A new optical microscopy technique, termed high spatial and temporal resolution synthetic aperture phase microscopy (HISTR-SAPM), is proposed to improve the lateral resolution of wide-field coherent imaging. Under plane wave illumination, the resolution is increased by twofold to around 260 nm, while achieving millisecond-level temporal resolution. In HISTR-SAPM, digital micromirror devices are used to actively change the sample illumination beam angle at high speed with high stability. An off-axis interferometer is used to measure the sample scattered complex fields, which are then processed to reconstruct high-resolution phase images. Using HISTR-SAPM, we are able to map the height profiles of subwavelength photonic structures and resolve the period structures that have 198 nm linewidth and 132 nm gap (i.e., a full pitch of 330 nm). As the reconstruction averages out laser speckle noise while maintaining high temporal resolution, HISTR-SAPM further enables imaging and quantification of nanoscale dynamics of live cells, such as red blood cell membrane fluctuations and subcellular structure dynamics within nucleated cells. We envision that HISTR-SAPM will broadly benefit research in material science and biology.
KEYWORDS: Digital holography, Microscopy, Real time imaging, Measurement devices, Imaging systems, Imaging devices, Microscopes, Phase imaging, 3D scanning, Profilometers
High performance commercial 3D scanners and profilometers are complex and expensive for individual users. On the other hand, there are many onsite applications that require handheld portable devices to reliably measure 3D structures with high accuracy and sensitivity, such as inspecting flat mechanical surfaces and testing water quality. Here, we present a portable quantitative phase microscopy (QPM) system to tackle such onsite applications and laboratory needs. Our portable QPM system integrates digital holography, optical microscopy, and smartphone technologies into one single platform to allow for a broader user accessibility. Unlike the current prevailing smartphone imaging techniques, our method does not require heavy computations and complicated algorithms. Our image retrieval algorithm, particularly phase retrieval based on digital holographic measurements, is simply that allows for achieving real-time imaging on mobile devices. Our system is an effort on miniaturizing a compact QPM system and reducing their cost. With this portable system, we are currently exploring various applications ranging from characterizing microfluidic devices to imaging microscopic structures in water. We envision our QPM unit will be widely available to individual users for more exciting applications in the near future.
Optical diffraction tomography (ODT), using an interferometric microscopy technique, can quantitatively measure the three-dimensional (3-D) refractive index (RI) distribution in transparent samples. ODT features unique advantages such as non-invasive, label-free, and high-resolution imaging; these capabilities have been increasingly explored recently, particularly in the field of cell biology. Normally, the RI map is reconstructed by solving the inverse scattering problem using more than one hundred holograms, which correspond to various angles of illumination. Current reconstruction methods all require that each hologram is created by only one illumination angle. Therefore, the number of measurements must be equal to the number of needed illumination angles, thus limiting ODT for video-rate or real-time imaging applications. To overcome this issue, we propose a new ODT system together with a new reconstruction algorithm. In the proposed optical system, the illumination is multiplexed by coding a digital micromirror device to display a series of Lee holograms, each of which corresponds to a plane wave of a specific incident angle in the sample plane. On the other hand, the reconstruction algorithm uses the beam propagation method to model the sample scattering process, as well as the error propagation method to train the artificial neural network which represents the RI distribution of the sample. This novel method is expected to reduce the measurement time by a factor of 4-6, which is crucial for video rate or even real time tomography imaging applications such as label-free 3-D imaging cytometry.
Most of the quantitative phase microscopy systems are unable to provide depth-resolved information for measuring complex biological structures. Optical diffraction tomography provides a non-trivial solution to it by 3D reconstructing the object with multiple measurements through different ways of realization. Previously, our lab developed a reflection-mode dynamic speckle-field phase microscopy (DSPM) technique, which can be used to perform depth resolved measurements in a single shot. Thus, this system is suitable for measuring dynamics in a layer of interest in the sample. DSPM can be also used for tomographic imaging, which promises to solve the long-existing “missing cone” problem in 3D imaging. However, the 3D imaging theory for this type of system has not been developed in the literature. Recently, we have developed an inverse scattering model to rigorously describe the imaging physics in DSPM. Our model is based on the diffraction tomography theory and the speckle statistics. Using our model, we first precisely calculated the defocus response and the depth resolution in our system. Then, we further calculated the 3D coherence transfer function to link the 3D object structural information with the axially scanned imaging data. From this transfer function, we found that in the reflection mode excellent sectioning effect exists in the low lateral spatial frequency region, thus allowing us to solve the “missing cone” problem. Currently, we are working on using this coherence transfer function to reconstruct layered structures and complex cells.
Due to the large number of available mirrors, the patterning speed, low-cost, and compactness, digital-micromirror devices (DMDs) have been extensively used in biomedical imaging system. Recently, DMDs have been brought to the quantitative phase microscopy (QPM) field to achieve synthetic-aperture imaging and tomographic imaging. Last year, our group demonstrated using DMD for QPM, where the phase-retrieval is based on a recently developed Fourier ptychography algorithm. In our previous system, the illumination angle was varied through coding the aperture plane of the illumination system, which has a low efficiency on utilizing the laser power. In our new DMD-based QPM system, we use the Lee-holograms, which is conjugated to the sample plane, to change the illumination angles for much higher power efficiency. Multiple-angle illumination can also be achieved with this method. With this versatile system, we can achieve FPM-based high-resolution phase imaging with 250 nm lateral resolution using the Rayleigh criteria. Due to the use of a powerful laser, the imaging speed would only be limited by the camera acquisition speed. With a fast camera, we expect to achieve close to 100 fps phase imaging speed that has not been achieved in current FPM imaging systems. By adding reference beam, we also expect to achieve synthetic-aperture imaging while directly measuring the phase of the sample fields. This would reduce the phase-retrieval processing time to allow for real-time imaging applications in the future.
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