Low-cost and portable UV holographic microscope for high-contrast protein crystal imaging Cite as: APL Photonics 4, 030804 (2019); https://doi.org/10.1063/1.5080158 Submitted: 06 November 2018 . Accepted: 31 January 2019 . Published Online: 01 March 2019 Mustafa Ugur Daloglu, Aniruddha Ray, Michael J. Collazo, Calvin Brown , Derek Tseng, Blanca Chocarro-Ruiz, Laura M. Lechuga, Duilio Cascio, and Aydogan Ozcan ARTICLES YOU MAY BE INTERESTED IN Adiabatic mode converters for silicon photonics: Power and polarization broadband manipulators APL Photonics 4, 030803 (2019); https://doi.org/10.1063/1.5080247 Nonlinear optics in carbon nanotube, graphene, and related 2D materials APL Photonics 4, 034301 (2019); https://doi.org/10.1063/1.5051796 Inter-modal Raman amplification of OAM fiber modes APL Photonics 4, 030802 (2019); https://doi.org/10.1063/1.5051794 APL Photonics 4, 030804 (2019); https://doi.org/10.1063/1.5080158 4, 030804 © 2019 Author(s). APL Photonics ARTICLE scitation.org/journal/app Low-cost and portable UV holographic microscope for high-contrast protein crystal imaging Cite as: APL Photon. 4, 030804 (2019); doi: 10.1063/1.5080158 Submitted: 6 November 2018 • Accepted: 31 January 2019 • Published Online: 1 March 2019 Mustafa Ugur Daloglu,1,2,3,a) Aniruddha Ray,1,2,3,b) Michael J. Collazo,4,5,6,7,8,c) Calvin Brown,1,2,3,d) Derek Tseng,1,2,3,e) Blanca Chocarro-Ruiz,9,f) Laura M. Lechuga,9,g) Duilio Cascio,4,5,6,7,8,h) and Aydogan Ozcan1,2,3,10,i) AFFILIATIONS 1 Electrical and Computer Engineering Department, University of California, Los Angeles, California 90095, USA 2 Bioengineering Department, University of California, Los Angeles, California 90095, USA 3 California NanoSystems Institute (CNSI), University of California, Los Angeles, California 90095, USA 4 Howard Hughes Medical Institute, University of California, Los Angeles, California 90024, USA 5 Department of Energy Institute, University of California, Los Angeles, California 90024, USA 6 Biological Chemistry Department, University of California, Los Angeles, California 90024, USA 7 Chemistry and Biochemistry Department, University of California, Los Angeles, California 90024, USA 8 Molecular Biology Institute, University of California, Los Angeles, California 90024, USA 9 Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, BIST, and CIBER-BBN, Campus UAB, Bellaterra, 08193 Barcelona, Spain 10 Department of Surgery, David Geffen School of Medicine, University of California, Engr. IV 68-119, 420 Westwood Plaza, Los Angeles, California 90095, USA Note: This paper is part of an APL Photonics Special Topic on Computational Optical Imaging. a) mudaloglu@ucla.edu b) rayani@ucla.edu c) mcollazo@mbi.ucla.edu d) calvinbrown@g.ucla.edu e) delike@ucla.edu f) blanca.chocarro@icn2.cat g) laura.lechuga@icn2.cat h) cascio@mbi.ucla.edu i) Author to whom correspondence should be addressed: ozcan@ucla.edu. Tel.: +1(310)825-0915. Fax: +1(310)206-4685. ABSTRACT Imaging protein crystals and distinguishing them from salt crystals is an important task for protein crystallographers. The conventional tool used for this purpose is a dual-mode microscope composed of bright-field and ultraviolet (UV) induced fluo- rescence modes. The distinction between a protein and a salt crystal is made based upon the fluorescence response to the UV excitation, where most protein crystals absorb the UV excitation and emit fluorescence, unlike salt crystals. These dual-mode optical microscopes are sensitive; however, they are relatively bulky and expensive as they require UV-grade optics. As an alter- native, here we demonstrate that on-chip UV holographic imaging offers a low-cost, portable, and robust technique to image and distinguish protein crystals from salt crystals, without the need for any expensive and bulky optical components. Only composed of a UV light-emitting-diode at 280 nm and a consumer-grade complementary metal–oxide–semiconductor image sensor de- capped and interfaced to a Raspberry Pi single-board computer, the necessary information from the crystal samples (placed very close to the sensor active area) is captured in the form of in-line holograms and extracted through digital back-propagation. In these holographic amplitude reconstructions, protein crystals appear significantly darker compared to the background due to the APL Photon. 