Microfluidic devices have undergone rapid development in recent years and provide a lab-on-a-chip solution for many biomedical and chemical applications. Optical imaging techniques are essential in microfluidics for observing and extracting information from biological or chemical samples. Traditionally, imaging in microfluidics is achieved by bench-top conventional microscopes or other bulky imaging systems. More recently, many novel compact microscopic techniques have been developed to provide a low-cost and portable solution. In this review, we provide an overview of optical imaging techniques used in microfluidics followed with their applications. We first discuss bulky imaging systems including microscopes and interferometer-based techniques, then we focus on compact imaging systems that can be better integrated with microfluidic devices, including digital inline holography and scanning-based imaging techniques. The applications in biomedicine or chemistry are also discussed along with the specific imaging techniques.

We report the implementation of an on-chip microscope system, termed fluorescence optofluidic microscope (FOFM), which is capable of fluorescence microscopy imaging of samples in fluid media. The FOFM employs an array of Fresnel zone plates (FZP) to generate an array of focused light spots within a microfluidic channel. As a sample flows through the channel and across the array of focused light spots, the fluorescence emissions are collected by a filter-coated CMOS sensor, which serves as the channel's floor. The collected data can then be processed to render fluorescence microscopy images at a resolution determined by the focused light spot size (experimentally measured as 0.65 μm FWHM). In our experiments, our established resolution was 1.0 μm due to Nyquist criterion consideration. As a demonstration, we show that such a system can be used to image the cell nuclei stained by Acridine Orange and cytoplasm labeled by Qtracker®.

The optofluidic microscope (OFM) is a lensless, low-cost and highly compact on-chip device that can enable high-resolution microscopy imaging. The OFM performs imaging by flowing/scanning the target objects across a slanted hole array; by measuring the time-varying light transmission changes through the holes, we can then render images of the target objects at a resolution that is comparable to the holes' size. This paper reports the adaptation of the OFM for imaging Giardia lamblia trophozoites and cysts, a disease-causing parasite species that is commonly found in poorquality water sources. We also describe our study of the impact of pressure-based flow and DC electrokinetic-based flow in controlling the flow motion of Giardia cysts -rotation-free translation of the parasite is important for good OFM image acquisition. Finally, we report the successful microscopy imaging of both Giardia trophozoites and cysts with an OFM that has a focal plane resolution of 0.8 microns.

We have developed a new microscopy design that can achieve wide field-of-view (FOV) imaging and yet possesses resolution that is comparable to a conventional microscope. In our design, the sample is illuminated by a holographically projected light-spot grid. We acquire images by translating the sample across the grid and detecting the transmissions. We have built a prototype system with an FOV of 6 mm × 5 mm and acquisition time of 2:5 s. The resolution is fundamentally limited by the spot size-our demonstrated average FWHM spot diameter was 0:74 μm. We demonstrate the prototype by imaging a U.S. Air Force target and a lily anther. This technology is scalable and represents a cost-effective way to implement wide FOV microscopy systems. © 2010 Optical Society of America OCIS codes: 170.0110, 090.2890, 170.5810.Automated, high-resolution, and cost-effective wide fieldof-view (FOV) microscopy is highly sought for many applications, such as high-throughput screening [1] and whole-slide digital pathology diagnosis [2]. In a conventional microscope, the FOV is inversely related to the microscope objective's resolution due to the critical requirement of aberration correction for the whole viewing area. Commercial products for accomplishing wide FOV imaging typically raster scan the target samples under microscope objectives and reconstitute full-view images from multiple smaller images. This approach requires precise mechanical actuation along two axes. Scaling up the FOV for such an approach requires a linear cost increase (add more objectives) or longer scan time. Recently, exciting in-line holography methods [3,4] demonstrated the potential to cover a wide FOV image very cost effectively and without requiring sophisticated optics and mechanical scanning. In-line holography does require excellent raw data quality, as data noise can significantly distort the computed image and deteriorate resolution. To our knowledge, in-line holography's demonstrated resolution for simple objects is about 1 μm [3]. In this Letter, we report a microscopy technique that employs holography concepts in a different fashion to accomplish wide FOV imaging. Our technique, termed holographic scanning microscopy (HSM), uses a specially written hologram to generate a grid of tightly focused light spots and uses this grid as illumination on the target sample to perform parallel multifocal scanning while the sample is translated across the grid. In comparison to inline holography, the resolution here is fundamentally determined by the focused spot size. Unlike in-line holography, this approach does require mechanical scanning, but the scanning format is a simple one-dimensional (1D) translation. This approach is readily scalable, as we would simply use a large hologram with more projection light spots to accomplish wider FOV imaging.Our HSM prototype demonstration, as shown in Fig. 1(a), used a laser (Excelsior-532-200-CDRH, Spectra Physics, with wavelength of 532 nm and power of 200 mW) as light source. The laser was attenuated, spatial filtered...

