Displacement measurement of the depth migration of transparent cellsMakoto Yoshida, Ichirou Ishimaru, Katsumi Ishizaki, Toshiki Yasokawa, Shigeki Kuriyama, Tsutomu Masaki,Seiji Nakai, Kaoru Takegawa, and Naoyuki Tanaka Citation: Applied Physics Letters 89, 241102 (2006); doi: 10.1063/1.2405396 View online: http://dx.doi.org/10.1063/1.2405396 View Table of Contents: http://scitation.aip.org/content/aip/journal/apl/89/24?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Microscopic modulation of mechanical properties in transparent insect wings Appl. Phys. Lett. 104, 063702 (2014); 10.1063/1.4865202 Reply to “Bessel beams and glory scattering: Comment on ‘Generation of Bessel beams using a 4 - f spatialfiltering system,’” by Philip L. Marston [Am. J. Phys.77(11), 1084–1084 (2009)] Am. J. Phys. 77, 1085 (2009); 10.1119/1.3133091 Simple fiber-optic confocal microscopy with nanoscale depth resolution beyond the diffraction barrier Rev. Sci. Instrum. 78, 093703 (2007); 10.1063/1.2777173 Translational velocity measurement for single floating cell based on optical Fourier transform theory Appl. Phys. Lett. 88, 101114 (2006); 10.1063/1.2183747 Transflective spatial filter based on azo-dye-doped cholesteric liquid crystal films Appl. Phys. Lett. 87, 011106 (2005); 10.1063/1.1990248

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Displacement measurement of the depth migration of transparent cellsMakoto Yoshida,a� Ichirou Ishimaru, Katsumi Ishizaki, and Toshiki YasokawaFaculty of Engineering, Kagawa University, 2217-20 Hayashi-cho, Takamatsu, Kagawa 761-0396, Japan

Shigeki Kuriyama, Tsutomu Masaki, and Seiji NakaiFaculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan

Kaoru Takegawa and Naoyuki TanakaFaculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan

�Received 22 May 2006; accepted 9 November 2006; published online 11 December 2006�

This letter reports a method for displacement measurement of the depth migration of transparentcells. This proposed optical spatial filtering method allows visualization of the transparent cells anddetermination of depth migration as a horizontal displacement positive or negative first orderdiffracted light on the detector surface. When the sample is displaced upward or downward from thefocal plane, first and negative first order diffracted light form images at a different point as a lightcircle. The coordinates of these two light circles on the detector surface change places when thedisplacement of depth migration moves to the opposite direction. © 2006 American Institute ofPhysics. �DOI: 10.1063/1.2405396�

We have proposed the visualization and translational dis-placement measurement of transparent cells using an opticalspatial filtering method.1 The conventional imaging process-ing technologies for cell tracking2–9 can measure the transla-tional movement of cells with high-contrast texture. How-ever, the depth movement of transparent cells cannot bemeasured. Here, we propose a method for depth displace-ment measurement of transparent cells. These translationaland depth displacement measurements allow for long-termtime-lapse observations of apoptotic cells without flourescentlabeling.10 First, the improvement in the visualized cell im-age by removing the fringe pattern caused from stray light isdescribed. Then, the principle of the depth displacementmeasurement and experimental results are presented.

Previous reports define the periodic span of the or-ganelles, which forms a complex refractive index distribu-tion, as the virtual phase grating. We have installed a pinholeas a spatial filter on the optical Fourier transform plane toselect the specific component of spatial frequency. On theimaging surface, a fringe is formed by interference betweenthe selected frequency component and the zeroth order dif-fracted light. Thus, we introduce white light of low coher-ence as a light source, because a laser as a light sourcecauses fringe resulting from the interference of stray light,which affects the desired periodic light intensity. However,the intensity of white light is low, so the high contrast of thefringe pattern does not occur. The zeroth order diffractedlight that includes reflected light from the cover glass, calledstray light, deteriorates the image. Thus, the low coherencelight is effective in preventing the influence of interferenceof the reflected light. The positive or negative first orderdiffracted light that does not include specular light from thecover glass was chosen. In this case, the transparent cells canbe visualized as the fringe pattern that is interfered betweenpositive and negative first order diffracted light. Moreover,this allows introduction of a laser, a high-intensity lightsource, to increase the contrast.

Figure 1�a� shows the initial image while Fig. 1�b�shows the improved image. Human cancerous cells wereused as the sample. In order to obtain the resolution of0.35 �m, the distance between two holes is set to about4 mm. The white line of the fringe pattern in Fig. 1�b� can bedistinguished clearly in comparison with Fig. 1�a�. More-over, because Fig. 1�b� is the dark field image, the blackbackground leads to greater contrast.