4, 030804 (2019); doi: 10.1063/1.5080158 4, 030804-1 © Author(s) 2019 APL Photonics ARTICLE scitation.org/journal/app strong UV absorption, unlike salt crystals which do not show any contrast, enabling us to clearly distinguish between them. We believe that the on-chip UV holographic microscope could serve as a low-cost, sensitive, and robust alternative to conventional lens-based UV-microscopes used in protein crystallography. © 2019 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/1.5080158 I. INTRODUCTION the need for imaging lenses or other bulky optical compo- nents, only using consumer-grade image sensor chips25 and Protein crystallographers rely on a dual-mode optical simple light sources like light-emitting diodes (LEDs) open- microscope composed of bright-field and ultraviolet (UV) ing up new frontiers for portability.26–29 The ever decreasing induced fluorescence modes to image protein crystals as well peak emission wavelengths and increasing efficiencies of LED as to distinguish them from salt crystals that could form dur- technologies made the deep UV LEDs (240 nm-360 nm)30–33 ing the crystallization process. This distinction is mainly based more available and affordable. This, when integrated with the on the response to the UV illumination, where most protein capabilities of portable and cost-effective on-chip holographic crystals absorb the UV light and emit fluorescence through microscopy tools, enables a wide range of studies related to tryptophan residues, unlike most salt crystals.1,2 In addition proteins, particularly in the field of crystallography. to UV fluorescence,3,4 the strong absorption of UV light within Here we present a portable on-chip holographic imag- organic materials5,6 has been utilized as an inherent contrast ing platform (Fig. 1) that is composed of a UV-LED operat- agent in imaging tissue samples,7 cells,8 intracellular nucleic ing at 280 nm peak wavelength and a de-capped comple- acids and proteins,9–12 viruses,13 and protein aggregates,14 mentary metal–oxide–semiconductor (CMOS) image sensor making UV microscopy15 an important tool for researchers. connected to a Raspberry Pi single-board computer.34,35 The However, conventional lens-based UV microscopy is a rela- sample is placed very close (∼300-400 µm) to the image sen- tively expensive imaging modality, requiring the use of rel- sor and the light source further above, where the interference atively bulky optical components that are specially designed between the light scattered from the target crystals and the for UV wavelengths, in addition to UV light sources and UV- background illumination creates in-line holograms that are sensitive cameras for bright-field imaging,2 adding up to sig- digitized/recorded by using the image sensor. The captured nificant costs (e.g., $35k-200k).16 Furthermore, inherent lim- frames are then digitally processed to extract the informa- itations of lens-based microscopy also apply to conventional tion about the target objects that is encoded in the holograms, UV microscopes, where the trade-off between the field-of- resulting in UV transmission images over a large FOV that is view (FOV) and resolution limits the total sample area that can only limited by the sensor active area (>10 mm2 ). be imaged. The utility of this platform in crystallography was demon- As an alternative, on-chip holographic imaging17–20 offers strated by imaging different protein crystals including pro- a cost-effective option21–24 for such microscopy tasks without teinase K (Figs. 2 and 3), the RING1B complex (Fig. 4), maltose FIG. 1. (a) Our portable holographic on-chip imaging platform operates at 280 nm illumination wavelength. The UV LED is spectrally filtered using a band- pass filter to block the side-bands, let- ting through pure UV light with 280 nm peak wavelength and a 10 nm band- width toward the sample, which is placed very close to the image sensor (∼300- 400 µm), utilizing the full active area as the imaging FOV (>10 mm2 ). (b) A sim- plified CAD model of the same portable on-chip imaging platform. APL Photon. 