Although fluorescence microscopy is the gold standard tool for biomedical research and clinical applications, their use beyond well-established laboratory infrastructures remains limited. The present study investigated a novel on-chip cell analysis platform based on contact fluorescence microscopy and microfluidics. Combined use of a contact fluorescence imager based on complementary metal-oxide semiconductor technology and an ultra-thin glass bottom microfluidic chip enabled both to observe living cells with minimal image distortion and to ease controlling and handling of biological samples (e.g. cells and biological molecules) in the imaged area. A proof-of-concept experiment of on-chip detection of cellular response to endothelial growth factor demonstrated promising use for the recently developed on-chip cell analysis platform. Contact fluorescence microscopy has numerous desirable features including compatibility with plastic microfluidic chips and compatibility with the electrical control system, and thus will fulfill the requirements of a fully automated cell analysis system.
A wide variety of sophisticated fluorescent probes enables the high sensitivity and specificity of cellular activity detection. Fluorescence microscopy is a powerful tool and remains the gold standard for biomedical research and clinical applications. However, conventional tabletop-type fluorescence microscopes are relatively bulky and expensive, due to the series of lenses and mechanical structures required for focusing lenses. Their use beyond well-established laboratory infrastructures with well-trained technicians remains limited.1
Recent rapid development in semiconductor image sensor technology, including a charge coupled device and a complementary metal-oxide semiconductor (CMOS) image sensor, have led to the rise of on-chip contact (lens-free) imaging techniques, which aim to overcome the limitations of conventional table-top type microscopes. On-chip imaging techniques are highly attractive, particularly in the application fields of automated systems, for high-throughput biological and medical screening due to their advantages compared with conventional tabletop-type microscopes, including (i) ease of scale-up of field of view with large numbers of pixels and smaller pixel sizes, (ii) compact hardware geometry, cost effectiveness and mass producibility, (iii) and simple usability without careful selection and focusing of the lenses.2–8 Automated system using microfluidic technology has also demonstrated great potential for biological and medical analysis,9,10 drug screening,11,12 and cell processing.13,14 Thus, using on-chip fluorescence imaging techniques and a microfluidic chip platform provides a fully automated system from sample handling to detect of cellular activity.
The present study investigated a novel on-chip cell analysis platform for integrating contact fluorescence microscopy and disposable microfluidic chips. A chip-scale CMOS fluorescence imager and an ultra-thin glass bottom microfluidic chip were developed for the on-chip cell analysis platform and enabled contact fluorescence imaging with a spatial resolution of ∼20 μm in microfluidic chips. To demonstrate the potential use of this on-chip cell analysis platform, the time course of cellular response to endothelial growth factor (EGF) was determined in disposable microfluidic chips. Proof-of-concept experiments revealed a promising use of on-chip platform for biomedical research and drug development.
An integrated platform of microfluidics and on-chip microscopy has been intensively developed based on bright field (shadow or diffraction) imaging techniques.15–19 However, implementation of contact fluorescence microscopy in a microfluidic chip platform is not simple, due to certain technical obstacles that arise from the characteristic features of fluorescence imaging. First, the fluorescence signal intensity and spatial resolution decreases at a greater rate as a function of the vertical distance between samples and the detection region (e.g. photodiodes of an image sensor chip), compared with bright field imaging.20 Therefore, it is essential to locate the samples in a microfluidic chip in close proximity to the image sensor chip. Second, fluorescent light has a wide range of incident angles in contact fluorescence imaging. As a result, standard interference filters that are used in conventional table-top type fluorescence microscopes would not function properly, as their transmission spectra fundamentally depend on the incident light angle.21
Schematics of on-chip cell analysis platform
Figure 1a presents a schematic illustration of the on-chip cell analysis platform with the ultra-thin glass bottom microfluidic chip and the contact CMOS fluorescence imager. The ultra-thin glass bottom microfluidic chip was designed for culturing cells, delivering biological molecules to the cultured cells, and performing contact fluorescence imaging, using the contact CMOS fluorescence imager as presented in Fig. 1b. Ultra-thin glass with a thickness of 10 μm was used as the bottom substrate of the microfluidic chips to minimize the vertical distance between the observing targets (e.g. fluorescent cells) and the contact imager. A thin-film absorption filter, which consisted of dye molecules and polymer matrix, was coated on the CMOS image sensor to remove excitation light for fluorescence imaging. Aiming to protect the dye-based absorption filter layer from mechanical damage due to contact with the microfluidic chips, the flat surface of the fiber optic plate (FOP) was placed on the filter layer. To perform contact fluorescence imaging, the ultra-thin glass bottom microfluidic chip was directly placed on to the FOP surface of the contact CMOS fluorescence imager.
