Márton Kiss
(Supervisor: Dr. Szabolcs Tőkés) kisma1@digitus.itk.ppke.hu
137 Abstract—A single-shot, volumetric and fluorescent digital
holographic microscope setup is introduced here. The aim is to develop a microscope that is able to detect fluorescent and freely flowing microscopical objects. This is why single-shot exposure is needed. The presented setup is based on a Hariharan-Sen interferometer. In this presentation the relation between the place of the target and the quality of the hologram is introduced.
Keywords: Incoherent or self referenced Digital Holographic Microscope, single-shot exposure, bifocal optical system, Hariharan-Sen interferometer, volumetric imaging
I. INTRODUCTION
Nowadays it is a frequently asked question whether the water we use for drinking and swimming is clean enough or not. The quality of water can be measured physically, chemically and biologically. After the first impression that comes from the water's physical properties we usually ask that what kinds of living organisms are in it? Because the living organisms are indicators of the quality of the water, in many cases their presence gives enough information about the water and chemical measurement is not needed. The main indicators in the water are the living bacteria, algae, cells, worms and other micro-organisms that usually can be seen only with microscope. The living cells can also be detected by the help of their fluorescence capability, and also it helps to separate them from the debris. Measurement without any preparation of the sample and volumetric imaging can supports fast, real time and automatic measurements. This is the background of our aim, which is to build a microscope for real time water measurements and monitoring.
The self-referenced digital holographic microscopy is a type of microscopes that can have the advantage of volumetric viewing and fluorescent imaging. The first holographic setup [1] that was invented used a (color and spatial filtered lamp's light as) coherent light, and its theoretical background was also based on the attribute of the coherent light. At the self-referenced or in other name incoherent holography the theoretical basis is the same: with two beams (reference and target), which are coherent with each other, an interference
fringe system called hologram is created. If we illuminate the hologram with the known beam (the reference one), the target beam that can draw the image of the target can be produced. At self-referenced holography the reference and target beams light sources are the same target point, which is self-luminous or just reflects the light that has a short coherent length. That is why the optical path difference of the self-referenced setup has to be smaller than the coherent length of the used light. In self -referenced holography color filter is usually used, because decreasing the bandwidth of the scattered or emitted light the coherent length can be increased, and also if many lights with different wavelength create hologram from the same object point to the same plane their interference fringes may disturb each other. And also one hologram is reconstructed numerically with one wavelength.
At the beginning of the self-referenced holography the main idea was to create Fresnel zone-plate (FZP) from the interference fringes, which is the coherent summation of two beams with different radius emitted by the same object point. If this FZP is back lighted, it will focus the light. They used it for example in astronomy [2]. There are mainly two kinds of methods to separate the light, modulate it in different way and interfere them. The first one when an interferometer is used for example Linnik interferometer [3], Hariharan-Sen interferometer [4], and the second one when a bifocal lens [5]
or a spatial light modulator is used [6]. The setup with bifocal lens is more compact and stable than the setup with other interferometers.
Digital holography has the degree of freedom to numerically modulate and modify the hologram and also the reconstruction beam, and the advantage to automatically evaluate the hologram that contains the needed information from the viewed volume. This is why at 3D incoherent holographic imaging could grow up. In these new techniques incoherent holographic imaging can be assisted with tomography [7] or scanning [8].
FINCH is a mature technique of nowadays that ignores these possibilities to create a fast setup but also it gives a high quality image [9]. Because FINCH uses three exposures to retrieve the complex hologram through phase shifting, this method is not able to create images from freely moving samples.
Digital holographic microscopy for single-shot, volumetric and fluorescent measurements
Márton Kiss
(Supervisor: Dr. Szabolcs Tőkés) kisma1@digitus.itk.ppke.hu
M. Kiss, “Digital holographic microscopy for single-shot, volumetric and fluorescent measurements,”
in Proceedings of the Interdisciplinary Doctoral School in the 2012-2013 Academic Year, T. Roska, G. Prószéky, P. Szolgay, Eds.
Faculty of Information Technology, Pázmány Péter Catholic University.
Budapest, Hungary: Pázmány University ePress, 2013, vol. 8, pp. 137-140.
Here I present my setup that is based on a Hariharan-Sen interferometer. It uses only one exposure to get an intensity hologram. But two self-luminescence points that are not coherent with each other could be reconstructed from their own holograms that were captured to the same image by a CCD sensor.
II. SELF REFERENCED HOLOGRAPHY WITH AN INTERFEROMETER
A. self-referenced holographic setup based on a Hariharan-Sen interferometer
Hariharan-Sen interferometer is a triangular shaped optical setup, where the entrance and exit gate for the light is one beam splitter cube. This cube divides the incoming light and then the separated lights go around in the optical path (that is puckered to a triangular by two mirrors) on the same path, but in opposite direction, and than this cube combines them too.
Because the beams have the same optical path, there is no difference between them. If this triangular is made asymmetric by properly inserting a lens, the exiting beams will have different wave fronts. This will generate interference fringes.
Figure 1 shows this asymmetric interferometer completed with an objective (olympus LUCPLFLN 20X), a tube lens (Bi-Convex lens, f=100mm), a polarizer filter that can set the intensity ratio between the two beams, and a detector (Lumenera).
