Confocal Bioluminescence Imaging for Living Tissues with a Caged

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Confocal bioluminescence imaging for living tissues with a caged substrate of luciferin Mitsuru Hattori, Genki Kawamura, Ryosuke Kojima, Mako Kamiya, Yasuteru Urano, and Takeaki Ozawa Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04142 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 25, 2016

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Analytical Chemistry

Confocal bioluminescence imaging for living tissues with a caged substrate of luciferin

Mitsuru Hattori1, Genki Kawamura1, Ryosuke Kojima2, Mako Kamiya3, Yasuteru Urano2,3 and Takeaki Ozawa*1

1. Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Bunkyo-ku, Hongo, Tokyo 113-0033, Japan 2. Laboratory of Chemistry and Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Bunkyo-ku, Hongo, Tokyo, 113-0033, Japan 3. Laboratory of Chemical Biology and Molecular Imaging, Graduate School of Medicine, The University of Tokyo, 7-3-1 Bunkyo-ku, Hongo, Tokyo 113-0033, Japan

*Correspondence should be addressed to T. O. Email: [email protected] Tel.: 81-3-5841-4351 Fax: 81-3-5802-2989

1 ACS Paragon Plus Environment


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Abstract Fluorescence imaging can elucidate morphological organization and coordinal networks, but its background luminescence degrades the image contrast. Our confocal bioluminescence imaging system uses a luciferase caged substrate, with light passing through multi-pinhole arrays, causing bioluminescence at a focal plane. After a charge-coupled device camera captures luminescence, the imaging system acquires confocal images of multi-layered cells with depth information, supporting quantitative analysis of spatial cellular localization in living tissues.


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Introduction A spatial arrangement of individual cells in living tissue is regulated stringently in various

physiological

events.

In

developing

embryos,

cell-to-cell

interactions

spatiotemporally control the induction of cell differentiation.1,2 Particularly, precise locations of neurons and their connections are fundamentally important for the construction of intricate neural networks.3 Solid tumor cells show collective invasion into an adjacent tissue, which is led by arrangements of cancer cells that express characteristic genes.4 Visualization of such cellular localization requires imaging techniques with high depth resolution. An approach to obtaining spatial information related to multilayered cells is the use of confocal fluorescence microscopy, which enables the isolation of fluorescence signals at a depth position of interest with high optical resolution. The depth of acquired images is, in principle, dependent on the tissue permeability of excitation or emission light. The wavelength, mostly less than 600 nm, is subject to being absorbed by different biomaterials in living tissues.5,6 To overcome these issues, two-photon fluorescence microscopy adopting nonlinear optical phenomena is often used.7-9 Its excitation wavelength greater than 1,000 nm is advantageous for tissue penetration. However, its observation range is restricted to depth of a few hundred micrometers from a tissue surface. One reason is the background fluorescence originating from intracellular compartments such as mitochondria10 and extracellular matrixes comprising collagen, fibronectin, and elastin.11,12 In addition, vertebrate tissues are



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filled by hemoglobin-containing blood, which absorbs light effectively with a broad range of wavelengths. Aside from fluorescence imaging techniques, bioluminescence imaging becomes a crucially important method for the analysis of gene expression, protein–protein interactions, and enzymatic activities.13-17 Bioluminescence imaging includes various benefits such as a high signal-to-background ratio, broad dynamic ranges, no excitation light, and versatile molecular design of luciferases and their substrates. Because of such benefits, the imaging techniques are often used for animal organs and plants that emit strong auto-fluorescence. However, the depth resolution for those obtained images is much lower than that of fluorescence images because of the lack of potential methodologies. Herein, we present a novel technique of confocal bioluminescence imaging with an engineered luciferase: NanoLuc. We developed a caged furimazine as its substrate and a laboratory-built bioluminescence microscopic system with an attached microarray pinhole (Figure 1). The combination of confocal light irradiation with the bioluminescence reaction of the substrate facilitated confocal three-dimensional imaging of multilayer tissue samples with no background fluorescence.


