Johns Hopkins engineers have created a new lens-free ultra-miniaturized endoscope, the size of a few human hairs in width, that is less bulky and can produce higher quality images.
Their findings were published today in Science Advances.
“Usually, you have sacrifice either size or image quality.
We’ve been able to achieve both with our microendoscope,” says Mark Foster, an associate professor of electrical and computer engineering at The Johns Hopkins University and the study’s corresponding author.
Intended for examining neurons firing off in the brains of animals such as mice and rats, an ideal microendoscope should be small to minimize brain tissue damage yet powerful enough to produce a clear image.
Currently, standard microendoscopes are about half a millimeter to a few millimeters in diameter, and require larger, more invasive lenses for better imaging.
While lensless microendoscopes exist, the optical fiber within that scans an area pixel by pixel frequently bends and loses imaging ability when moved.
In their new study, Foster and colleagues created a lens-free ultra-miniaturized microendoscope that, compared to a conventional lens-based microendoscope, increases the amount researchers can see and improves image quality.
The researchers achieved this by using a coded aperture, or a flat grid that randomly blocks light creating a projection in a known pattern akin to randomly poking a piece of aluminum foil and letting light through all of the small holes.
This creates a messy image, but one that provides a bounty of information about where the light originates, and that information can be computationally reconstructed into a clearer image. In their experiments, Foster’s team looked at beads in different patterns on a slide.
“For thousands of years, the goal has been to make an image as clear as possible. Now, thanks to computational reconstruction, we can purposefully capture something that looks awful and counterintuitively end up with a clearer final image,” says Foster.
Additionally, Foster and team’s microendoscope doesn’t require movement to focus on objects at different depths; they use computational refocusing to determine where the light originated from in 3 dimensions.
This allows the endoscope to be much smaller than a traditional one that requires moving the endoscope around to focus.
Looking forward, the research team will test their microendoscope with fluorescent labeling procedures in which active brain neurons would be tagged and illuminated, to determine how accurately the endoscope can image neural activity.
Micro-optical Components for in vitro Bioimaging
Recent advances in photonics and imaging techniques offer new possibilities for optical imaging systems and sensor devices for fundamental studies and future biomedical applications. Ongoing developments suggest that an impressive diversity of biological and medical questions can be answered using optical microscopy. Advanced optical techniques have been proposed for achieving high-resolution imaging of sample of interest. For example, multi-photon microscopes can investigate cortical micro-architecture in animals’ brain with single cell resolution . Also in regard to detecting levels of the tumor suppressing protein p53 in cells, fluorescence microscopy has been used to image cancerous tissues that were stained by specially designed oncolytic adenoviruses, the latter being programmed to replicate if the cellular p53 level is low [56,57].
However, these techniques are frequently accompanied by the need for high-end, bulky and expensive optical set-ups that are rather cumbersome to operate and to maintain, especially for untrained users. Such drawbacks have motivated researchers to minimize and integrate optical components in imaging systems for in situ analysis and observation of bio-samples under in vitro environment. MOEMS or Optical MEMS technology has recently demonstrated a strong potential in biomedical imaging applications due to its outstanding advantages including, for instance, low cost, high operating frequency and convenience of batch fabrication.
Integrating micro-optical components with optical imaging setups for in vitro observation and analysis has been demonstrated by many researchers. Here, we will review several works as examples, showing the capability and potential of the miniaturized optical components. Diekmann et al. demonstrated the use of a waveguide-integrated optical chip, which hosts the biological sample and utilizes the waveguide as the illumination source, and a standard low-cost microscope to acquire super-resolved images of single molecules via two different approaches .
The waveguides composed of a material with high refractive-index contrast can provide a strong evanescent field that is used for single-molecule switching and fluorescence excitation, thus enabling chip-based single-molecule localization microscopy. Additionally, multimode interference patterns can induce spatial fluorescence intensity variations that enable fluctuation-based super-resolution imaging .
The waveguides on the chip are used for total-internal-reflection fluorescence excitation, and the detection signal are collected and recorded by conventional optical microscope. By achieving visualization of fenestrations in liver sinusoidal endothelial cells with feature size smaller than the diffraction limit, the setup shows its great potential on upgrading standard optical microscopes to super-resolution microscopic tools .
Yokokawa et al. reported a micro-device that is equipped with an optical fiber, a microlens and a micro-prism for in situ observation and analysis of cells . The optical components were used to compose a TIR-based chip to perform TIR fluorescence microscopy on adherent cells. The cells were cultured under continuous medium perfusion in a microfluidic channel that is located on top of the TIR-based chip (Figure 8a,b). The device was evaluated by monitoring the location of insulin granules in mouse pancreatic cells. The system allowed a higher imaging signal-to-noise ratio than obtained with epifluorescence microscopy.
