We have built a simple holographic microscope completely out of consumer components. We obtain at least 2.8 micrometer resolution and depth of field greater than 200 micrometers from an instrument costing less than $1000.Engineering and Applied SciencesPhysic

Dimiduk, Thomas Gwilym

Gardel, Emily Jeanette

Kaz, David

Kosheleva, Ekaterina Alexeevna

Manoharan, Vinothan N.

McGorty, Ryan

Harvard University - DASH 

 A Simple, Inexpensive Holographic Microscope  (Article begins on next page)The Harvard community has made this article openly available.Please share how this access benefits you. Your story matters.Citation Dimiduk, Thomas G., Ekaterina A. Kosheleva, David Kaz, RyanMcGorty, Emily J. Gardel, and Vinothan N. Manoharan. 2010. Asimple, inexpensive holographic microscope. In DigitalHolography and Three-Dimensional Imaging, OSA TechnicalDigest (CD), paper JMA38.http://www.opticsinfobase.org/abstract.cfm?URI=DH-2010-JMA38.Published Version http://www.opticsinfobase.org/abstract.cfm?URI=DH-2010-JMA38Accessed February 19, 2015 9:24:28 AM ESTCitable Link http://nrs.harvard.edu/urn-3:HUL.InstRepos:8300209Terms of Use This article was downloaded from Harvard University's DASHrepository, and is made available under the terms and conditionsapplicable to Open Access Policy Articles, as set forth athttp://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#OAPA Simple, Inexpensive Holographic MicroscopeThomas G. Dimiduk1, Ekaterina A. Kosheleva1, David Kaz1, Ryan McGorty2,Emily J. Gardel2, Vinothan N. Manoharan1,21Department of Physics, Harvard University2School of Engineering and Applied Sciences, Harvard Universitytdimiduk@physics.harvard.eduAbstract: We have built a simple holographic microscope completely out of consumer com-ponents. We obtain at least 2.8µm resolution and depth of field greater than 200µm from aninstrument costing less than $1000.OCIS codes: (090.0090) Holography, (180.0180) Microscopy.1. IntroductionHolographic microscopes have the potential to avoid some of the complexities of conventional microscopes. A holo-graphic microscope focuses numerically, reducing the need for mechanical translation. Holography is inherently threedimensional, a capability otherwise requiring confocal or other complicated apparatus. Holography intrinsically pos-sesses phase contrast without a specialized illumination system. Finally, recent advances in semiconductor technologyhave now combined to make it possible to build a holographic microscope out of mass-market consumer components.Combined with the simplicity inherent in holography, this gives the possibility of a three dimensional imaging systemwhich is simple and inexpensive, very inexpensive relative to other scientific microscopes. Such a microscope thenbecomes a module that can be cheaply and easily added to another apparatus instead of being a dominating focus ofdesign. Cost reduction by an order of magnitude or more will also allow such microscopes to be used in places wheremicroscopes were not even considered previously, particularly in industry, medicine, and education.We present an example of such a simple microscope. The core of our apparatus is a consumer digital camera, abattery powered diode laser, a microcontroller and a generic microscope objective. Even with supporting optics andstructure, the total component cost is well under $1000. With this minimal instrument we are still able to achieveresolution in excess of 3µm and depth of field in excess of 200µm.2. Apparatus and MethodsWe are primarily interested in transparent colloidal and biological samples, so we can make use of inline holographyto keep complexity down. Our apparatus, Fig. 1, consists of, from top to bottom, a Canon EOS 300D digital camera,a Newport 20x 0.4 NA microscope objective, a slide holder mounted to a 3 axis micrometer stage, a beam telescope,a 785 nm laser diode, and an Arduino microcontroller board. The Canon EOS 300D is a 6.3 megapixel camera witha 3088x2056 pixel 22.7 mm x 15.1 mm CMOS sensor. The microscope objective sufficiently magnifies hologramfringes for adequate resolution by the camera’s 7.35µm square pixels. Illumination from the 785 nm ADL 78901 laserdiode is expanded by the