Optofluidic Microscopy Research Articles

Super-resolution optofluidic scanning microscopy.

Optofluidics enables visualizing diverse anatomical and functional traits of single-cell specimens with new degrees of imaging capabilities. However, the current optofluidic microscopy systems suffer from either low resolution to reveal subcellular details or incompatibility with general microfluidic devices or operations. Here, we report optofluidic scanning microscopy (OSM) for super-resolution, live-cell imaging. The system exploits multi-focal excitation using the innate fluidic motion of the specimens, allowing for minimal instrumental complexity and full compatibility with various microfluidic configurations. The results present effective resolution doubling, optical sectioning and contrast enhancement. We anticipate the OSM system to offer a promising super-resolution optofluidic paradigm for miniaturization and different levels of integration at the chip scale.

Optical Time Stretch for High-Speed and High-Throughput Imaging—From Single-Cell to Tissue-Wide Scales

Initially developed for high-speed optical communication, optical time stretch has recently been adopted for ultrafast and sensitive optical imaging at an unprecedented speed. In this paper, we highlight the essential concepts as well as the enabling elements of this ultrafast technology. More importantly, we review the recent developments of optical time-stretch imaging, especially in the context of 1) quantitative optofluidic microscopy for high-content single-cell phenotyping at an imaging throughput ~100 000 cells/s; 2) all-optical multi-MHz (>10 MHz) swept-source optical coherence tomography (OCT) for high-speed in vivo anatomical and functional 3-D tissue imaging. We also discuss the current technological challenges in time-stretch imaging. In particular, generating the enormous data in real time, this technology could uniquely create new insights of data-driven science in clinical diagnostics and basic biological research.

Dual characterization of biological cells by optofluidic microscope and resistive pulse sensor.

Label-free detection technique has emerged as a powerful platform for biomedical applications since it can avoid laborious multi-step sample preparation. In this paper, we demonstrate a dual analysis of biological cells using a single microfluidic system combining optofluidic microscopy and resistive pulse sensing. Both red blood cells (RBCs) and circulating tumor cells (CTCs) have been used to validate the concept of dual analysis and also to test the performance of the microfluidic device. The cell characterization by resistive pulse sensing is in good agreement with the analysis by optofluidic microscopy, further verified by the commercial Beckman-Coulter® FC500 flow cytometry. The present system has attractive merits such as simple fabrication, easy integration, high portability, and low cost. This study has great potentials for the development of innovative on-chip flow cytometry with concurrent imaging sensing and resistive sensing.

Three-dimensional refractive index reconstruction of red blood cells with one-dimensional moving based on local plane wave approximation

To simplify the reconstruction optical system, an improved method is developed to reconstruct the three-dimensional (3D) quantitative refractive index of live cells based on a local plane wave approximation. In this method, the cell is illuminated by a convergent beam whose cross section is much larger than the cell, so the beam passing through the cell can be treated as a local plane wave. With the one-dimensional moving of cells in the beam cross section, multi-directional phase projections can be obtained using a Mach–Zehnder interferometer and the Hilbert transform phase retrieval algorithm. An inverse Radon–Radon iterative algorithm is used to reconstruct the 3D refractive index distribution which is verified by a simulation reconstruction. The corresponding set-up is applied to obtain the multi-directional phase projections and reconstruct the 3D refractive index of red blood cells (RBCs). The results show that only with a simple device could the method measure the 3D refractive index of cells with high precision. The reconstruction method could also be applied in opto-fluidic microscopy to fabricate a compact on-chip opto-fluidic tomographic microscope.

Automation Highlights from the Literature

Automation Highlights from the Literature

Color Capable Sub-Pixel Resolving Optofluidic Microscope and Its Application to Blood Cell Imaging for Malaria Diagnosis

Miniaturization of imaging systems can significantly benefit clinical diagnosis in challenging environments, where access to physicians and good equipment can be limited. Sub-pixel resolving optofluidic microscope (SROFM) offers high-resolution imaging in the form of an on-chip device, with the combination of microfluidics and inexpensive CMOS image sensors. In this work, we report on the implementation of color SROFM prototypes with a demonstrated optical resolution of 0.66 µm at their highest acuity. We applied the prototypes to perform color imaging of red blood cells (RBCs) infected with Plasmodium falciparum, a particularly harmful type of malaria parasites and one of the major causes of death in the developing world.