Figure 1. The built self-referenced holographic setup based on a Hariharan-Sen type interferometer. At the way of "a" the beam is going throw an afocal optical
system.
One of the two optical ways of the system is afocal. The afocal system has the advantages that the magnification is independent of the target distance, and target and image distance has a linear connection. These are different in a common focal system. Figure 3 displays focal and afocal system's characteristic.
B. Light Source
At the experiment a stabile target was needed. That is why the target points were fiber ends in a same connector with a distance of 128 µm. Light from one red LED was coupled into these fibers, but leaving the fibers they couldn't create any interference fringes, because their coherent length was small enough. This target can be seen in figure 2.
Figure 2. Target was simulated with two fiber coupled red LED light. They couldn't interfere with each other, they added only in intensity.
C. Holograms
In my measurement I was interested in the connection between the target place and the created holograms, and I also wanted to know what kind of image can be reconstruct from them. All the other parameters were fixed. The target was moved from the distance 5 mm from the focal plane of the objective to close to the objective that was 4 mm far from the focal plane. The detector was set after the beam splitter with 20mm, as close to the beam splitter as it was possible. In this case and when the target was in the objective's focal plane, the afocal systems image was before the detector and the focal systems image was after the detector. Moving the target through the above explained area, I founded six sub-areas separated with 5 times.
These areas can be seen in figure 3. In the 1st and the 6th the illumination was quite homogenous, because the beams radius was nearly detector size. In this case the holograms were as big as to overlap each other and that is why moiré effect could be seen between the two interference fringes. In the 2nd and 3rd the target is moving a bit, the interference fringes changed so fast, and also they had only a few fringes. At the separating points III. and IV. one of the beams was focused to the detector. So there were no interference fringes and around those separating points the high intensity level disturbs the small holograms.
Figure 3. The place of the target before the objective will define the images of the same target point, and also the size and shape of the interference fringes.
In the area of 4 and 5 we got nice interference fringes: they didn't overlap each other and they can be seen clearly. It is shown in figure 4.
Figure 4. Interference fringes from the 4th area. (See figure 3.)
The separating points II. and V. show that case when a target-point's two images are in the same image plane. In these cases at the detector the curvature of the two beams are the same, so the interference fringes are not concentric but parallel lines.
D. Reconstruction
Angular spectrum method, which is a plane wave propagation method is used to reconstruct the holograms. This method calculates the scalar electric field in this way:
{ }
{ }
1 2 ( , )
( , , ) ( , ,0)
i u v zE x y z = F
−F E x y ⋅ e
π ω⋅ ⋅ , where E is the electromagnetic field, F and F-1are the Fourier and inverse Fourier transforms, ω is the transfer function and the z is the propagating distance.At the measurement, when the hologram belonged to a target that was at the separating points as it can be seen in figure 3, propagation didn't give any result. In the cases of II. and V.
parallel fringes were just moving across the plane because parallel fringes do not focus the plane wave, and in the I. and the III. case the points were already in focus.
At the 2nd to 5th areas of the hologram's reconstruction the problem was that when there were some interference fringes, the reconstructed point cannot be seen at the reconstructed image, because the background intensity overruns it. It can be also possible to compensate the intensity on the hologram (before propagating) to get a higher contrast, but we should see that the better the contrast of the hologram, the better the light efficiency, and at fluorescent imaging, what the final application will be, we should use the light in the best way because it is few.
In the 1st and 6th areas the reconstructed points can be clearly seen as figure 5 and figure 6 shows. When the two point's hologram was propagated at the same time the reconstructed image background was more flat, than when the points hologram were captured and propagated separately, but the contrast became smaller. It also can be seen that the two intensity holograms do not disturb each other, they don’t change each other’s propagation distance and the place and
magnification of the image. The density of the moiré fringes created by the two intensity hologram, gives information about magnification. The closer the fringes are the bigger is the magnification. Two intensity holograms do not disturb each other. It is a question that without any phase retrieves how many point sources can constitue a target. Comparing the 1st and the 6th areas the later has the advantage that it is closer to the objective, so the optical setup can gather more light from the object.
Figure 5. Here a reconstructed image can be seen, where the A and B points that are in the same plane were in the 1st area (see figure 3.)
Figure 6. Here a reconstructed image can be seen, where the A and B points that are in the same plane were in the 6th area (see figure 3.)
139 Here I present my setup that is based on a Hariharan-Sen
interferometer. It uses only one exposure to get an intensity hologram. But two self-luminescence points that are not coherent with each other could be reconstructed from their own holograms that were captured to the same image by a CCD sensor.
II. SELF REFERENCED HOLOGRAPHY WITH AN INTERFEROMETER
A. self-referenced holographic setup based on a Hariharan-Sen interferometer
Hariharan-Sen interferometer is a triangular shaped optical setup, where the entrance and exit gate for the light is one beam splitter cube. This cube divides the incoming light and then the separated lights go around in the optical path (that is puckered to a triangular by two mirrors) on the same path, but in opposite direction, and than this cube combines them too.