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Experimental methods and Materials Materials DNA restriction and modification enzymes were obtained from TaKaRa Bio Inc. (Japan). Rapamycin and blasticidin were purchased from Wako Pure Chemical Industries Ltd. (Japan). Fluorescence beads in resin for assessment of spatial resolution were obtained from Olympus Corp. (Japan). General chemicals for chemical synthesis were purchased from commercial suppliers (Wako Pure Chemical, Tokyo Chemical Industries, Aldrich Chemical Company and Dojindo) and were used without further purification. Synthesis of caged furimazine. For synthetic protocols of caged furimazine, see the supplementary information. 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCEIII 400 (400 MHz for 1H and 101 MHz for 13C) with chemical shifts (δ) reported in ppm relative to the solvent residual signals of CDCl3 (7.26 ppm for 1H). High-resolution mass spectra (HRMS) were measured on a Bruker micrOTOFII with electron spray ionization (ESI). Plasmid construction The cDNA of NanoLuc (GenBank: JQ437370.1) was synthesized using oligonucleotides by polymerase chain reaction (PCR) with PrimeSTAR HS DNA polymerase (TaKaRa Bio Inc.). For expression of the recombinant NanoLuc protein, the PCR product was inserted into a pCold I bacterial expression vector (TaKaRa Bio Inc.) with restriction enzyme sites. In the



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transient expression of NanoLuc in NIH3T3 cells, a signal peptide and an N-terminal sequence of mouse proacrosin (Pro; MVEMLPTVAVLVLAVSVVAKDNTT) and a GPI attachment signal peptide of mouse prion protein (PrP; SSSTVLFSSPPVILLISFLIFLIVG) were synthesized using PCR. These PCR products were connected with both ends of the cDNA of NanoLuc (Pro-NanoLuc-PrP) and were inserted into a mammalian expression vector, pcDNA3.1(+) (Invitrogen Corp., CA), with restriction enzyme sites. As a control experiment, the NanoLuc sequence of the expression vector was replaced by the sequence of enhanced green fluorescence protein (EGFP). To express NanoLuc stably in NIH3T3 and normal human epidermal keratinocytes (NHEK) cells, the inserted sequence of Pro-NanoLuc-PrP was transferred to a retrovirus vector: pMX. All PCR fragments were sequenced using a genetic analyzer (ABIPRISM 310; Life Technologies Inc., CA). Expression vectors were purified using PureLink HiPure Plasmid Midiprep Kit (Life Technologies Inc.). In vitro characterization of caged furimazine E. coli BL21 (DE3) cells including the NanoLuc expression vector were cultured in LB medium until the OD 600 nm value reached 0.6. The expression of NanoLuc was induced by cold-shock for 30 min under 1 mM IPTG. After the induction, E. coli cells were cultivated for 24 h at 16 °C. The cells were destructed with sonication and addition of 0.1% (v/v) Tween-20 for solubilization. Recombinant NanoLuc proteins in the soluble fraction were purified using


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His SpinTrap columns (GE Healthcare, UK). The molecular weight of the NanoLuc proteins was confirmed using SDS-PAGE electrophoresis and CBB staining. We quantified the concentrations of the recombinant protein using BCA Protein Assay Reagent (Thermo Fisher Scientific Inc., USA). For characterization of the caged furimazine, 100 µL of a caged-furimazine solution (1 µM caged furimazine, 100 mM HEPES-NaOH, pH 7.5, 10 mM MgSO4, 1 mM DTT, and 0.5% glycerol) was illuminated with an LED (385 nm; Optocode Corp., Japan) for 1 min. Next, the caged-furimazine solution was mixed with a 250 µL solution of NanoLuc (2.5 µg/ml of NanoLuc protein and 0.1% BSA in Opti-MEM, GIBCO). Luminescence from the mixed solution was measured using a luminometer (LB9507; Berthold Technologies, Germany) with a 5 µL portion of the mixed solution with 0.2 s exposure at each time point. Ratio of the bioluminescence intensity to the background one was shown in the graphs as “Normalized intensity”. Cell cultures and induction of DNAs For the transient expression of NanoLuc or GFP, NIH3T3 cells were sub-cultured in Dulbecco’s modified Eagle’s medium (Wako Pure Chemical Industries Ltd.) supplemented with 10% fetal bovine serum (FBS) (GIBCO), 100 unit/mL penicillin and 100 µg/mL streptomycin (GIBCO) at 37 °C in an incubator with 5% CO2. The plasmids were introduced into the cell using Lipofectamine 3000 reagent (Life Technologies Inc.). In the case of stable gene expression, retrovirus was collected according to a procedure presented in a previous