The ePetri dish, an on-chip cell imaging platform based on subpixel perspective sweeping microscopy (SPSM)

We report a chip-scale lensless wide-field-of-view microscopy imaging technique, subpixel perspective sweeping microscopy, which can render microscopy images of growing or confluent cell cultures autonomously. We demonstrate that this technology can be used to build smart Petri dish platforms, termed ePetri, for cell culture experiments. This technique leverages the recent broad and cheap availability of high performance image sensor chips to provide a low-cost and automated microscopy solution. Unlike the two major classes of lensless microscopy methods, optofluidic microscopy and digital in-line holography microscopy, this new approach is fully capable of working with cell cultures or any samples in which cells may be contiguously connected. With our prototype, we demonstrate the ability to image samples of area 6 mm × 4 mm at 660-nm resolution. As a further demonstration, we showed that the method can be applied to image color stained cell culture sample and to image and track cell culture growth directly within an incubator. Finally, we showed that this method can track embryonic stem cell differentiations over the entire sensor surface. Smart Petri dish based on this technology can significantly streamline and improve cell culture experiments by cutting down on human labor and contamination risks.

Lensfree Optofluidic Microscopy and Tomography

Microfluidic devices aim at miniaturizing, automating, and lowering the cost of chemical and biological sample manipulation and detection, hence creating new opportunities for lab-on-a-chip platforms. Recently, optofluidic devices have also emerged where optics is used to enhance the functionality and the performance of microfluidic components in general. Lensfree imaging within microfluidic channels is one such optofluidic platform, and in this article, we focus on the holographic implementation of lensfree optofluidic microscopy and tomography, which might provide a simpler and more powerful solution for three-dimensional (3D) on-chip imaging. This lensfree optofluidic imaging platform utilizes partially coherent digital in-line holography to allow phase and amplitude imaging of specimens flowing through micro-channels, and takes advantage of the fluidic flow to achieve higher spatial resolution imaging compared to a stationary specimen on the same chip. In addition to this, 3D tomographic images of the same samples can also be reconstructed by capturing lensfree projection images of the samples at various illumination angles as a function of the fluidic flow. Based on lensfree digital holographic imaging, this optofluidic microscopy and tomography concept could be valuable especially for providing a compact, yet powerful toolset for lab-on-a-chip devices.

Fluorescence microscopy imaging with a Fresnel zone plate array based optofluidic microscope.

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(®).

Holographic opto-fluidic microscopy

Over the last decade microfluidics has created a versatile platform that has significantly advanced the ways in which micro-scale organisms and objects are controlled, processed and investigated, by improving the cost, compactness and throughput aspects of analysis. Microfluidics has also expanded into optics to create reconfigurable and flexible optical devices such as reconfigurable lenses, lasers, waveguides, switches, and on-chip microscopes. Here we present a new opto-fluidic microscopy modality, i.e., Holographic Opto-fluidic Microscopy (HOM), based on lensless holographic imaging. This imaging modality complements the miniaturization provided by microfluidics and would allow the integration of microscopy into existing on-chip microfluidic devices with various functionalities. Our imaging modality utilizes partially coherent in-line holography and pixel super-resolution to create high-resolution amplitude and phase images of the objects flowing within micro-fluidic channels, which we demonstrate by imaging C. elegans, Giardia lamblia, and Mulberry pollen. HOM does not involve complicated fabrication processes or precise alignment, nor does it require a highly uniform flow of objects within microfluidic channels.

Pixel level optical-transfer-function design based on the surface-wave-interferometry aperture.

The design of optical transfer function (OTF) is of significant importance for optical information processing in various imaging and vision systems. Typically, OTF design relies on sophisticated bulk optical arrangement in the light path of the optical systems. In this letter, we demonstrate a surface-wave-interferometry aperture (SWIA) that can be directly incorporated onto optical sensors to accomplish OTF design on the pixel level. The whole aperture design is based on the bull’s eye structure. It composes of a central hole (diameter of 300 nm) and periodic groove (period of 560 nm) on a 340 nm thick gold layer. We show, with both simulation and experiment, that different types of optical transfer functions (notch, highpass and lowpass filter) can be achieved by manipulating the interference between the direct transmission of the central hole and the surface wave (SW) component induced from the periodic groove. Pixel level OTF design provides a low-cost, ultra robust, highly compact method for numerous applications such as optofluidic microscopy, wavefront detection, darkfield imaging, and computational photography.

