2000 Supergraphic Workstation 13, where numerical image reconstruction is performed. The reconstructed image can then be displayed by a monitor 14, or printed by a Tektronix Copy Processor 15.

Experimental Study of an LHM of
A picture of one certain ascaris cell, chosen for particular study, obtained with a high-performance Nikon COM 10×100 1.25 ⁄ with immersion oil and green filter is given in Figure 4, Figure 5 shows the image of the same cell and approximately equal magnification obtained with the LHM.

Conclusions
1. The contrast of the image obtained with the LHM is considerably higher than by the COM. The LHM allows observing distinctly a stripped structure of the cytoplasm, micromorphology of the nucleus, and transmission stage from the nucleus to the cytoplasm. The ultrahigh contrast of the LHM is assumed to be explained by the high coherence of the laser radiation, which allows separating neighbor structures with various resonance absorption frequencies.
2. The high quality of the images obtained with LHM, which are completely agree to electron microscopes ones [3,4], confirms the absence of the speckle-noise.
Other biological samples were also studied with LHN. The results listed above are consistent with them too. The author have chosen the results for ascaris as the most visual.