4, 030804 (2019); doi: 10.1063/1.5080158 4, 030804-2 © Author(s) 2019 APL Photonics ARTICLE scitation.org/journal/app FIG. 2. Using the setup shown in Fig. 1, proteinase K crystals appear significantly darker compared to the background level due to the strong UV absorption at 280 nm, unlike salt crystals. (a) Bright- field microscope images of proteinase K, sodium chloride, and ammonium sul- fate crystals. (b) Amplitude reconstruc- tions of the same FOVs in (a), proteinase K crystals appearing significantly darker. (c) X-ray diffraction data from the pro- teinase K, sodium chloride, and ammo- nium sulfate crystal samples, with largest spacing values of 30 Å, 3.25 Å, and 3.25 Å, respectively. binding protein (Fig. 4), and salt crystal samples which include image sensor. These holographic images are then digitally pro- sodium chloride (Fig. 2), ammonium sulfate (Fig. 2), lithium cessed to extract the amplitude information encoded in the acetate (Fig. 3), and lithium sulfate (Fig. 3). Our mobile holo- interference patterns from the target objects. graphic platform can clearly differentiate between protein We tested our portable on-chip imaging platform to verify crystals and salt crystals due to the strong contrast in the the effect of strong UV absorption in our amplitude recon- protein crystal images which appear significantly darker com- structions, imaging protein crystals (Fig. 2). Samples were pre- pared to the background. Without the need for fine align- pared by constructing imaging chambers using UV fused silica ment and temperature stability, unlike its lens-based counter- glass slides and pieces of ACLAR protein crystallization covers, parts, this platform would be a low-cost, robust, and portable a standard material used by protein crystallographers which alternative to conventional UV microscopes used by protein is also UV transparent. It is important to note that additional crystallographers. consideration has to be given in the transparency of mate- rials for coherent imaging modalities like holography, as the irregularities in the material volume could result in strong II. RESULTS AND DISCUSSION background distortions. The standard UV transparent ACLAR Our portable on-chip imaging platform (Fig. 1) is built used by protein crystallographers is suitable for holographic upon the versatile Raspberry Pi 3 board,35 with its readily imaging, only creating a faint modulation in the background available 8 Megapixel CMOS camera, all housed within a cus- [Fig. 2(b)] which does not affect our imaging quality. tom designed and 3D printed shell which also holds a UV LED Our imaging platform (Fig. 1) was first tested by prepar- operating at 280 nm peak wavelength with a band-pass filter ing a sample with proteinase K, a serine protease readily to block the side-band emissions.31,32,36 The CMOS camera available for crystallization, which contains aromatic amino is de-capped, removing the lens module, and the sample is acids that fluoresce when excited at 280 nm light, and two placed very close (∼300-400 µm) to the active area of the salt crystal samples (sodium chloride and ammonium sulfate) image sensor chip that is fully utilized as the imaging FOV [Figs. 2(a) and 2(b)], where the respective X-ray diffraction [>10 mm2 , Fig. 3(a)]. The filtered UV light is scattered through data are shown in Fig. 2(c). Individual crystals were pipetted the sample, generating in-line holograms through its interfer- along with ∼1 µl of solution onto the ACLAR piece (sticky side ence with the background light that is captured by using the facing up), and a UV fused silica was used to seal the droplet APL Photon. 