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report. The viruses were used to infect NIH3T3 and NHEK cells; the NanoLuc or EGFP was expressed in the cells. Immunostaining NIH3T3 cells transiently expressing NanoLuc were cultured on a cover glass. Cells were fixed for 15 min with DMEM containing 3.7% formaldehyde at 37 °C. We then washed the cells with PBS buffer and permeabilized them with 0.2% TritonX-100 in phosphatase buffer saline (PBS) for 5 min. The cells were washed again three times with PBS. A blocking reaction was done with 0.2% fish skin gelatin (FSG) in PBS overnight at 4 °C. The buffer was exchanged to PBS (0.2% FSG) containing 1/5000 dilution of mouse Anti-myc antibody (Cell Signaling Technology Inc., MA). Then it was incubated for 1 h at room temperature with gentle shaking. After washing with PBS, we reacted the cells with a donkey anti-mouse IgG labeled with AlexaFluoro 488 (Molecular Probes Inc., USA) for 1 h at room temperature. We washed the cells and fixed them on a cover glass with FluorSave reagent (Calbiochem, CA). We observed samples using confocal fluorescence microscopy (FV-1000; Olympus Corp.). Construction of 3D cell population For a cell layer model of NIH3T3 cells, trypsinized cells were coated with collagen and fibronectin (CellFeuille Kit; Sumitomo Bakelite Co. Ltd., Japan) according to the manufacturer’s protocol. Coated cells were allowed for stacking on a polycarbonate


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membrane in a cell-culture insert and were incubated overnight at 37 °C with 5% CO2. For an epidermal tissue model, three-dimensional NHEK cell culture was performed using a LabCyte EPI-KIT (JTEC Corp. Ltd., Japan) according to the manufacturer’s protocol. Live cell imaging We constructed an imaging system for bioluminescence, fluorescence, and bright-field images using an inverted fluorescence microscope (IX-81; Olympus Corp.) with a 20 × objective lens (UPLSAPO20XO; Olympus Corp.). The light source for stimulation and excitation was a metal-halide light source (Photo Fluor II; 89 North, VT). A dichroic mirror (455 nm) separated stimulation light (0.1 mW) and the resultant bioluminescence. For fluorescence imaging, a band-pass excitation filter (460–495 nm), dichroic mirror (488–594 nm), and long-pass emission filter (510 nm) were used. We set multi-pinhole arrays (Olympus Corp.) in the optical path with an electric motor to control the position. The spatial resolution of the system was measured by 60 × objective lens (UPLSAPO60XO; Olympus Corp.). All images were taken using a cooled EM-CCD camera (ImagEM; Hamamatsu Photonics, Japan). The total imaging system was located in a dark box, of which the temperature was set at 22 °C. Buffer solutions of living cells or tissue samples were exchanged into a Hanks’ Balanced Salt Solution (HBSS; GIBCO) in the presence or absence of the caged furimazine (10 µM). We set the cells or tissue samples on the stage of the laboratory-build bioluminescence



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microscope. For samples cultured in a cell-culture-insert, the insert was put directly on the cover glass and was filled with buffer. For confocal bioluminescence imaging, we conducted each irradiation step for 500 ms every 1 min. The bioluminescence was detected before and after the irradiation for 30 s exposure. We used software (Metamorph; Molecular Devices Corp., USA) to control all image acquisition procedures, including light stimulation, data acquisition, and subsequent moving of the multi-pinhole array.

Results and Discussion

Basic principle of confocal bioluminescence imaging

Confocal bioluminescence imaging is composed of a combination of conventional fluorescence microscopy with multi-pinhole arrays. To activate the bioluminescence reaction with a caged furimazine at an interest depth position, we developed our laboratory-built confocal imaging system (Figure 2). A multi-pinhole array is set in the optical path of the microscopy, by which caged furimazine at multiple spots of the focal plane in a sample is stimulated specifically by light. The uncaged furimazine immediately emits bioluminescence from the spot area, which is detected by an EM-CCD camera through the corresponding pinholes. When the microscope stage moves for the x–y plane in a stepwise manner,


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bioluminescence of the whole area at the focal plane is collected. Finally, all the acquired images are overlaid to produce an integrated bioluminescence image.