Contact CMOS fluorescence imager
The contact CMOS fluorescence imager is mainly composed of three elements: A CMOS image sensor chip, a thin-film absorption filter, and FOP, as presented in Fig. 2a. The CMOS image sensor chip was fabricated using the 0.35-μm 2-poly, 4-metal standard CMOS technology (Table S1 of the supplementary material). Figure 2b presents a microscopic image of the fabricated CMOS image sensor chip with a width of 1.0 mm and a length of 2.7 mm. The CMOS image sensor chip had 120 × 268 pixels (pixel size: 7.5 μm × 7.5 μm). The thin-film fluorescence filter was a 500 nm-long pass filter, as presented in Fig. 2c. The FOP is important for protection the thin-film filter layer from mechanical damage and maintenance of the flat surface in order to closely place the microfluidic chips without any degradation of fluorescence signal intensity and resolution. The individual fibers (3 μm in diameter) of the FOP are smaller than the pixel size of the CMOS image sensor. To fulfill the aforementioned requirements, the thickness of the FOP was reduced to 400 μm and carefully polished (Fig. S1 of the supplementary material). Subsequently, a fluorescence imaging test was performed. Fluorescent microspheres of 10-μm diameters (F-13080; Molecular Probes, Inc., Eugene, OR, USA) were placed at the imaging region of the contact CMOS fluorescence imager. Figures 2d presents a bright image and fluorescent images obtained using a table-top type microscope and the contact CMOS fluorescence imager. Figure 2e presents a representative fluorescence intensity profile of the fluorescent microsphere (ϕ = 10 μm) obtained by the contact CMOS fluorescence imager. The average value of the full widths at half maximums (FWHMs) of observed fluorescent microspheres within the entire imaging region was 19.0 ± 0.7 μm (mean ± standard deviation; n = 12). This low value of standard deviation of the average FWHM indicated that entire uniformity of the resolution within the imaging region was achieved, because the imaging plane at the surface of the FOP was successfully maintained as a flat surface with no tilt.
Ultra-thin glass bottom microfluidic chips
To improve the coupling efficiency of fluorescence signals and the resolution of contact fluorescence imaging, ultra-thin glass 10 μm in thickness was used at the bottom of the microfluidic chips (Figs. S2a-c of the supplementary material). The flexible ultra-thin glass can be rolled similar to a ribbon, as presented in Fig. 3a. Figure 3b presents the fabrication process of the ultra-thin glass bottom microfluidic chips. As observed in the third assembly step, the lower part of the microfluidic chip was a 10 μm-thick ultra-thin glass and the top part was poly(dimethylpolysiloxane) (PDMS) with microchannels. Figure 3c presents the fabricated ultra-thin glass bottom microfluidic chip. An inlet port and an outlet port of each microchannel can be used for introducing culture medium, cells, and chemical compounds into the microchannels. In order to avoid excessive sheer stress to cultured cells inside the microchannels, numerical fluid simulation was performed and the fluid flow range in changing medium solution and delivering chemicals to cultured cells was determined so as to not exceed 100 μL/min (Fig. S3 of the supplementary material).
On-chip fluorescence imaging and detection of cellular activity
The present study investigated the contact fluorescence imaging of cultured cells in the ultra-thin glass bottom microfluidic chips. As illustrated in Fig. 4a, Hela cells with or without fluorescent probes in nuclei were cultured inside microchannels. Hela cells can adhere, spread, and proliferate normally in the microchannels due to collagen treatment of the ultra-thin glass (Fig. 4b). Figures 4c and 4d present representative fluorescent images obtained using a tabletop-type microscope and the developed on-chip cell analysis platform. It is noteworthy that (i) the ultra-thin glass (10 μm in thickness) and thin-film absorption filter (15 μm in thickness) minimized the vertical distance between observing targets (e.g., fluorescent cells) and the image sensor, and (ii) the flat surface of the FOP enabled close contact with the microfluidic chips. As a result, as shown in Fig. 4d, fluorescent signals from the nuclei of the cultured cells were obtained using the on-chip cell analysis platform.
