Image plane holograms for holographic microscopy

Mary E. Cox and Kerry J. Vahala

We have studied the use of image holograms in holographic microscopy. Reducing the degree of spatial co-herence in the reconstructing light source reduces the speckle in the reconstructed real image field. A care-ful choice of recording parameters is necessary to retain the resolution needed to study microscopic objects.Sample photomicrographs of test targets, blood cells, and human hairs are included.

Image plane holograms have long been known tobe capable of reconstructing an object field using largeilluminating sources with little spatial coherence.1 -3 Asthe image distance approaches zero (that is, when theimage is formed in the plane of the photosensitive re-cording medium), the degree of spatial coherence canbe made small, still retaining the resolution in theimage. Since the degree of spatial coherence is directlyrelated to the amount of speckle in the reconstructedimage, reducing the spatial coherence during recon-struction can also reduce the speckle originating duringreconstruction.

For microscopic objects, speckle can degrade thequality of the reconstructed image. This often obscuresthe detail one seeks to examine in a holographic mi-croscope. We have studied the use of image plane ho-lograms in holographic microscopy. Our goal is to re-duce the amount of speckle by reducing the degree ofspatial coherence needed to reconstruct the image, whileretaining the resolution needed to study microscopicobjects.

We record the image plane hologram in the conven-tional manner. 3 In our studies we use USAF test tar-gets, stained blood smears, and human hairs arrangedin three layers. We transilluminate the object, col-lecting the scattered light with a lens having a 3.1-cmfocal length and a 2.5-cm diam. We attempt to keep theimage of the object laterally magnified no more than tentimes. With this geometry, objects larger than 3 gm canbe resolved using a cw He-Ne laser operating at 0.6328

When this work was done both authors were with University ofMichigan-Flint, Department of Physics & Astronomy, Flint, Michi-gan 48503. M. E. Cox is on leave at University of Oxford, PhysicsDepartment, Clarendon Laboratory, Oxford, U.K., and K. J. Vahalais a student at California Institute of Technology, Pasadena, Cali-fornia 91103.

Received 13 August 1977.0003-6935/78/0501-1455$0.50/0.©0 1978 Optical Society of America.

gim. The reference beam is collimated and is incidenton the film at an angle of 100 to the normal to the filmplane.

We use Kodak SO-253 film with a bias exposure of 2.0ergs/cm 2 when developed in D-19 at 251C for 5 min.After processing we remove the residual blue dye witha 1:1 mixture of methanol and water. The film is airdried before viewing. With this technique we found lessthan 1% shrinkage when making sine gratings with twocollimated beams, each incident at 100 from the normalto the film plane.

To reconstruct the image from the processed holo-gram, we use a collimated laser beam (X = 0.6328 gim).To reconstruct the real image, the reconstruction beamis incident at 100 to the normal to the back of the filmplane, i.e., 1800 from the direction of the original ref-erence beam. To remove any aberrations introducedby the lens during recording, we reintroduce the lens inthe same location it had during recording.4 5 We ex-amine the recontructed real image, which has now beendemagnified to its original size by the lens, with a con-ventional microscope. Photomicrographs of this imageare taken through this conventional microscope.

Paraxial optics predicts that the longitudinal mag-nification is proportional to the lateral magnificationsquared. Thus, a 10-gm thick object, when magnifiedlaterally by a factor of 10, should be 1000 ,m = 1 mm indepth. Only the center of this object can be in the planeof the film. However, by keeping the reconstructingbeam as parallel as possible, so the distance from thereconstructing source to the hologram is essentiallyinfinite (compared with a few millimeters), we have seenno loss in resolution in the image due to object pointsimaged in front of or behind the film. We used a sourcehaving a diameter of 4 cm to illuminate fully the holo-gram.

Our initial reconstructions were made with the whitelight from a slide projector. At low magnifications withthe conventional microscope the reconstructions wereclear and reasonably bright. However, at higher mag-nifications (>100X) the images could hardly be seen.

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(a)

4 k w' . n §

t ii + k#Ff-iiX = .' i m {-I1

-~ ~ ~ I t : 3f.U 3;

(b)

Fig. 1. Photomicrographs of reconstructed real image of USAF testtarget. The resolution limit on the original negatives is group 7, line3, which has an original linewidth of 3.10 im. Photomicrographs weretaken with nonspatially coherent light (a) and spatially coherent light

(b) through a conventional microscope.

Fig. 2. Photomicrographs of reconstructed real image of stainedhuman blood cells with human hair for reference. The blood cellshave diameters from 5,um to 10,um. Photomicrographs were takenwith nonspatially coherent light (a) and spatially coherent light (b)

through a conventional microscope.

(a) (b) (c)

Fig. 3. Photomicrographs of reconstructed real image of human hairs at three depths. The original hairs were mounted on three microscopeslides about 1 mm thick. These photomicrographs were all taken with nonspatially coherent light. The horizontal hair near the top is in

focus in (a), the vertical hair is in focus in 'b), and the horizontal hair near the middle is in focus in (c).

Using a filtered mercury spectral source (X = 0.5961 gim)increased the brightness for viewing with the eye, butnot enough for taking photomicrographs without longexposure times. To take photomicrographs with ex-posure times less than 10 min using Polaroid negativefilm, we use a 5-mW He-Ne laser. To reduce the spatialcoherence of this reconstructing beam, yet keep itslongitudinal coherence large (its bandwidth small) and

retain the brightness in the field of the microscope, weplace a moving diffuser in a plane between the lens thatis collimating the expanded laser beam and the holo-gram. For depths up to 2 mm, image resolution remainsas theoretically predicted. Speckle in the image is re-duced dramatically, even as higher microscope magni-fications (large numerical apertures) are used.