Optical Setup of Bioluminescence imaging system The confocal bioluminescence imaging system designed for the analysis of cell distribution requires precise regulation of the bioluminescence reaction at a focal point in living tissues. A bright luminescence luciferase, NanoLuc, has been applied for single-cell and in vivo bioluminescence imaging.18,19 Because the luminescence intensity of NanoLuc is more than 100-fold higher than that of Firefly luciferase,18 we inferred that the use of NanoLuc shortens the total time to acquire a single bioluminescence image. To control the bioluminescence reaction spatially, a new caged-furimazine for NanoLuc was developed as a photo-activatable substrate (Figure 3A). Caged furimazine was developed by introducing photo-labile caged group to furimazine. 4,5-Dimethoxy-2-nitrobenzyl group was chosen as a caged group due to the capability of uncaging by relatively longer wavelength UV illumination, and was conjugated to furimazine through enolized keto group of the C-3 carbon to develop caged furimazine. First, we investigated the potential of caged furimazine to recover the luminescence property by light irradiation with 385 nm. Recombinant NanoLuc proteins were mixed with the caged furimazine and the luminescence intensities were measured thereafter. The intensities increased rapidly 80-fold over the background luminescence upon light irradiation, and decreased gradually to the baseline because of the



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consumption of uncaged furimazine (Figure 3B). Next, to investigate the photoreaction in living cells, caged furimazine was introduced into NIH3T3 cells transiently expressing NanoLuc. The expressed NanoLuc was located on the outside of the plasma membrane by fusion with a pro-acrosin signal20 and a GPI anchor peptide21 for increasing the frequency of the

enzymatic

reaction

with

furimazine

(Supporting

Information

Figure

S1).

Bioluminescence from living cells was obtained using a bioluminescence microscope with a CCD camera. Cells expressing NanoLuc showed rapid increases in the luminescence after light stimulation followed by decreases to nearly the initial level within 2 min (Figures 4A and 4B). There was no bioluminescence from the medium region surrounding cells. Furthermore, repeated irradiation showed the same pattern of bioluminescence changes, indicating the influx of caged furimazine from non-irradiated areas (Figure 4C). These results demonstrated that the caged furimazine is applicable for temporal regulation of the bioluminescence activity in living cells by external light. Development of Confocal Bioluminescence Imaging System We constructed a laboratory-built confocal bioluminescence imaging system (Figure 5). Multi-pinhole arrays (64 × 64 pinholes; 26 µm øD; 128 µm interval) were developed on a Cr-coated glass plate (Supporting Information Figure S2A). A CCD camera composed of 512 × 512 pixels was attached to the imaging system. On a region of 2 × 2 pixels of the CCD camera, it captured light passing through a pinhole (Supporting Information Figure S2B).


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Therefore, 4 × 4 steps of the imaging were necessary for the completion of a single confocal image at a Z position. We used artificial fluorescence beads to assess the spatial resolution of this system. Then x–y scanning of the multi-pinhole arrays was conducted at different Z positions; we stacked the resultant images in layers to produce a 3D fluorescence image (Supporting Information Figure S3). The calculated half width of the fluorescence spot in the XY direction was within 0.3 µm. That in the Z direction was the same value, indicating that the resolution was sufficiently high to trace the cell morphology. Consequently, this bioluminescence imaging system with multi-pinhole arrays has sufficiently high spatial resolution to enable visualization of the distribution of individual cells in living tissues. Next, we evaluated the ability to visualize living cells using confocal bioluminescence imaging with the caged furimazine. To investigate the temporal variation of the bioluminescence intensity, we dipped the cells into buffer with caged furimazine and irradiated them with blue light under the microscopic system. Bioluminescence was achieved immediately from the cells through the pinholes (Supporting Information Figure S4A). Repetitive irradiation with a fixed pinhole-array position showed almost identical bioluminescence patterns. The intensities increased rapidly and decreased gradually thereafter (Supporting Information Figure S4B), demonstrating that light irradiation through the confocal pinholes induced instantaneous bioluminescence from the focal position.



