Smartphone-based multi-contrast microscope using color-multiplexed illumination

www.nature.com/scientificreports OPEN Received: 5 May 2017 Accepted: 3 July 2017 Published: xx xx xxxx Smartphone-based multi-contrast microscope using color-multiplexed illumination Daeseong Jung 1, Jun-Ho Choi2, Soocheol Kim1, Suho Ryu Lee2 & Chulmin Joo1 1 , Wonchan Lee1, Jong-Seok We present a portable multi-contrast microscope capable of producing bright-field, dark-field, and differential phase contrast images of thin biological specimens on a smartphone platform. The microscopy method is based on an imaging scheme termed “color-coded light-emitting-diode (LED) microscopy (cLEDscope),” in which a specimen is illuminated with a color-coded LED array and light transmitted through the specimen is recorded by a color image sensor. Decomposition of the image into red, green, and blue colors and subsequent computation enable multi-contrast imaging in a single shot. In order to transform a smartphone into a multi-contrast imaging device, we developed an add-on module composed of a patterned color micro-LED array, specimen stage, and miniature objective. Simple installation of this module onto a smartphone enables multi-contrast imaging of transparent specimens. In addition, an Android-based app was implemented to acquire an image, perform the associated computation, and display the multi-contrast images in real time. Herein, the details of our smartphone module and experimental demonstrations with various biological specimens are presented. Microscopes constitute one of the most commonly employed equipment in biology and medicine1. Continued advances in microscopy have introduced many new modalities; however, the relatively large sizes of microscopes, along with their complexity and cost, often limit the utility of these devices in general population and diverse field settings. Recent advances in smartphone technology have made a significant impact on microscopy2–9, transforming the bulky and expensive microscope into a portable and low-cost device. Smartphones are equipped with high computing power, high-performance sensors, and wireless network connectivity. Further, the camera modules in smartphones, in particular, employ state-of-the-art image sensors with small pixel sizes and high pixel counts. Microscopes using these built-in camera modules facilitate compact and portable imaging, providing a wide range of opportunities with regard to education, telemedicine, and remote diagnostics. Diverse imaging modalities have thus been implemented on the smartphone platform. Among the many modalities, label-free imaging on a portable platform is particularly useful in resource-limited settings, as it does not require expensive and time-consuming sample preparation procedures. Bright-field (BF) microscopy is the simplest and most common form of label-free microscopy. However, this technique is unsuitable for the observation of translucent specimens such as unlabeled cell monolayers and thin tissue sections, as these specimens do not exhibit strong attenuation in visible light. Dark-field (DF)10, 11 and phase contrast microscopes12, 13, on the other hand, offer higher contrast and detailed structural information of unlabeled specimens compared to BF imaging. Therefore, these modalities are extensively utilized for various studies in laboratory settings. Recently, Philips et al.14 demonstrated a multi-contrast microscope on a smartphone platform capable of generating BF, DF, and differential phase contrast15–20 (DPC) images. In this method, a programmable light-emitting-diode (LED) array in a domed arrangement illuminates the specimen at different angles. Synchronized image acquisition with the smartphone camera module and subsequent computation then facilitate BF, DF, and DPC imaging. The application of this method, however, requires synchronized operation of the external LED light source and image sensor, corresponding to relatively high implementation complexity. For multi-contrast imaging, this method also requires sequential acquisition of multiple images with different illumination patterns, necessitating a minimum of three images in order to obtain BF, DF, and DPC images. 1 Yonsei University, School of Mechanical Engineering, Seoul, 03722, Republic of Korea. 2Yonsei University, School of Integrated Technology & Yonsei Institute of Convergence Technology, Incheon, 21983, Republic of Korea. Correspondence and requests for materials should be addressed to C.J. (email: ) Scientific REPOrtS | 7: 7564 | DOI:10.1038/s41598-017-07703-w 1 www.nature.com/scientificreports/ Figure 1. Operating principle of color-coded LED microscopy. Patterned illumination with a color LED array and subsequent computation enable acquisition of bright-field (BF), dark-field (DF), and differential phasecontrast (DPC) images in a single shot. Summation of the images in red (R) and blue (B) colors produces BF images. The image in green (G) corresponds to the DF image. The DPC image is obtained by evaluating the difference between the images in red and B, with subsequent division of the difference by the BF image. OBJ: Objective lens. Here, we present a simpler and more cost-effective strategy for smartphone-based multi-contrast imaging. The proposed method is capable of producing BF, DF, and DPC images in a single shot and thus, the synchronized operation of the LED array and smartphone camera module is not required. The enabling device consists of a lightweight (~250 g) opto-mechanical smartphone attachment and a custom-developed Android application (“App”) for acquisition, computation, and display of the imaging results. The basic strategy of our method involves color-coded LED illumination, in which red, green, and blue colors correspond to different illumination angles. Image acquisition with a built-in smartphone color image sensor, and subsequent decomposition and computation using the images in each color channel produces BF, DF, and DPC images in a single shot. Here, we describe the implementation of our smartphone-based multi-contrast microscope and demonstrate its imaging capability by presenting multi-contrast images of various biological specimens. Having a compact design and exhibiting robust performance, our proposed microscope is likely to constitute an ideal imaging tool for educational purposes as well as diagnostics in resource-limited settings. Results and Discussion Color-coded LED microscopy (cLEDscope). Operation of our mobile multi-contrast microscope is based on the previously demonstrated microscopy scheme termed “color-coded LED microscopy (cLEDscope)21”. (Fig. 1) In cLEDscope, a color LED array is used as the light source, and is placed at a certain distance from the specimen plane, such that its position is approximately located in the Fourier plane of the specimen plane. Light transmitted through the specimen is then imaged by a color image sensor. In order to obtain BF, DF, and DPC images in a single shot, the LED array is patterned as illustrated in Fig. 1. (...truncated)