The principle of the depth displacement measurements isexplained as follows. Figure 2 shows the geometric opticsmodel of diffracted light when the sample moves to greaterdepth. The hole diameter of the spatial filter is the same asthe focal diameter of diffracted light whose diameter is0.5 mm. When the sample is at the focal plane as shown inFig. 2�b�, the parallel pencil of the diffracted rays condensesat the optical Fourier transform plane by the objective lens.These selected diffracted light rays interfere at the imagingplane by the imaging lens, forming a fringe pattern �Fig.2�b��. This fringe pattern is equivalent to that shown in Fig.1�b�. In the case of movement toward the left, as shown inFig. 2�a�, the traveling direction of the positive or negativefirst order diffracted light is changed and these diffractedlight rays form images at different points as a light circle.The coordinates of these two light circles on the detectorsurface change places, as shown in Fig. 2�c�, where the po-sition of the sample moves to the opposite direction than that

a�Electronic mail: s06g535@stmail.eng.kagawa-u.ac.jp

FIG. 1. Fringe pattern of diffracted light: �a� initial image; �b� improvedimage. Human cancerous cells were used as the sample. The white line ofthe fringe pattern in �b� is distinguished clearly in comparison with �a�.

APPLIED PHYSICS LETTERS 89, 241102 �2006�

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shown in Fig. 2�a�. Therefore, depth migration can be de-tected as the horizontal displacement positive or negativefirst order diffracted light on the detector surface. However,if the diameters of both holes of the spatial filter are equal,the light intensity observed on the detector also is equal.Thus, the direction of the depth movement cannot be recog-nized. Therefore, the relative intensity of the positive ornegative order diffracted light is made different by varyingthe hole diameters of the spatial filter, as shown in Fig. 2.When the sample moves toward the left, the brighter lightcircle moves to the upper part of the imaging area, as shownin Fig. 2�a�. For the reverse case, the brighter light circlemoves downward and the darker light circle moves upward,as shown in Fig. 2�c�. Therefore, the light intensity ratiobetween the upper and lower areas changes in accordancewith the direction of depth migration, which allows determi-nation of the direction and displacement of depth movementsimultaneously.

As shown in Fig. 3, a high-response split photodiode�frequency response: 80 MHz� can be applied to the mea-surement. Light intensity distribution in minute areas can bemeasured separately as two regions. A HeNe laser �MellesGriot, type: 05LHR121, output: 5.5 mW, wavelength:632.8 nm� is used as a light source. Human cancerous cellswere used as sample transparent objects.

The sample set on the stage �Siguma Koki, type: TSD-602S, resolution: 5 �m� moves up and down relative to thefocal plane. For securing the controllability of the cell track-ing, the linearity of the depth measurement should be accom-plished. As shown on the left of Fig. 4, when the area of A isbrighter than the area of B, the light intensity ratio is ob-tained by A /B−1. For the right side, the area of B is brighterthan the area of A. In this situation, the light intensity ratio isobtained by −�B /A−1�. For example, when the samplemoves 10 �m downward, the measuring ratio is about 1.7. Ifthe sample moves 10 �m upward, the measuring ratio isabout −1.1. These experimental results confirm the good lin-earity of the depth measurements.

FIG. 2. Geometric optics model of diffracted light when the sample movesto greater depth. The direction of the positive or negative first order dif-fracted light changes and forms the images at a different point as a lightcircle. �a� The sample moves toward the left. �b� The sample is at the focalplane. �c� The sample moves toward the right.

FIG. 3. The light intensity distributionin minute areas can be measured sepa-rately as two regions using a split pho-todiode. A HeNe laser was used as alight source.

241102-2 Yoshida et al. Appl. Phys. Lett. 89, 241102 �2006�

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In conclusion, we propose depth displacement measure-ment to achieve three-dimensional tracking of transparentcells. The proposed optical spatial filtering allows detectionof depth migration as horizontal displacement of the positiveor negative order diffracted light on the detector surface.Depth movement can be measured as the light intensity ratio

of the split photodiode. Good linearity of the depth displace-ment measurement for the transparent cells was verified.

This study was supported by the 2004 Industrial Tech-nology Research Grant Program from New Energy and In-dustrial Technology Development Organization �NEDO� ofJapan.

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FIG. 4. Confirmation of the good linearity of the depth measurements fromexperimental results. As shown on the left side of the figure, the area of A isbrighter than the area of B. As shown on the right side of the figure, the areaof B is brighter than the area of A.

241102-3 Yoshida et al. Appl. Phys. Lett. 89, 241102 �2006�

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