A novel photolithography method to build aligned patterns of two different proteins is presented. Chessboard patterns of 125 microm x 125 microm squares are constructed on a silicon dioxide substrate, using standard photoresist chemistries in combination with low-temperature oxygen plasma etching. Low-melting-point agarose (LMPA) is used to protect underlying protein layers and, at the appropriate stage, the digestive enzyme GELase (EPICENTRE) is used to selectively remove the prophylactic LMPA layers. Two antibodies, mouse-IgG and human-IgG, were immobilized and patterned by this procedure. The patterned antibodies maintained the specificity of their antigen-antibody binding, as demonstrated by fluorescence microscopy. In addition, normalized fluorescence intensity profiles illustrate that the patterned proteins layers are uniform (standard deviations below 0.05). Finally, a trypsin activity test was conducted to probe the effect of the patterning protocol on immobilized enzymes; the results imply that this photolithographic process using LMPA as a protection layer preserves 70% of immobilized enzyme activity.

A novel self-aligned method to selectively immobilize proteins on a silicon dioxide surface is developed in conjunction with a standard lift-off patterning technique of a PEG layer. The approach is designed to photolithographically pattern regions that specifically bind target proteins and particles, surrounded by regions that suppress non-specific attachment of bio-species. The physical and biological properties of the derivatized surfaces at the end of the fabrication process are characterized.

Optofluidic microscopy (OFM) is a novel technique for lowcost, high-resolution on-chip microscopy imaging. In this paper we report the use of the Fresnel zone plate (FZP) based projection in OFM as a costeffective and compact means for projecting the transmission through an OFM's aperture array onto a sensor grid. We demonstrate this approach by employing a FZP (diameter = 255 μm, focal length = 800 μm) that has been patterned onto a glass slide to project the transmission from an array of apertures (diameter = 1 μm, separation = 10 μm) onto a CMOS sensor. We are able to resolve the contributions from 44 apertures on the sensor under the illumination from a HeNe laser (wavelength = 633 nm). The imaging quality of the FZP determines the effective field-of-view (related to the number of resolvable transmissions from apertures) but not the image resolution of such an OFM system -a key distinction from conventional microscope systems. We demonstrate the capability of the integrated system by flowing the protist Euglena gracilis across the aperture array microfluidically and performing OFM imaging of the samples. E. Di Fabrizio, F. Romanato, M. Gentili, S. Cabrini, B. Kaulich, J. Susini, and R. Barrett, "High-efficiency multilevel zone plates for KeV X-rays," Nature 401, 895-898 (1999). 8.R. M. Henkelman and M. J. Bronskill, "Imaging extended objects with a Fresnel-zone-plate aperture," J. Opt. Soc. Am. 64, 134-137 (1974). 9.M. Young, "Zone plates and their aberrations," J. Opt. Soc. Am. 62, 972-976 (1972). 10. F. Wyrowski, "Diffractive optical elements: iterative calculation of quantized, blazed phase structures," J.

In this letter, the authors present a novel quadrature interferometry method based on the use of a harmonically matched shallow grating pair. Unlike a simple beam splitter or single shallow grating, the grating pair can confer a nontrivial interference phase shift ͑other than 0°or 180°͒ between the output ports of the interferometer. Using the grating pair as the beam splitter/combiner, the authors implement a homodyne quadrature full field phase interferometer and demonstrate the system's capability to acquire phase and amplitude images. © 2007 American Institute of Physics. ͓DOI: 10.1063/1.2722685͔Full field phase based imaging techniques 1-4 are important for a wide range of applications, such as microscopy and metrology. These methods generally involve interferometry and incorporate some form of nontrivial encoding ͑in time, space, or polarization͒ for phase extraction. The encoding process typically entails a more complicated experimental scheme, computationally intensive postprocessing, or some sacrifice in the imaging field of view. In this context, a full field interferometry scheme where the resulting interference outputs are naturally in or close to quadrature can, in principle, simplifies the phase imaging process. However, this requirement is nontrivial. In fact, the outputs of any two-port nonlossy interferometer scheme, including Michelson, Mach-Zehnder, and Sagnac schemes, are constrained to be 180°shifted ͑trivial͒ by energy conservation. We recently demonstrated that a quadrature free-space phase interferometer, termed as the G1G2 interferometer, can be created with a pair of harmonically matched shallow diffraction gratings. 5 In this letter, we report the following: ͑1͒ the creation of the harmonically matched grating pair on a single holographic plate, ͑2͒ the use of this single optical element in place of a beam splitter in a modified Mach-Zehnder interferometer and the observation of nontrivial phase between the outputs, and ͑3͒ a demonstration of full field phase imaging, which additionally illustrates the utility of phase imaging for flow dynamics studies.A single shallow diffraction grating can be used to create a multiport ͑n ജ 3͒ interferometer. However, the outputs of such an interferometer are trivially related in phase. In comparison, the interference between diffractions from the two gratings in a G1G2 interferometer can give rise to nontrivial phase shifts between the outputs. To better explain this concept, we listed the phase of each diffraction order of interest and interference term for a single grating interferometer and a G1G2 interferometer in Fig. 1. In the figure, G1 and G2 are single gratings and their periods ⌳ 1 , ⌳ 2 satisfy ⌳ 2 =2⌳ 1 . x 1 and x 2 are the displacements of the single gratings G1 and G2 with respect to the origin. The phase shift of the mth diffracted order from a shallow grating is given by 5where x 0 is the displacement of the grating from the origin. From Fig. 1͑a͒, we can see that a single grating interferometer can only give rise to a trivial phas...