Subsequently, the present study used the on-chip cell analysis platform for the evaluation of cellular activity alterations induced by extracellular agents. In order to detect cellular response, we used a Förster resonance energy transfer (FRET) probe, which comprised two fluorescent proteins, an enhanced cyan fluorescent protein (ECFP) and a yellow fluorescent protein for energy transfer (YPet) and to detect extracellular signal-regulated kinase (ERK), named EKAREV.22 The EKAREV probe increases fluorescent intensity of YPet as a consequence of the FRET efficiency increase following detection of the extracellular signal. Thus, excitation light (425–445 nm) to ECFP was used and fluorescent light (>500 nm) from YPet was detected using the contact CMOS fluorescence imager. FRET based fluorescent probes demonstrate large stokes shift due to intramolecular energy transfer, and thus, were selected for use in the on-chip fluorescence detection system with dye-based absorption filter in the present study.23 Cellular response to extracellular agents was induced by delivering EGF. As presented in Fig. 4e, EKAREV expressed Hela cells were cultured inside microchannels and solutions without or with extracellular agents were delivered into each microchannel (EGF, 0 and 10 ng/mL, respectively). Figures 4f and 4g revealed the fluorescence signal intensity alters of EKAREV-expressing cultured cells without or with stimulation from EGF, respectively. Following delivery of solutions, only cultured cells with EGF demonstrated an increase in fluorescence intensity (Fig. 4g). These results revealed that delivering extracellular molecules and detection of cellular response using the on-chip cell analysis platform was possible, in addition to a promising use for this platform in biological research and drug screening applications.
In order to obtaining high resolution images by contact imaging, it is useful to utilize computation-based decoding techniques. Although novel image reconstruction algorithms with specific optical designs have previously been well investigated for contact bright field imaging, these techniques would not be applicable to contact fluorescence imaging due to incoherence of fluorescence emission.24–27 Recently, image reconstruction techniques in contact fluorescence imaging have been investigated using compressive decoding of sparse objects.28 This image reconstruction technique is considered to be feasible in microfluidic chip platforms. We would like to also point out that the localized illumination technique would improve the resolution in contact fluorescence imaging. The use of a near-field optical probe for illumination, which is a technique from fluorescence near-field scanning optical microscopy,29 could be beneficial for achieving spatial resolution beyond the diffraction limits.
Regarding the signal-to-noise ratio (SNR) of contact fluorescence imaging, improvements in absorption filter performance by concentration increase of dispersed dye molecules in polymer matrix and progress in dye molecular synthesis may aid improvements to contact fluorescence imaging with high SNRs. In addition, absorption filters with micro-sized structures, including silo-shaped30 or light-pipe31 filters, may be applied for avoiding optical cross-talk with neighboring pixels.
Furthermore, combined use of filter patterning techniques of absorption dye filter, similar to a color CMOS image sensor, may address contact multi-color fluorescence imaging and ratiometric imaging of the FRET signal, which may extend the use of contact fluorescence microscopy to various biomedical research and clinical applications. Due to ultra-compactness and cost-effectiveness of the CMOS image sensor chips, implantable devices for the evaluations of biological information inside living bodies may be promising applications of contact fluorescence microscopy, particularly for brain research,32,33 glucose monitoring,34,35 and optical theranostics.36
In conclusion, we have developed an on-chip cell analysis platform with the integration of contact fluorescence microscopy and microfluidics. The contact CMOS fluorescence imager and ultra-thin glass bottom microfluidic chips were newly developed for performing contact fluorescence imaging of cultured cells inside microchannels. The present study demonstrated the detection of cellular activity by delivering biological molecules to cultured cells on the newly developed on-chip platform. On-chip fluorescence imaging techniques have numerous desirable characteristics, including compatibility with plastic microfluidic chips, low-cost mass production, and integratability between the electrical control and cellular incubation system.
MATERIALS AND METHODS
Fabrication of the contact CMOS fluorescence imager
The CMOS image sensor chip, which had four bonding pads, three input pads (VDD, GND, and CLK), and one output pad, was fixed on a printed circuit board and the bonding pads were electrically connected via Al wires. The Al wires were covered with epoxy resin (EPOTEK 730; Epoxy Technology Inc., Billerica, MA, USA). A FOP with the thin-film fluorescence filter was prepared as follows: A FOP thickness (J5734; Hamamatsu Photonics K.K., Japan) was reduced to 400 μm and polished using a polishing apparatus (MA-150; Musashino Denshi Corporation, Ltd., Japan). A thin-film fluorescence filter based on a yellow dye was prepared as follows: The dye solution was prepared by mixing dye molecules (Valifast yellow 3150; Orient chemical industrial Co., Ltd., Japan) and cyclopentanone (Tokyo chemical industrial Co., Ltd., Japan) with a 1:1 ratio by weight of dye:cyclopentanone. The dye solution was dispersed in the polymer matrix (GA-R-1 GA-H-1; Canon chemical Inc., Tokyo, Japan) with a 1:5 ratio by weight of dye solution:adhesive material. In order to form a thin-film filter, spin-coating was performed at 200 rpm for 60 s, and subsequently the polymer matrix with dye was cured at C for 3 h. The thin-film fluorescence filter was carefully transferred on to the FOP. Finally, the FOP with the thin-film fluorescence filter was immobilized by epoxy resin (EPOTEK 730, Epoxy Technology Inc.) onto the CMOS image sensor chip at C for 3 h. The contact CMOS fluorescence imager was controlled using a personal computer.