4, 030804 (2019); doi: 10.1063/1.5080158 4, 030804-3 © Author(s) 2019 APL Photonics ARTICLE scitation.org/journal/app FIG. 3. A low-cost, portable, and high-throughput alternative to lens-based UV microscopes used by protein crystallographers to distinguish between protein and salt crystals. (a) The full FOV (>10 mm2 ) image captured by our on-chip microscopy platform (Fig. 1) where the red rectangle shows a typical FOV possible using a lens-based UV microscope with a 5× objective lens. (b) Samples containing proteinase K and salt crystals (lithium acetate and lithium sulfate) in the same chamber, imaged by using the lens-based UV microscope in both brightfield and UV fluorescence modes. The proteinase K crystals strongly absorb the 280 nm UV light and emit fluorescence, where the salt crystals do not. (c) Protein crystals show a significantly stronger contrast compared to salt crystals (p < 0.02). The standard deviation bars represent the variation of the contrast within the rectangular regions used to calculate the average contrast of the target objects. (d) The amplitude reconstructions of the same FOVs captured by our on-chip UV imaging platform. The same crystals that emitted fluorescence in (b) appear significantly darker compared to the background due to the stronger absorption, while the crystals that did not emit fluorescence do not show statistically significant contrast in our amplitude reconstructions. into the chamber. The amplitude reconstructions of our holo- significantly stronger contrast (p < 0.02) compared to the grams clearly show that the protein crystals appear much salt crystals [Fig. 3(c)]. We calculated the contrast (C) in darker compared to the background, unlike the salt crys- our UV holographic amplitude reconstructions by subtract- tals [Fig. 2(b)]. The amplitude reconstruction results shown ing the average amplitude signal of the target crystals (Sc ) in Fig. 2(b) could be further improved using various phase- from the average background signal value (Sb ) and dividing this retrieval techniques17 to mitigate twin-image artifacts, at the difference by the average background signal value,14,37 i.e., cost of additional computation and/or holographic measure- Sb − Sc ments. C= , (1) Sb To further evaluate our portable on-chip holographic imaging platform, we imaged mixed-samples containing both where Sc is calculated within the largest rectangular region protein (proteinase K) and salt crystals (lithium acetate and that fits inside the target crystal and Sb is calculated within a lithium sulfate) within the same FOV [Figs. 3(a), 3(b), and 3(d)], clear region of the FOV that does not contain any objects. prepared by sandwiching droplets of ∼1 µl buffer solution To further test the imaging capabilities of our portable containing the crystals between a UV fused silica slide and on-chip holographic imaging platform for protein crystallog- a piece of ACLAR sheet. The crystals were grown in their raphy, additional experiments were performed using (1) the respective buffer solutions and then individually fished by a RING1B complex, which is associated with the nuclear mem- microloop and deposited in the droplet before sealing. The brane and participates in histone ubiquitination in humans,38 samples were first imaged with the lens-based microscope, and (2) the maltose binding protein, which breaks down mal- which is able to distinguish between the protein and the todextrins in Escherichia coli and also forms UV active crys- salt crystal through the fluorescence of the protein crystal tals39 (Fig. 4). All the samples under test were imaged by using [Fig. 3(b)], where the protein crystal is observed to be emitting the lens-based UV microscope first and then by using our on- fluorescence, while the salt crystal remains dark. The sam- chip UV microscope [Fig. 4(a)]. The capabilities of our portable ples were then imaged with our on-chip microscopy platform UV imaging platform in distinguishing between protein and [Figs. 3(a) and 3(d)], where the proteinase K crystals showed a salt crystals were further verified with these different types APL Photon. 