Optofluidic microscope with 3D spatial resolution

This paper reports on-chip based optical detection with three-dimensional spatial resolution by integration of an optofluidic microscope (OFM) in a microfluidic pinched flow fractionation (PFF) separation device. This setup also enables on-chip particle image velocimetry (PIV). The position in the plane perpendicular to the flow direction and the velocity along the flow direction of separated fluorescent labeled polystyrene microspheres with diameters of 1 microm , 2.1 microm , 3 microm and 4 microm is determined by the OFM. These results are bench marked against those obtained with a PFF device using conventional fluorescence microscope readout. The size separated microspheres are detected by OFM with an accuracy of <or=0.92 microm . The position in the height of the channel and the velocity of the separated microspheres are detected with an accuracy of 1.4 microm and 0.08 mm/s respectively. Throughout the measurements of the height and velocity distribution, the microspheres are observed to move towards the center of the channel in regard to its height.

Implementation of a color-capable optofluidic microscope on a RGB CMOS color sensor chip substrate.

We report the implementation of a color-capable on-chip lensless microscope system, termed color optofluidic microscope (color OFM), and demonstrate imaging of double stained Caenorhabditis elegans with lacZ gene expression at a light intensity about 10 mW/cm(2).

Sub-pixel resolving optofluidic microscope for on-chip cell imaging

We report the implementation of a fully on-chip, lensless, sub-pixel resolving optofluidic microscope (SROFM). The device utilizes microfluidic flow to deliver specimens directly across a complementary metal oxide semiconductor (CMOS) sensor to generate a sequence of low-resolution (LR) projection images, where resolution is limited by the sensor's pixel size. This image sequence is then processed with a pixel super-resolution algorithm to reconstruct a single high resolution (HR) image, where features beyond the Nyquist rate of the LR images are resolved. We demonstrate the device's capabilities by imaging microspheres, protist Euglena gracilis, and Entamoeba invadens cysts with sub-cellular resolution and establish that our prototype has a resolution limit of 0.75 microns. Furthermore, we also apply the same pixel super-resolution algorithm to reconstruct HR videos in which the dynamic interaction between the fluid and the sample, including the in-plane and out-of-plane rotation of the sample within the flow, can be monitored in high resolution. We believe that the powerful combination of both the pixel super-resolution and optofluidic microscopy techniques within our SROFM is a significant step forwards toward a simple, cost-effective, high throughput and highly compact imaging solution for biomedical and bioscience needs.

Optofluidic microscope shrinks to fit on a chip

Optofluidic microscope shrinks to fit on a chip

The application of on-chip optofluidic microscopy for imaging Giardia lamblia trophozoites and cysts

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 poor-quality 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.

The application of Fresnel zone plate based projection in optofluidic microscopy

Optofluidic microscopy (OFM) is a novel technique for low-cost, 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 cost-effective 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 microm, focal length = 800 microm) that has been patterned onto a glass slide to project the transmission from an array of apertures (diameter = 1 microm, separation = 10 microm) 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.

An optical tweezer actuated, nanoaperture-grid based Optofluidic Microscope implementation method

We report a novel grid based Optofluidic Microscope (OFM) method where a closely spaced 2D grid of nanoapertures (diameter = 100 nm, separation = 2.5 mum) provided patterned illumination. We achieved a one-to-one mapping of the light transmissions through the nanoapertures onto a high-speed CCD camera. By optically tweezing a targeted sample across the grid in a controlled fashion and recording the time varying light reception from the nanoapertures, we were able to generate high-resolution images of the sample. The achievable resolution limit of the prototype was ~ 110 nm (Sparrow's criterion) under optimal conditions. We demonstrated the technique by imaging polystyrene beads and pollen spores.

Optofluidic microscopy—a method for implementing a high resolution optical microscope on a chip

We report a novel microfluidics-based lensless imaging technique, termed optofluidic microscopy (OFM), and demonstrate Caenorhabditis elegans imaging with an OFM prototype that gives comparable resolution to a conventional microscope and a measured resolution limit of 490 +/- 40 nm.