Advanced microelectronics and emerging computational microscopy techniques have provided another scheme to bypass various limitations of conventional optical microscopy. Typically, bioimaging is performed at the microscopic scale and this usually requires lenses integrated in a microscopic system. Conventional optical microscope can be downsized to a portable device by utilizing commercial electronic devices. Breslauer et al. developed a microscope attachment for cell-phones that is capable of both bright-field and fluorescent imaging . This microscope utilizes trans-illumination configuration with standard microscope eyepieces and objectives; magnification and resolution can be adjusted using different objectives. This cellphone microscope shows promising results for clinical use by imaging P. falciparum-infected and sickled red blood cells in bright-field mode, and M. tuberculosis-infected sputum samples in fluorescent mode with light-emitting diode (LED) excitation.
Furthermore, lens-free imaging has matured as a modality competitive with traditional lens-based microscopy. By placing an image sensor (CMOS/CCD) beneath a biological sample and using it to record light transmission pixel by pixel, a diffraction pattern resulting from the sample is recorded directly on the image sensor without being optically imaged or magnified by any lens elements, traditional lens components in a microscopic system can be abandoned .
Such lens-free (or lensless) scheme shows great flexibility on the integration format, and offers great possibilities to provide a compact and non-diffraction-limited imaging technique with large field-of-view, of the same size as the sensor size and a numerical aperture close to 1, since the large-area detector is placed very close to the sample. In the last decade, multiple lens-free on-chip microscope modus have been proposed to perform imaging on microorganisms  and cells  etc., showing exciting breakthroughs in point-of-care applications. Yang’s group demonstrated an optofluidic microscope (OFM) for lens-free contact imaging on Caenorhabditis elegans . The OFM mobilizes the specimen along a microfluidic channel by laminar flow, and the channel is positioned directly over the image sensor.
A tilted array of metallic apertures is patterned directly over the image sensor. Each aperture is carefully positioned at the center of a pixel of the image sensor, so that shadows of the specimens can be sampled by these sub-micron apertures as they flow along the microfluidic channel. By using OFM, automated phenotype characterization of different Caenorhabditis elegans mutant strains, as well as imaging of spores and single cellular entities, have been demonstrated .
The same group later on presented the implementation of color OFM prototypes color imaging of red blood cells infected with Plasmodium falciparum, a particularly harmful type of malaria parasites and one of the major causes of death in the developing world . Ozcan’s research group has developed different lens-free holographic imaging platforms for on-chip cytometry and diagnostics, with either fluorescent imaging format  or shadow imaging format . Zhu et al. demonstrated a compact platform that integrated imaging cytometry and fluorescent microscopy and could be attached to a cell phone .
The resulting device could be used to rapidly image bodily fluids for cell counts or cell analysis (Figure 8c). Wei et al. proposed using gold and silver nanobeads as specific labels to identify and count the number of CD4 and CD8 cells in a cell suspension . CD4 and CD8 cells are specific types of T lymphocytes, and their relative populations are important for evaluating the stage of human immunodeficiency virus (HIV) infection or acquired immune deficiency syndrome (AIDS), as well as for evaluating the efficacy of antiretroviral treatment.
Counting the relative populations of these cells can be challenging, however, because the only significant difference between these cells is in the types of proteins expressed on their membranes. Under a conventional optical microscope, both types of cells look virtually identical. However, by using gold nanoparticles functionalized with anti-CD4 antibodies to label CD4 cells and silver nanoparticles functionalized with anti-CD8 to label CD8 cells, the different spectral response of the labelled cells can be used to discriminate these two types of cells with greater than 95% accuracy using a machine learning algorithm.
Micro-optical Components for in vivo Bioimaging
In vitro imaging of biological samples remains as one of the main tools for diagnosis of disease. However, it typically requires invasive sample collection (e.g., biopsy) steps as well as time consuming sample preparation protocols (e.g., fixation and staining) . On the other hand, in vivo optical imaging techniques enable real-time visualization and might provide accurate information for diagnostics, making them highly desirable for diagnostics. In this section, we will briefly review two main in vivo optical imaging techniques, i.e., microendoscopy and OCT, that hold various integration format with the micro-optical components.
Microendoscopy is a promising approach for in vivo imaging. It combines conventional intravital microscopy and miniature endoscopy, using a narrow-diameter optical probe that provides minimally invasive access to internal organs that are otherwise difficult to reach with conventional instruments .
Optical microendoscopy provides spatial resolution that can approach that of a conventional water-immersion objective lens ; is compatible for use with multiple contrast modalities including epifluorescence, two-photon excited fluorescence, and second-harmonic generation; and has been used in both live animals and humans . Kim et al. developed an in vivo confocal and multiphoton microendoscopic imaging system which integrated with gradient index lenses .