4, 030804 (2019); doi: 10.1063/1.5080158 4, 030804-4 © Author(s) 2019 APL Photonics ARTICLE scitation.org/journal/app FIG. 4. High contrast imaging capabilities of our portable on-chip holographic imag- ing platform further demonstrated by imaging RING1B complex and maltose binding protein crystals, where lithium acetate crystals were used as control. (a) The protein crystals showed fluores- cence when imaged by the lens-based UV imaging platform, while the salt crys- tals did not show any significant fluores- cence. In the same manner, amplitude reconstructions of the protein crystals showed a significant contrast, while the salt crystals did not. (b) This significant contrast difference (p < 0.003) clearly shows the efficacy of the on-chip UV imaging platform in differentiating protein crystals from salt crystals. The standard deviation bars represent the variation of the contrast within the rectangular regions used to calculate the average contrast of the target objects. APL Photon. 4, 030804 (2019); doi: 10.1063/1.5080158 4, 030804-5 © Author(s) 2019 APL Photonics ARTICLE scitation.org/journal/app of proteins, which showed a significantly higher contrast C. Sample preparation (p < 0.003) compared to salt crystals [Fig. 4(b)]. UV compatible materials which include UV fused silica In summary, recent improvements in UV LED technolo- slides (10 mm × 10 mm, 0.2 mm thick, MTI Corp., CA, USA) gies have made deep UV LEDs with short peak emission wave- and pieces of standard protein crystallization covers made lengths and improved efficiencies more accessible, enabling of ACLAR composed of poly-chloro-trifluoroethylene (Grace the design of a portable and low-cost on-chip UV imag- Bio-Labs ProCrystal Cover 875238, OR, USA) were used to con- ing platform as a potential alternative to the expensive and struct the sample chambers holding the crystal samples. A bulky dual-mode UV microscopes used by protein crystallog- 0.8-1 µl droplet containing the crystals and the corresponding raphers. We expect that our portable on-chip UV holographic buffer solution was deposited onto an ACLAR piece containing imaging platform could be even further strengthened with one well, sticky side facing up. A UV fused silica slide was then near real-time imaging capabilities, driven by future improve- gently used to cover the well, sealing the droplet. It is note- ments in deep UV LED power output efficiencies, enabling worthy that the ACLAR standard protein crystallization cover the use of lower sensor integration times and the increasing material was suitable for coherent imaging and only resulted in availability of embedded graphics processing units (GPUs) for a faint background modulation [Fig. 2(b)] which did not affect single-board computers. our imaging quality. III. MATERIALS AND METHODS D. Protein and salt crystallization A. UV on-chip imaging platform A TTP LabTech mosquito (TTP Labtech, Inc., MA, USA) was used to generate 96-well hanging drop crystallization setups. Our portable on-chip microscopy platform (Fig. 1) is com- All protein crystals were grown in a manner of days using posed of a de-capped 8 Mega-pixel (3280 horizontal × 2464 vapor diffusion. Proteinase K (VWR catalog number 97062- vertical, with an active area of ∼10.14 mm2 ), 1.12 µm pixel 238, PA, USA) was crystallized by dissolving lyophilized pow- pitch, CMOS image sensor (IMX219, Sony Corporation, Tokyo, der in water to obtain a 50 mg/ml stock. The stock solu- Japan) interfaced to a Raspberry Pi 3B single-board computer, tion was mixed 1:1 with 1.5 M ammonium sulfate and 0.1 M a deep UV LED operating at 280 nm peak wavelength (TH- Tris-HCl pH 7.5. Maltose binding protein 80 mg/ml in 20 mM UV280J9-C-H-B, Tianhui Optoelectronics Co., Ltd, Guang- Tris–HCl pH 8.0 and 50 mM NaCl was crystallized by mix- dong Province, China), and a UV band-pass filter with a ing 1:1 with 0.2M magnesium chloride hexahydrate, 0.1M center wavelength of 280 nm and a bandwidth of 10 nm (FF01- 4-Morpholineethanesulfonic acid pH 6.0%, and 20% w/v 280/10-25, Semrock, NY, USA) to block the side-band emis- Polyethylene glycol 6000. Oligomerization regions of RING1B, sions31,32,36 from the UV LED. All of these components are polycomb group RING finger protein 4, chromobox 8, and housed within a custom designed and 3D printed acrylonitrile polyhomeotic homolog 1 were supplied by the Chemistry and butadiene styrene (ABS) (Stratasys, Dimension Elite) shell. A Biochemistry Department at UCLA. This sample was mixed custom Python script was used to capture, extract, and save 1:1 with 0.7M sodium formate pH 7.0% and 20% w/v PEG the raw frames/holograms from the sensor to the onboard 3350. All 300 nl drops were equilibrated over 100 µl of the storage of the Raspberry Pi 3B. corresponding crystallization solutions. 1.0M sodium chloride, 2.0M ammonium sulfate, 1.0M B. Data processing lithium acetate, and 1.0 lithium sulfate were dispensed in µl Since the green pixels of the image sensor were most aliquots and allowed to evaporate in air while being observed responsive to the UV illumination,14 the values in the red in a stereomicroscope. Crystals that formed by dehydration and blue pixels of the raw frames were replaced with in aqueous solution were manually harvested using 50 µm the average of their neighboring green pixels. The frames microloops (Mitegen M5-L18SP-50LD, NY, USA) and placed in containing holographic projections were then digitally back- 1 µl of the stock salt solution. These solutions, containing crys- propagated using the angular spectrum approach,40 numeri- tals, were placed on the ACLAR surface by using a pipette for cally solving the Rayleigh-Sommerfeld integral by multiplying imaging. the Fourier transform of the hologram with the transfer E. Lens-based UV microscopy function of wave propagation,17,26 generating the amplitude images of the sample. The complete data processing takes A dual-mode UV microscope (Korima PRS-1000, CA, USA) ∼1 minute using a standard desktop computer (Dell Optiplex was used for comparison with the holographic imaging plat- 9010, Intel i7, 32 GB RAM) operating MATLAB (MathWorks, MI, form. Samples were imaged with the UV microscope first USA). and then holographically imaged with our mobile instrument. The statistical significance of the increased contrast in Crystals were exposed to 280 nm light for no more than 5 s, the amplitude reconstructions of protein crystals compared and the images taken were compared with the corresponding to salt crystals was verified using a t-test41 with two sepa- holographic images [Fig. 4(a)]. rate experiments for proteinase K [Fig. 3(d)] and three sep- arate experiments for the additional set of proteins which F. X-ray diffraction included the RING1B complex and the maltose binding protein To further distinguish protein crystals from salt, diffrac- [Fig. 4(a)]. tion images were taken. Individual crystals from the target APL Photon. 4, 030804 (2019); doi: 10.1063/1.5080158 4, 030804-6 © Author(s) 2019 APL Photonics ARTICLE scitation.org/journal/app sample were harvested and placed in their mother liquor REFERENCES with 33% glycol added to resist the formation of crys- 1 S. Desbois, S. A. Seabrook, and J. Newman, “Some practical guidelines for talline water. A rotating anode generator (Rigaku FRE+, Tokyo, UV imaging in the protein crystallization laboratory,” Acta Crystallogr., Sect. Japan) and an imaging plate detector (Rigaku HTC, Tokyo, F: Struct. Biol. Cryst. Commun. 69, 201–208 (2013). Japan) were employed for X-ray data collection. Macro- 2 C. S. Lunde et al., “UV microscopy at 280 nm is effective in screening molecule crystals are distinguishable from salt crystals by for the growth of protein microcrystals,” J. Appl. Crystallogr. 38, 1031–1034 lower resolution reflections that occur as the result of (2005). 3 larger spacing between symmetric elements of the crystal I. Vayá, T. Gustavsson, F.-A. Miannay, T. Douki, and D. Markovitsi, “Fluo- rescence of natural DNA: From the femtosecond to the nanosecond time [Fig. 2(c)]. scales,” J. Am. Chem. Soc. 132, 11834–11835 (2010). 