Because the beams have the same optical path, there is no difference between them. If this triangular is made asymmetric by properly inserting a lens, the exiting beams will have different wave fronts. This will generate interference fringes.
Figure 1 shows this asymmetric interferometer completed with an objective (olympus LUCPLFLN 20X), a tube lens (Bi-Convex lens, f=100mm), a polarizer filter that can set the intensity ratio between the two beams, and a detector (Lumenera).
Figure 1. The built self-referenced holographic setup based on a Hariharan-Sen type interferometer. At the way of "a" the beam is going throw an afocal optical
system.
One of the two optical ways of the system is afocal. The afocal system has the advantages that the magnification is independent of the target distance, and target and image distance has a linear connection. These are different in a common focal system. Figure 3 displays focal and afocal system's characteristic.
B. Light Source
At the experiment a stabile target was needed. That is why the target points were fiber ends in a same connector with a distance of 128 µm. Light from one red LED was coupled into these fibers, but leaving the fibers they couldn't create any interference fringes, because their coherent length was small enough. This target can be seen in figure 2.
Figure 2. Target was simulated with two fiber coupled red LED light. They couldn't interfere with each other, they added only in intensity.
C. Holograms
In my measurement I was interested in the connection between the target place and the created holograms, and I also wanted to know what kind of image can be reconstruct from them. All the other parameters were fixed. The target was moved from the distance 5 mm from the focal plane of the objective to close to the objective that was 4 mm far from the focal plane. The detector was set after the beam splitter with 20mm, as close to the beam splitter as it was possible. In this case and when the target was in the objective's focal plane, the afocal systems image was before the detector and the focal systems image was after the detector. Moving the target through the above explained area, I founded six sub-areas separated with 5 times.
These areas can be seen in figure 3. In the 1st and the 6th the illumination was quite homogenous, because the beams radius was nearly detector size. In this case the holograms were as big as to overlap each other and that is why moiré effect could be seen between the two interference fringes. In the 2nd and 3rd the target is moving a bit, the interference fringes changed so fast, and also they had only a few fringes. At the separating points III. and IV. one of the beams was focused to the detector. So there were no interference fringes and around those separating points the high intensity level disturbs the small holograms.
Figure 3. The place of the target before the objective will define the images of the same target point, and also the size and shape of the interference fringes.
In the area of 4 and 5 we got nice interference fringes: they didn't overlap each other and they can be seen clearly. It is shown in figure 4.
Figure 4. Interference fringes from the 4th area. (See figure 3.)
The separating points II. and V. show that case when a target-point's two images are in the same image plane. In these cases at the detector the curvature of the two beams are the same, so the interference fringes are not concentric but parallel lines.
D. Reconstruction
Angular spectrum method, which is a plane wave propagation method is used to reconstruct the holograms. This method calculates the scalar electric field in this way:
{ }
{ }
1 2 ( , )
( , , ) ( , ,0)
i u v zE x y z = F
−F E x y ⋅ e
π ω⋅ ⋅ , where E is the electromagnetic field, F and F-1are the Fourier and inverse Fourier transforms, ω is the transfer function and the z is the propagating distance.At the measurement, when the hologram belonged to a target that was at the separating points as it can be seen in figure 3, propagation didn't give any result. In the cases of II. and V.
parallel fringes were just moving across the plane because parallel fringes do not focus the plane wave, and in the I. and the III. case the points were already in focus.
At the 2nd to 5th areas of the hologram's reconstruction the problem was that when there were some interference fringes, the reconstructed point cannot be seen at the reconstructed image, because the background intensity overruns it. It can be also possible to compensate the intensity on the hologram (before propagating) to get a higher contrast, but we should see that the better the contrast of the hologram, the better the light efficiency, and at fluorescent imaging, what the final application will be, we should use the light in the best way because it is few.
In the 1st and 6th areas the reconstructed points can be clearly seen as figure 5 and figure 6 shows. When the two point's hologram was propagated at the same time the reconstructed image background was more flat, than when the points hologram were captured and propagated separately, but the contrast became smaller. It also can be seen that the two intensity holograms do not disturb each other, they don’t change each other’s propagation distance and the place and
magnification of the image. The density of the moiré fringes created by the two intensity hologram, gives information about magnification. The closer the fringes are the bigger is the magnification. Two intensity holograms do not disturb each other. It is a question that without any phase retrieves how many point sources can constitue a target. Comparing the 1st and the 6th areas the later has the advantage that it is closer to the objective, so the optical setup can gather more light from the object.
Figure 5. Here a reconstructed image can be seen, where the A and B points that are in the same plane were in the 1st area (see figure 3.)
Figure 6. Here a reconstructed image can be seen, where the A and B points that are in the same plane were in the 6th area (see figure 3.)
III. CONCLUSION
A self-referenced digital holographic microscopy was created with a modified Hariharan-Sen interferometer. The tests showed that this setup is able to create the hologram from a target that is in a large volume and illuminating an incoherent
A self-referenced digital holographic microscopy was created with a modified Hariharan-Sen interferometer. The tests showed that this setup is able to create the hologram from a target that is in a large volume and illuminating an incoherent