Fabrication of the ultra-thin glass bottom microfluidics
The top section of the PDMS was prepared by soft lithography.37 In order to form a microchannel mold, photoresist (SU-8 3050, Nippon Kayaku Co., Ltd., Japan) was spin-coated on a Si substrate and exposed to ultraviolet light through a chrome photomask using a mask aligner (MA-10; Mikasa Co., Ltd., Japan) and developed. A PDMS prepolymer (Sylgard 184; Dow Corning Co., MI, USA) was cured at C for 4 h on the microchannel mold. The slab of PDMS was peeled off the mold, and the inlets and outlets were formed by biopsy punch (PB-10F, Kai Corp., Japan). The PDMS slab and the ultra-thin glass sheet were bonded by exposure to O2 plasma (100 W, O2, 25 Pa) for 10 s using a plasma asher (Plasma system FA-1; Samco Inc., Japan). The ultra-thin glass sheet (Nippon Electric Glass Co. Ltd., Japan) was cut using laser cutting apparatus (VLS 2.30; Yokohama systems Co., Ltd., Japan). In order to improve cell adhesion to the lower surface of the microchannels, collagen treatment of the ultra-thin glass was performed as follows: Microchannels were filled with 100 ng/mL collagen from calf skin (C8919; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and stored at C for 12 h. Prior to cell seeding in the microchannel, the microfluidic chips were incubated at C in a humidified atmosphere with 5% CO2 for 2 h.
Cell culture and fluorescent imaging
Hela cells were cultured in Minimum Essential Medium Eagle (M4655; Sigma-Aldrich; Merck KGaA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin (Wako Pure Chemical Industries, Ltd., Japan) at C in a humidified atmosphere with 5% CO2. The ultra-thin glass bottom microfluidic chip was filled with the culture medium. The cells were seeded onto the ultra-thin glass bottom microfluidic chips and cultured at C for ≥24 h for attachment onto the glass bottom. Prior to fluorescence imaging, culture medium in the microchannel was replaced with no phenol red Hank’s Balanced Salt Solution (14025-092; Thermo Fisher Scientific Inc., Waltham, MA, USA). Commercially available light-emitting diodes (EP-U1311B-A3; Epileds Technologies, Inc., Taiwan) with a band-pass filter from 425 to 445 nm (Olympus Corp., Tokyo, Japan) were used as an excitation light source. Fluorescence images were obtained using the contact CMOS fluorescence imager with 4.4 frames per second. The fluorescent imaging was also performed using a table top type microscope (BX51WI; Olympus Corp.), equipped with objective lenses (MPlanN 5×/0.10 and LUMPlanFL 40×/0.83W, Olympus Corp.) and a fluorescent filter set (Ex:425-445HQ and Em:495-540HQ; both Olympus Corp.).
Ethics and dissemination
Research ethics approval is not required for this study.
See supplementary material for more details about thinning and polishing process of FOP, comparison between conventional glass bottom and ultra-thin glass bottom microfluidic chips, and numerical simulation of shear stress to cultured cells in the ultra-thin glass bottom microfluidic chips.
We thank Prof. M. Matsuda of Kyoto University (Kyoto, Japan) for providing the EKAREV-expressing cells. This research was supported by the Nakatani Foundation for advancement of measuring technologies in biomedical engineering, Tateishi Science and Technology Foundation, CREST, Japan Science and Technology Agency (JST), Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science (grant no. 26249051 and 15K21164) of Japan, Project for Promotion of Researches toward Creation of Humanophilic Science and Technology, and VLSI Design and Education Center (The University of Tokyo) in collaboration with Cadence Corporation and Mentor Graphics Corporation.
The authors declare no competing financial interests.