Figures 1-3 show representative samples of our re-

1456 APPLIED OPTICS / Vol. 17, No. 9 / 1 May 1978

(b)

(a)

sults to date. All photomicrographs are taken using a10X flat, photo eyepiece, and a 1oX objective (0.25 =NA). The actual magnification at the plane of thenegative in the camera is 87X. The photomicrographsof the USAF target [Figs. 1(a) and 1(b)] are shown toobtain an accurate scale since, in printing, we cannotguarantee an accurate demagnification. Group 5, line1 has a linewidth of 15.63 gm on the original; group 6,line 1 has a linewidth of 7.81 gim; group 7, line 1 has alinewidth of 3.91 gim. The limit of resolution on theoriginal negative is group 7, line 3, which has a width of3.10 gim. Figure 1(a) was taken with diffused laser light;Fig. 1(b) was taken with nondiffused laser light. Asidefrom the obvious removal of speckle due to the spatialcoherence of the laser, Fig. 1(a) also shows some otherinteresting properties. Dirt, in the form of dust and finefibers, must have been on the microscope or cameraoptics. This can be seen and identified in Fig. 1(a); itcannot be distinguished from the speckle in Fig. 1(b).Newton's rings can also be seen in Fig. 1(a) and are dueto the temporal coherence of the laser.

Figure 2 shows photomicrographs of the recon-structed real image of stained human blood cells, witha human hair (originally 50 gm in width) for reference.Figure 2(a) was taken with diffused laser light; Fig. 2(b)was taken with nondiffused laser light. Again some dirtfrom the microscope and/or camera can be identifiedin Fig. 2(a). Also, Newton's rings can be seen. Thelarger diameter circles at the top of Fig. 2(a) are due totwo-beam interference between the front and backsurfaces of the microscope slide on which the blood cellsare mounted and were recorded on the original holo-gram.

Figure 3 shows photomicrographs of the recon-structed real image of human hairs mounted at threedepths on microscope slides. All the photomicrographswere taken with diffused laser light. The three views(a), (b), and (c) show hairs at different depths in focus.The vertical hair (originally about 80 gm in width) isfocused in the plane of the hologram. The hair farthestfrom the hologram during recording (originally about80 gm in width), i.e., closest to the lens, appears near thetop of all three views and is in focus of Fig. 3(a). Thehair closest to the hologram during recording (originallyabout 95 ,gm in width), i.e., farthest from the lens, ap-pears near the middle of all three views and is in focusin Fig. 3(c). The gross fringe structure seen in thebackground on all three views originated in multiplebeam interference between the glass microscope slidesurfaces during recording. The fine fringes surroundingthe out-of-focus hairs originates from diffraction andinterference in the original hologram recording. Thedirt and Newton's rings come from the microscopeand/or camera optics while recording the photomicro-graphs.

In recording the image plane holograms of the hairs,the original separation in depth between adjacent layersis the thickness of a standard microscope slide, about1 mm. This results in a separation in depth betweenadjacent layers of 9 cm on magnification of about 1OXto record the hologram. Using a collimated reference

beam at an angle of 100, the relative bandwidth neces-sary to reconstruct objects 3 gm in size and 9 cm fromthe hologram plane is about 1.7 X 10-6, which is easilyachieved using atomic line sources. Although mostbiologic objects are not this thick, it is interesting to notethat good quality images can be obtained over a depthchange of 2 mm. On reconstruction and demagnifica-tion the images of the hairs were within a depth of 2mm.

These results are important for the development ofholographic microscopy in three ways. First, thequality of the image seen through the microscope issuperior to other techniques. To locate and identifymicrostructure in a macroscopic sample, the image mustbe as free from unwanted noise as possible. By recon-structing the hologram with a light source having a verylow degree of spatial coherence, the image appears morelike that obtained from a conventional microscope.The out-of-focus gross fringe structure is not bother-some when viewing the images, because the detail in theimages is easily identified. Second, the reconstructedimage is easily seen and aligned for maximum clarity.No random apertures, spatial filters, or other devicesusual in image processing are needed. The image seenthrough the microscope is bright, clean, and free fromaberrations. Third, resolution and depth are preserved.Most microscopic objects can be transilluminated andimaged with a simple, inexpensive lens. With a rela-tively simple, stable apparatus setup, most biologicobjects can be imaged and studied.

We have demonstrated that image plane hologramscan be used in holographic microscopy. The quality,resolution, and depth in the reconstructed image fieldwhen studied with a conventional microscope are ex-cellent. Since most biologic samples are thin and dilute,this technique should provide an impetus to micro-scopists to evaluate holographic microscopy for theirapplications.

Portions of the equipment used for these experimentswere acquired from an unrestricted grant from theNational Institutes of Health to the University ofMichigan. Other equipment had been obtained pre-viously from a grant from the Office of Naval Re-search.

References1. L. Rosen, Appl. Phys. Lett. 9, 337 (1966).2. G. W. Stroke, Phys. Lett. 23, 325 (1966).3. R. J. Collier, C. B. Burkhardt, and L. H. Lin, Optical Holography

(Academic, New York, 1971), pp. 204-206.4. L. Toth and S. A. Collins, Jr., Appl. Phys. Lett. 13, 7 (1968).5. M. E. Cox, R. G. Buckles, and D. Whitlow, Appl. Opt. 10, 128

(1971).

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