Space- and time-resolved spectrophotometry in microsystems Nicolae Damean, Samuel K. Sia, Vincent Linder, Max Narovlyansky, and George M. Whitesides* Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138 Contributed by George M. Whitesides, June 6, 2005 This work describes a simple optical method for obtaining, in a We used a commercially available transmission grating, made of single still-capture image, the continuous absorbance spectra of an index-matched epoxy on glass, (92 grooves per mm; Edmund samples at multiple locations of microsystems. This technique uses Industrial Optics, Barrington, NJ) in which the pitch was suffi- an unmodified bright-field microscope, an array of microlenses, ciently small (11 ␮m) to disperse different wavelengths of visible and a diffraction grating to disperse the light transmitted by light into resolvable spatial positions. According to the manu- samples of 10- to 500-␮m dimensions. By analyzing in a single facturer’s specifications, the distribution of light at 632 nm image the first-order diffracted light, it is possible to collect the full through this grating is 45% for 0 order and 20% for Ϫ1 and for and continuous absorbance spectra of samples at multiple loca- ϩ1 orders. tions (to a spatial resolution of Ϸ8 ␮m) in microwells and micro- A Leica (Vienna) DMRX optical microscope served both as channels to examine dynamic chemical events (to a time resolution light source and detector. The microscope was operated in the of <10 ms). This article also discusses the optical basis of this transmission mode by using a L ϫ40͞0.60 D Plan ϱ͞0 objective method. The simultaneous resolution of wavelength, time, and lens (Leitz). A tungsten-halogen lamp acted as the light source, space at a scale <10 ␮m provides additional capabilities for and a black-and-white charge-coupled device (CCD) camera chemical and biological analysis. (Hamamatsu ORCA-ER, Hamamatsu Photonics, Hamamatsu City, Japan), which typically has higher sensitivity than color poly(dimethylsiloxane) ͉ array of microlenses ͉ spectrophotometer ͉ cameras, captured the image containing the diffraction spectra. image processing The time resolution of this experiment was determined by the acquisition time, which was 1–10 ms (depending on the level of his article describes an optical method that resolves wave- illumination of the light source). The images collected by this length, time, and location simultaneously for samples in camera were analyzed with the METAMORPH software package T (Universal Imaging, Downingtown, PA). For clarity of visual- microsystems. This technique, which we call micropattern spec- ization, we also captured images with a color camera (Nikon trophotometry (␮PS), analyzes a continuous spectrum of wave- Digital Camera DXM1200). lengths (with best performance, in the system described here, in We characterized the spectral resolving power of the system by the range of 450 to 700 nm) at multiple positions in the field of using two lasers for the light source rather than broad-spectrum view of a microscope. This procedure provides a flexible method light from the microscope. Onto the stage of the microscope, we for analyzing the composition of samples at a number of points, placed a glass slide containing the cylindrical microlens, on top or in a number of samples, simultaneously and continuously. of which we placed a transmission grating with the grooves Microsystems are now ubiquitous in chemistry and biology (1). oriented along the long axis of the microlenses. We changed the Applications include analysis of chemical reactions (2), sorting of focus of the objective lens until we observed a clear image of the cells (3), and high-throughput screening (4). Because these diffraction spectra and captured an image with the black-and- systems often require separation (5), mixing (6, 7), and reaction white CCD camera. (8) of components with distinct optical profiles, they would Further details are given in Supporting Text, which is published CHEMISTRY benefit from a method that allows the components to be as supporting information on the PNAS web site. characterized optically in space and time. Advances in minia- turization of components used in spectrophotometric systems Spectral Measurements Using the Micropattern Spectrophotometer. have produced a number of useful microsystems: these systems We obtained quantitative absorbance values of samples over a typically work at a single wavelength at any one time (9, 10) or range of visible wavelengths by analyzing the first-order dif- perform measurements at a single spatial location (11–15), and fracted light. To measure the absorbance spectrum of a given most cannot be easily interfaced with microfluidic systems location of the sample in the field of view, we measured the pixel (16–18). Miniaturized systems for integrating microspectrom- intensities along a line placed orthogonal to the corresponding eters and microfluidics have been proposed but not demon- diffraction spectrum and converted them to absorbance