4 Hargis, Jr. et al., “Ultraviolet fluorescence identification of protein, DNA, IV. CONCLUSIONS and bacteria,” Proc. SPIE 2366, 147 (1995). 5 Y. Kumamoto, “Deep-ultraviolet microscopy and microspectroscopy,” in We designed and built a low-cost and portable on-chip Far- and Deep-Ultraviolet Spectroscopy, edited by Y. Ozaki and S. Kawata holographic imaging platform operating at the deep UV wave- (Springer, Japan, 2015), pp. 123–144. length of 280 nm for high-contrast imaging of protein crys- 6 F.-X. Schmid, Biological Macromolecules: UV-Visible Spectrophotometry tals. Without the need for sensitive, bulky, and costly compo- (eLS, 2001). 7 nents, our platform offers a low-cost, high-throughput, and E. H. Land et al., “A color translating ultraviolet microscope,” Science 109, robust alternative to the dual-mode optical microscopes com- 371–374 (1949). 8 posed of bright-field and ultraviolet (UV) induced fluorescence J. R. G. Bradfield and M. Errera, “Ultra-violet absorption of living cells,” Nature 164, 532–533 (1949). modes that are routinely used by protein crystallographers 9 D.-K. Yao, K. Maslov, K. K. Shung, Q. Zhou, and L. V. Wang, “In vivo label- to image protein crystals and to distinguish them from salt free photoacoustic microscopy of cell nuclei by excitation of DNA and RNA,” crystals. We tested the on-chip holographic imaging platform Opt. Lett. 35, 4139–4141 (2010). by imaging different protein crystals including proteinase K, 10 B. J. Zeskind et al., “Nucleic acid and protein mass mapping by live-cell maltose binding protein, and the RING1B complex in compar- deep-ultraviolet microscopy,” Nat. Methods 4, 567–569 (2007). 11 ison with several different salt crystals which include sodium M. C. Cheung, J. G. Evans, B. McKenna, and D. J. Ehrlich, “Deep ultraviolet chloride, ammonium sulfate, lithium acetate, and lithium sul- mapping of intracellular protein and nucleic acid in femtograms per pixel,” fate. While the amplitude reconstructions of the protein crys- Cytometry, Part A 79A, 920–932 (2011). 12 M. C. Cheung et al., “Intracellular protein and nucleic acid measured in tals appear much darker compared to the background, the eight cell types using deep-ultraviolet mass mapping,” Cytometry, Part A salt crystals do not show any contrast, clearly distinguish- 83A, 540–551 (2013). ing between the two types of crystals. We believe that our 13 J. E. Barnard, “Microscopical evidence of the existence of saprophytic portable on-chip holographic platform could aid protein crys- viruses,” Br. J. Exp. Pathol. 16, 129 (1935). tallographers as a low-cost and robust alternative platform 14 M. U. Daloglu et al., “Computational on-chip imaging of nanoparticles and to image protein crystals and to distinguish them from salt biomolecules using ultraviolet light,” Sci. Rep. 7, 44157 (2017). 15 crystals. J. Smiles, “Ultraviolet microscopy,” J. Appl. Bacteriol. 21, 137–142 (1958). 16 H. S. Gill, “Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals,” Acta Crys- ACKNOWLEDGMENTS tallogr., Sect. F: Struct. Biol. Cryst. Commun. 66, 364–372 (2010). 17 Z. Gorocs and A. Ozcan, “On-chip biomedical imaging,” IEEE Rev. Biomed. The Ozcan Research Group at UCLA acknowledges the Eng. 6, 29–46 (2013). support of NSF Engineering Research Center (ERC, PATHS- 18 A. Greenbaum et al., “Imaging without lenses: Achievements and remain- UP), the Army Research Office (ARO; Nos. W911NF-13-1-0419 ing challenges of wide-field on-chip microscopy,” Nat. Methods 9, 889–895 and W911NF-13-1-0197), the ARO Life Sciences Division, the (2012). 19 National Science Foundation (NSF) CBET Division Biopho- D. Tseng et al., “Lensfree microscopy on a cellphone,” Lab Chip 10, 1787 tonics Program, the NSF Emerging Frontiers in Research (2010). 