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To obtain three-dimensional bioluminescence images, we scanned the pinhole array at each Z position. The image acquisition fundamentally comprises four steps: 1) light irradiation, 2) the bioluminescence detection with the CCD camera, 3) duration of diminishing bioluminescence, and 4) moving the pinhole arrays to the next position (Figure 2). We investigated the irradiation light intensity, exposure time and the interval at each step (Supporting Information Figure S4C). Upon setting the light intensity lower than 0.1 mW, generated bioluminescence counts became lower and longer irradiation time was needed for clear confocal images. In such an experimental condition, there is an issue to be considered; diffusion of uncaged furimazine possibly effects on the spatial resolution of the imaging system. All the furimazine that is uncaged by light irradiation are not consumed at the irradiated region if the luciferase does not exist in the same region. The uncaged furimazine diffuses in the cytosol, where it reacts with the luciferase and produces a background luminescence. When light stimulation is repeated, the background luminescence might increases gradually, possibly because of accumulation of uncaged furimazine in the cells (Supporting Information Figure S4D). To eliminate the background luminescence, we took an additional image before light stimulation and used it for background correction. We compared the images stacked from each Z position to standard bioluminescence images without the use of the pinhole array (Figure 6A). Standard images of bioluminescence blurred when the position deviated from the focal plane (indicated as “0”). When we investigated the


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bioluminescence imaging using the same pinholes with the uncaged substrate (original substrate), contrast of the obtained images became blur and depth information was almost lost. This clearly indicates that combination of pinhole-passed light with the caged substrate is requisite for capturing the confocal bioluminescence images. Taken together, bioluminescence images with the pinhole arrays clearly exhibited a confocal effect at cell edges and the pseudopodium. When we compared between both images in the depth direction (Figure 6B), contrast of the pinhole-array-used images was improved sharply at the edge of cell bodies. Confocal Bioluminescence Imaging of Living Cell Layers and Tissues Finally, we demonstrated applicability of the confocal bioluminescence imaging system to living artificial cell layers of NIH3T3 cells. Cells expressing NanoLuc were mixed with the native cells. Subsequently, the mixed cells were coated repeatedly by collagen and fibronectin, and were piled up on polycarbonate membrane in a cell-culture insert. The cell layers in the presence of caged furimazine were visualized using the confocal bioluminescence imaging system. Focal positions were moved step-by-step from the layer bottom to the top. The bioluminescence originated from the caged furimazine in the culture medium was stable against repeated light irradiation over 3 h. We obtained integrated images at each Z position. Localization of individual bioluminescent cells was obtained clearly at each depth position (Figure 7A). When the images were compared with the standard



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bioluminescence images, the contrast and the resolution deep were markedly improved. Moreover, several peripheral regions of the cells were visualized at each Z position, suggesting that the imaging system enables to discriminate layer by layer in the living cells (Figure 7B and Supporting Information Figure S5). To demonstrate the benefits of bioluminescence imaging over fluorescence imaging, we prepared NIH3T3 cells expressing enhanced green fluorescent protein (EGFP) located on the cell surface (Supporting Information Figures S6A and S6B). Fluorescence images taken with the same system clearly portrayed the effects of pinholes on depth information without background fluorescence. However, when the multilayered cells generated with collagen and fibronectin were used, the images became blurred because strong auto-fluorescence from those proteins hampered the EGFP fluorescence detection. To apply the present imaging system for visualizing spatial distribution of differentiated cells, we cultured living epidermis tissue including luminescent cells. Reportedly, keratinocytes in the epidermal basal layer proliferate and differentiate into the prickle layer, granular layer, and horny layer.22,23 NanoLuc was expressed heterogeneously in the normal human epidermal keratinocytes (NHEK) cells. The cells were differentiated on a cell-culture insert. Localization of luminescent cells in the cultured tissues was observed diurnally using the confocal bioluminescence imaging system. Two days after the cell culture, we obtained an image of almost an entire single-cell layer including several bioluminescent cells (Figure 8,


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the cover glass length is “20 µm”). The tissues which had been cultured for 5 days showed other bioluminescent cells having different morphology located on the layer at some distance from the basal layer (Figure 8, “60 µm”), suggesting that NHEK cells at the basal layer proliferated to the outside direction to differentiate into prickle cells.24 Moreover, small bioluminescent cells appeared at the top layer 7 days after the cell culture. They were identified as granular cells (Figure 8, “100 µm”, Supporting Information Figure S7). All results described above demonstrated that the imaging system enabled visualization of spatiotemporal cellular distribution in living tissues over several days.