values strated (19, 20). Although fiber optic-based microspectropho- as a function of wavelength. To obtain absorbance values of tometers (21) have been described and microscope-based acceptable signal-to-noise, we used lines of 8-␮m thickness, spectrophotometers (22) are available, they are capable of which determined the spatial resolution of the method. We used analyzing only one or only a few samples at a time, and they scan another sample as a standard for calibration to convert the pixels the spectrum one wavelength at a time. Also, they require of the camera image to wavelength in nm. alignment of a fiber optic cable to the sample region. Further details are given in Supporting Text. The method described here collects spectral information at many wavelengths and for many samples simultaneously. Results and Discussion Design of the Micropattern Spectrophotometer. The technique we Materials and Methods describe here combines an array of convex microlenses, which Setup of the Micropattern Spectrophotometer. We fabricated an array of microlenses in an opaque background by reflowing photoresist (with an index of refraction of 1.59) followed by Abbreviations: ␮PS, micropattern spectrophotometry; CCD, charge-coupled device. electroplating of nickel around the microlenses (23–26). We *To whom correspondence should be addressed. E-mail: gwhitesides@gmwgroup. constructed the sample chamber from poly(dimethylsiloxane) harvard.edu. (which is optically transparent) by using soft lithography (27, 28). © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504712102 PNAS ͉ July 19, 2005 ͉ vol. 102 ͉ no. 29 ͉ 10035–10039 Downloaded by guest on October 1, 2021 Fig. 1. Setup of ␮PS. (A) Schematic diagram and photograph of the setup. (B) Illumination pathway of the optical setup in this study that leads to facile imaging of the diffraction spectrum by using a bright-field microscope. In a regular arrangement for observing diffraction in a microscope, the objective lens is focused on the grating; as a result, an image of the grating forms at the primary image plane (and can be observed via the eyepiece), and an image of the diffracted light forms at the rear focal plane of the objective lens (and can be observed by using a Bertrand lens). In this study, we removed the condenser of the microscope from the light path and used the microlenses as minicondensers. By focusing the objective lens near the microlenses rather than the grating, the optical plane of the condenser iris (which is conjugate to the rear focal plane; both conjugate planes are shown in blue) becomes nearly coplanar with the specimen plane (which is conjugate to the primary image plane; both conjugate planes are shown in orange). As a result, the image of the diffracted light, which is normally observed only in the rear focal plane, also forms on the primary image plane, and is easily captured by a black-and-white CCD camera in a bright-field microscope. (C) Determination of spectral resolution using lasers. (Upper) In the optical image of the diffraction spectrum, numbers correspond to the diffracted orders, and colors correspond to the wavelengths. (Lower) In the processed spectrum of intensity vs. pixel number, the arrows point to the full-width half-maxima that were measured as ⌬␭, and used to calculate R; the colors correspond to the wavelengths. concentrate light on specific points in the sample, with a plane (and its conjugate planes) (Fig. 1B). Our system has transmission grating, which disperses the light that leaves the significant advantages over previous methods for observing sample into a continuous spectrum (Fig. 1A). The focal distance diffracted light in a microscope (such as using a Bertrand lens): of the microlenses, as calculated from the equation in ref. 24, is it produces magnified and high-quality images of the diffracted 179 ␮m. The lenses concentrate the light into the sample placed light that are suitable for data processing (compared to low- directly above them. In addition, because the grating is Ϸ3mm magnification and spherically distorted images by direct obser- above the sample, well past the focal point of the microlenses, the vation of the rear focal plane), and it uses only a bright-field microlenses help to spread the light at the grating to illuminate microscope with no requirements for special optics. By analyzing a large number of grooves and hence increase the resolution of the intensity at each pixel of the image of the first-order the diffracted light. diffracted light, and converting the pixel number to wavelength, By focusing the objective lens near the microlenses, we capture we easily obtain, for any given spatial position of the sample, the the diffraction spectrum in the primary image plane; the dif- absorbance of the sample vs. wavelength in the visible spectrum. fraction spectrum is normally observed only at the rear focal The sample is normally placed between the microlenses and 10036 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504712102 Damean et al.