beam telescope to increase uniformity across the field of view.Control is centralized under the Amtel ATMega168 microcontroller based Arduino board from Adafruit Industries.It controls the camera’s external shutter release with a digital output and a transistor that powers the laser diode withan analog output. We synchronize the camera shutter with a laser pulse, allowing imaging with a 2.3 mW, 80µs pulse(power measured with a Thor Labs PM 100). The camera stores images internally on a flash memory card. Combinedwith battery power for all components, this allows the instrument to capture holograms in a completely self containedmanner.After an experiment, we transfer images to a standard computer for reconstruction. We reconstruct using the convo-lution method described by Schnars and Juptner [1] and extract both phase and amplitude information. We take intoaccount the action of the lens as described by Sheng [2]. We preform reconstructions using custom software written inPython with the SciPy numerical library and running on a 3 GHz Intel Core 2 Duo CPU.We determined the resolution of our instrument by imaging a USAF 1951 resolution test target manufactured byEdmund optics. We investigated imaging capabilities with 2µm Invitrogen sulfate latex particles embedded in KnoxGelatine. We made an ∼ 200µm thick sample using vacuum grease as a spacer between a slide and a number oneOSA / BIOMED/DH 2010       JMA38.pdf  Canon EOS 300D CameraArduino Board20x, 0.4 NANewportObjectiveSample Slide150 mm Lens40 mm lens785 nm Laser diode3 axis Micrometer16 cm (to detector)19 cm~ 1 mmFig. 1: Diagram of the microscope apparatus. Distances indicated can be varied by at least 10% without substantially affectingoperation.cover slip. We loaded gelatin hot, once cooled it solidified preventing sedimentation, keeping a sample interesting inthree dimensions.3. ResultsWe determined the resolution of our instrument to be at least 2.8µm. Fig. 2 shows the hologram and reconstruction weused to establish this resolution. The second smallest resolution element is discernible and contains elements spacedby 2.8µm. This resolution is approximately a factor of three worse than the ∼ 1µm diffraction limited resolution ofour 0.4 NA objective at 785 nm. We believe this resolution defect is from a combination of loss of fringes spacedtoo finely for the camera’s pixels and from obscurement of fringes by sensor noise, illumination inhomogeneity, andreflection and scattering from optics.(a) Hologram (b) ReconstructionFig. 2: Image of USAF 1951 resolution grid showing 2.8µm resolutionWe demonstrate three dimensional imaging and broad depth of field with 2µm sulfate latex colloids embedded ingelatin. From a single hologram, Fig. 3(a), we can reconstruct at many different planes and visualize distinct particles,example planes are shown in Figs. 3(b-d).OSA / BIOMED/DH 2010       JMA38.pdf  (a)Holo-gram(b) Amplitude Reconstruction z= 220µm (c) Amplitude Reconstruction z= 320µm(d) Amplitude Reconstruction z= 420µmFig. 3: Sulfate Latex Particles in gelatin4. ConclusionWe demonstrate the feasibility of constructing a simple, low cost holographic microscope with nonetheless usefulcapabilities. We show that a straightforward implementation can achieve at least 2.8µm resolution and at least 200µmdepth of field.With more advanced background subtraction and noise reduction algorithms and further optimization of the opticalpath, a sub $1000 microscope that approaches diffraction limited resolution should be possible. This design keepsdifficult optics to a minimum, facilitating implementation by those not traditionally involved in holography.[1] U. Schnars and W. P. Juptner, “Digital recording and numerical reconstruction of holograms” Measurement science and technology 13,85101(2002).[2] J. Sheng, E. Malkiel, and J. Katz, “Digital holographic microscope for measuring three-dimensional particle distributions and motions”Applied optics 45,38933901 (2006).OSA / BIOMED/DH 2010       JMA38.pdf  

A Simple, Inexpensive Holographic Microscope

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A Simple, Inexpensive Holographic Microscope

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