Conclusions We developed a confocal bioluminescence imaging system to visualize the localization of luminescent cells under a strong autofluorescence environment. To control the bioluminescence reaction spatiotemporally, caged furimazine was newly synthesized as a substrate of the luciferase. Combining the substrate and the imaging system, we conducted bioluminescence imaging with improved spatial resolution in the depth direction, which was sufficient for the discrimination of a luminescent cell in a cell population. Through the imaging procedure, background correction using an additional image taken before light stimulation alleviated an issue of the uncaged substrate diffusion. Additionally, we achieved confocal bioluminescence imaging for living cell layers, of which resolution was enough for



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discriminating luminescent cellular distribution and cell morphology. The imaging system improved the contrast of bioluminescence images of the cells, which is expected to increase the reliability of quantitative analyses as well as the spatial resolution. This technique with different luciferase reporters will be useful to analyze cellular activities such as gene expression and protein–protein interactions in specific living tissues.

Acknowledgments We are indebted to H. Takakura for supporting in vitro experiments of bioluminescence assays. We also thank Olympus Corp. for the development of imaging systems. This work has been supported by “SENTAN” the Japan Science and Technology Corporation (JST), Japan Society for the Promotion of Science (JSPS), and as a Basic Science Research Project from The Sumitomo Foundation and MEXT, Japan.

Supporting Information Available Structure and localization of NanoLuc used in this study (Figure S1). Structural model of developed multi-pinhole array (Figure S2). Resolution capacity of the imaging system. Fluorescent beads embedded in resin were observed using the system with excitation light (Figure S3). Demonstration of the photoreaction of caged furimazine used multi-pinhole array (Figure S4). Pseudo-color images of bioluminescence from living cell layers coated


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with extracellular matrixes (Figure S5). Structure and fluorescence images of GFP in the extracellular matrix (Figure S6). Fluorescence image of GFP in an epidermal tissue. NHEK cells including GFP expressing cells were cultured on a cell-culture insert for 7 days (Figure S7). Synthesis of caged furimazine (Scheme 1). This information is available free of charge via the internet at http://pubs.acs.org/ .



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(13) Misawa, N.; Kafi, A. K. M.; Hattori, M.; Miura, K.; Ozawa, T. Anal. Chem. 2010, 82, 2552-2560. (14) Tamaru, T.; Hattori, M.; Honda, K.; Benjamin, I.; Ozawa, T.; Takamatsu, K. PloS one 2011, 6, e24521. (15) Hattori, M.; Haga, S.; Takakura, H.; Ozaki, M.; Ozawa, T. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 9332-9337. (16) Evans, M. S.; Chaurette, J. P.; Adams, S. T., Jr.; Reddy, G. R.; Paley, M. A.; Aronin, N.; Prescher, J. A.; Miller, S. C. Nat. Methods 2014, 11, 393-395. (17) Porterfield, W. B.; Jones, K. A.; McCutcheon, D. C.; Prescher, J. A. J. Am. Chem. Soc. 2015, 137, 8656-8659. (18) Hall, M. P.; Unch, J.; Binkowski, B. F.; Valley, M. P.; Butler, B. L.; Wood, M. G.; Otto, P.; Zimmerman, K.; Vidugiris, G.; Machleidt, T.; Robers, M. B.; Benink, H. A.; Eggers, C. T.; Slater, M. R.; Meisenheimer, P. L.; Klaubert, D. H.; Fan, F.; Encell, L. P.; Wood, K. V. ACS. Chem. Biol. 2012, 7, 1848-1857. (19) Stacer, A. C.; Nyati, S.; Moudgil, P.; Iyengar, R.; Luker, K. E.; Rehemtulla, A.; Luker, G. D. Mol. Imaging 2013, 12, 1-13. (20) Nakanishi, T.; Ikawa, M.; Yamada, S.; Parvinen, M.; Baba, T.; Nishimune, Y.; Okabe, M. FEBS Lett. 1999, 449, 277-283.



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(21) Paladino, S.; Lebreton, S.; Tivodar, S.; Campana, V.; Tempre, R.; Zurzolo, C. J. Cell Sci. 2008, 121, 4001-4007. (22) Baroni, A.; Buommino, E.; De Gregorio, V.; Ruocco, E.; Ruocco, V.; Wolf, R. Clin. Dermatol. 2012, 30, 257-262. (23) Venus, M.; Waterman, J.; McNab, I. Surgery 2010, 28, 469-472. (24) Koehler, M. J.; Zimmermann, S.; Springer, S.; Elsner, P.; Konig, K.; Kaatz, M. Skin Res. Technol. 2011, 17, 479-486.