20 and Innovation (EFRI) Award, the NSF INSPIRE Award, NSF W. Luo, A. Greenbaum, Y. Zhang, and A. Ozcan, “Synthetic aperture-based on-chip microscopy,” Light Sci. Appl. 4, e261 (2015). Partnerships for Innovation: Building Innovation Capacity 21 E. McLeod and A. Ozcan, “Unconventional methods of imaging: Compu- (PFI:BIC) Program, the National Institutes of Health (NIH, No. tational microscopy and compact implementations,” Rep. Prog. Phys. 79, R21EB023115), the Howard Hughes Medical Institute (HHMI), 076001 (2016). Vodafone Americas Foundation, the Mary Kay Foundation, and 22 E. McLeod and A. Ozcan, “Microscopy without lenses,” Phys. Today 70(9), the Steven and Alexandra Cohen Foundation. The authors 50–56 (2017). acknowledge Zachary Scott Ballard and Yichen Wu for their 23 A. Ozcan and E. McLeod, “Lensless imaging and sensing,” Annu. Rev. help in obtaining some of the hardware components. We Biomed. Eng. 18, 77–102 (2016). 24 thank Isaijah Johnson and Trevor Sobol for their assistance E. McLeod, Q. Wei, and A. Ozcan, “Democratization of nanoscale imaging and sensing tools using photonics,” Anal. Chem. 87, 6434–6445 (2015). in crystallizing protein samples for evaluation. The UCLA X- 25 J. C. Contreras-Naranjo, Q. Wei, and A. Ozcan, “Mobile phone-based ray diffraction facilities are supported by the BER program of microscopy, sensing, and diagnostics,” IEEE J. Sel. Top. Quantum Electron. the Department of Energy Office of Science under Award No. 22, 1–14 (2016). DE-FC02-02ER63421. 26 O. Mudanyali et al., “Compact, light-weight and cost-effective microscope A.O. is the co-founder of a company that commercializes based on lensless incoherent holography for telemedicine applications,” Lab computational imaging and sensing technologies. Chip 10, 1417 (2010). APL Photon. 4, 030804 (2019); doi: 10.1063/1.5080158 4, 030804-7 © Author(s) 2019 APL Photonics ARTICLE scitation.org/journal/app 27 34 O. Mudanyali, C. Oztoprak, D. Tseng, A. Erlinger, and A. Ozcan, “Detec- Y.-C. Wu et al., “Air quality monitoring using mobile microscopy and tion of waterborne parasites using field-portable and cost-effective lensfree machine learning,” Light Sci. Appl. 6, e17046 (2017). microscopy,” Lab Chip 10, 2419 (2010). 35 C Edwards, “Not-so-humble Raspberry Pi gets big ideas,” Eng. Technol. 8, 28 30–33 (2013). T.-W. Su, A. Erlinger, D. Tseng, and A. Ozcan, “Compact and light-weight automated semen analysis platform using lensfree on-chip microscopy,” 36 J.-S. Park et al., “Origins of parasitic emissions from 353 nm AlGaN-based Anal. Chem. 82, 8307–8312 (2010). ultraviolet light emitting diodes over SiC substrates,” Jpn. J. Appl. Phys., 29 Part 2 45, 4083–4086 (2006). A. Feizi et al., “Rapid, portable and cost-effective yeast cell viability and concentration analysis using lensfree on-chip microscopy and machine 37 B. Yuan et al., “A system for high-resolution depth-resolved optical imag- learning,” Lab Chip 16, 4350–4358 (2016). ing of fluorescence and absorption contrast,” Rev. Sci. Instrum. 80, 043706 30 (2009). L. Krčmová et al., “Deep-UV-LEDs in photometric detection: A 255 nm LED on-capillary detector in capillary electrophoresis,” Analyst 134, 2394 38 R. Eskeland et al., “Ring1B compacts chromatin structure and represses (2009). gene expression independent of histone ubiquitination,” Mol. Cell 38, 452– 31 464 (2010). K. G. Kraiczek, R. Bonjour, Y. Salvadé, and R. Zengerle, “Highly flexible UV– vis radiation sources and novel detection schemes for spectrophotometric 39 O. K. Kellermann and T. Ferenci, “[75] Maltose-binding protein from HPLC detection,” Anal. Chem. 86, 1146–1152 (2014). Escherichia coli,” in Methods in Enzymology (Elsevier, 1982), Vol. 90, 32 pp. 459–463. M. S. Shur and R. Gaska, “III-nitride based deep ultraviolet light sources,” Proc. SPIE 6894, 689419 (2008). 40 J. W. Goodman, Introduction to Fourier Optics (Roberts & Co., 33 2005). Y. Li, P. N. Nesterenko, B. Paull, R. Stanley, and M. Macka, “Performance of a new 235 nm UV-LED-Based on-capillary photometric detector,” Anal. 41 D. S. Fay, “A biologist’s guide to statistical thinking and analysis,” Worm- Chem. 88, 12116–12121 (2016). Book 2013, 1–54. APL Photon. 4, 030804 (2019); doi: 10.1063/1.5080158 4, 030804-8 © Author(s) 2019