The cellular resolution of the system was demonstrated by imaging the micrometer-scale structures of the fluorescence labelled pollen grains. Besides, the boundaries of spherical starch granules in a sliced fresh potato were acquired with the second-harmonic generation imaging mode. Individual dendritic cells in the epidermis and dermis, blood vessels, as well as collagen fibrillar structure in live mice, were clearly imaged .
By using high-end micro-optical structures, such as high-resolution microlenses , the performance of the imaging system can be further improved. Barretto et al. demonstrated two-photon imaging of dendritic spines on hippocampal neurons and dual-color nonlinear optical imaging of neuromuscular junctions in live mice .
Many advances in microendoscopy have been reported in the last decade. Most endoscope designs involve three key components: optical fibers used for efficient laser pulse delivery and signal collection, micro-optics used for imaging, such as microlenses, and miniaturized scanning devices, such as micromirrors .
Rogers et al. demonstrated an integrated microendoscope and presented images of fixed biological samples acquired by the microendoscope to demonstrate its ability to image the cellular structure of tissue . To be useful as an endoscopic device, the instrument should be no more than a few millimeters in diameter. As shown in Figure 9, the objective includes a 1-mm plano-spherical glass lens mounted in a custom precision micromount and three aspheric microlenses printed via grayscale lithography in hybrid sol-gel glass.
The device incorporates a MEMS scanning grating, and the grating actuator is an electrostatic comb-drive actuator designed to scan the grating in resonance. The illumination is provided by a light-emitting diode (LED) that is coupled to a multimode fiber. The light from the fiber is collected by a 2-mm plano-spherical glass condenser lens mounted in a precision micromount. The device has a 250-µm-diameter field of view (FOV) and a working distance of 300 µm. The working distance allows for optical sectioning of epithelial cells below the tissue surface .
To verify the imaging ability of this optical system in relevant biological samples, such as cervix, oral epithelial cells and tissues were imaged by the system due to their structural and biomolecular similarity between the oral cavity and cervix. Human oral carcinoma cells were labelled and imaged, cell membranes were clearly visible and the results were comparable with a Zeiss optical microscope .
OCT is a 3D microscopic imaging technique that is especially suitable for in vivo imaging applications, such as biomedical tissue cross-sectional analysis. With an axial resolution of less than 15 μm and cross-sectional imaging to a depth of ~1–3 mm, it has significant potential in ophthalmology, cardiology, gastroenterology and oncology applications, etc. OCT is an interferometric imaging technique that employs light sources (typically in the near-infrared) with relatively large spectral bandwidths (e.g., ~10–300 nm), to achieve cross-sectional biomedical imaging. OCT systems in general employ a fiber-based Michelson interferometer to measure the backscattered light from an object. This back-scattered light is used to calculate the reflectivity or scattering potential profile of the biological sample along the probe beam direction. By scanning the probe beam in the transverse direction, 3D images of the object can be reconstructed .
With the increasing availability of micro-optical components, OCT becomes a promising non-invasive imaging technique that lends itself to a compact, relatively cost- effective and portable architecture for diagnostic imaging applications. Aljasem et al. presented a membrane-based microfluidic tunable microlens and an electrostatic 2D scanning micromirror which were fabricated using silicon and polymer-based MEMS technologies .
These components were assembled inside a 4.5 mm diameter probe of an endoscopic OCT system for beam focus and steering, and the system was used to test a slide of wood with a surface layer of varnish and a leek skin . Mu et al. demonstrated a prototype of an OCT bioimaging endoscopic probe utilizing a MEMS micromirror as the light beam manipulator .
To exert a large mirror platform tilt angle, electrothermal bimorph actuator was selected as the micromirror structure for its large deflection and low driving voltage, of which the latter is of particular importance for clinical applications. The MEMS micromirror and the silicon optical bench (SiOB) assembly were enclosed within a biocompatible, transparent and waterproof polycarbonate tube equipped with toroidal-lens for in vivo diagnostic applications. The muscle and skin next to the hind leg of a 6-week-old male mouse was tested by the OCT probe, epimysium, perimysium and endomysium layers were observed, and even the blood vessels that enclosed within the underneath layer of the epimysium can be easily distinguished from perimysium. Similarly, the superficial epidermis of the mouse leg skin tissue and the dermis layer were also successfully detected .
More information: “A minimally invasive lens-free computational microendoscope” Science Advances (2019). DOI: 10.1126/sciadv.aaw5595 , https://advances.sciencemag.org/content/5/12/eaaw5595