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Figure Captions

Figure 1. Schematic model of confocal bioluminescence imaging. A sample of the cells harbors a light-activated caged luciferin and a luciferase. First, the region of interest is stimulated by light that passes through the pinholes. Then, the light-stimulated region generates temporal bioluminescence at the focal plane. After turning off the light, the luminescence through the pinholes is detected using a CCD camera.

Figure 2. Procedure of confocal bioluminescence imaging at a single Z position.

Figure 3. Structure of caged furimazine and characterization. (A) Scheme of caged furimazine synthesis. (B) Time-course measurements of bioluminescence of NanoLuc with caged furimazine. The recombinant NanoLuc proteins were incubated with caged furimazine and were irradiated by light (385 nm) for 1 min. Bioluminescence with or without light irradiation was measured (n=3). Bioluminescence intensities were normalized against a count before irradiation. Green circles, irradiation for 1 min; blue circles, no irradiation. Error bars, s.d.



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Figure 4. Bioluminescence reaction of NanoLuc with caged furimazine in living cells. (A) Temporal changes in the bioluminescence images upon light irradiation. The cells were irradiated by blue light. Bioluminescence was visualized using a CCD camera. The exposure time was 1 s. Scale bar, 50 µm. (B) Temporal changes in bioluminescence in the cells. Eight regions of individual cells (A–H) were selected. Their luminescence intensities were measured. The bioluminescence intensities were normalized against the count before irradiation (Normalized intensity). (C) Reproducibility of bioluminescence upon repeated light irradiation. The irradiation was conducted at intervals of 2 min. Bioluminescence in a particular cell region was measured using 1 s exposure (n=3). The average normalized bioluminescence intensities are shown. Error bars, s.d.

Figure 5. Schematic models of confocal bioluminescence microscopy with a multi-pinhole array. Purple and blue colors respectively show the path of stimulation light and bioluminescence. Acquisition images are stacked using software to generate a single image.

Figure 6. Confocal bioluminescence imaging for living cells. (A) Confocal bioluminescence images of NIH3T3 cells expressing NanoLuc. The confocal images (Pinhole-based) were compared with those obtained without a multi-pinhole array (Standard). Images were taken at five Z positions. The image “0” was set at an arbitrary Z position in a cell region. Scale bar,


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50 µm. (B) Comparison of the resolution in the Z direction. Bioluminescence images in the depth direction with or without pinholes are presented. Data were constructed from the region shown by the red line. Pseudo-color images are indicated quantitatively. Scale bar, 20 µm (Y direction); 20 µm (Z direction).

Figure 7. Confocal bioluminescence imaging of living cell layers coated with extracellular matrixes. (A) Bioluminescence images of NIH3T3 cells. The layered cells were coated preliminarily with collagen and fibronectin. The confocal images (Pinhole-based) were compared with those obtained without the multi-pinhole arrays (Standard). Images were acquired at five Z positions. The image “0” was set at an arbitrary Z position in a cell region. Scale bar, 50 µm. (B) Observation of the cellular localization deep direction. Images are enlarged in the region of Figure 6A indicated by red open squares. Scale bar, 50 µm.

Figure 8. Confocal bioluminescence imaging of epidermal tissues. NHEK cells including NanoLuc-expressing cells were cultured on a cell-culture insert for 2, 5, or 7 days. Confocal bioluminescence images with caged furimazine were acquired each day. The bottom, middle, and tops of images were obtained, respectively, from the cell culture at 2, 5, and 7 days. Numbers to the right of images denote the distance from the cover glass. Scale bar, 50 µm.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Light source 23 24 25 26 Pinhole Pinhole 27 28 29 30 Mirror 31 32 Detector 33 34 Objective 35 lens 36 37 38 39 40 Sample 41 42 43 Luminescence 44 Light stimulation “OFF” state 45 46 47 48 49 50 51 52 53 54 55 ACS Paragon Plus Environment 56 57 58

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Confocal bioluminescence imaging for living tissues with a caged substrate of luciferin

Luminescence “ON” state

Figure1. M. Hattori et al.

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array


Multipoint stimulation to cell culture.

Luminescence temporally “ON”. Detection.

Moving the array.

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ACS Paragon Plus Environment

Figure2. M. Hattori et al.


A 385 nm light

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Figure 3. M. Hattori et al.

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Confocal Bioluminescence Imaging for Living Tissues with a Caged

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