Lensless Microscopy Research Articles

Achieving magnification smaller than 1 in lensless microscopy by illumination with a convergent wavefront.

Lensless or lensfree microscopy is now available commercially. With these new microscopes, it is possible to record images in contact mode with a magnification of 1 or in holographic mode with a magnification larger than 1. In this Letter, we present an original setup that allows us to record the image of an object whose surface is larger than that of the image sensor without placing an optical component between the object and the image sensor.

Lensless Imaging and Sensing

High-resolution optical microscopy has traditionally relied on high-magnification and high-numerical aperture objective lenses. In contrast, lensless microscopy can provide high-resolution images without the use of any focusing lenses, offering the advantages of a large field of view, high resolution, cost-effectiveness, portability, and depth-resolved three-dimensional (3D) imaging. Here we review various approaches to lensless imaging, as well as its applications in biosensing, diagnostics, and cytometry. These approaches include shadow imaging, fluorescence, holography, superresolution 3D imaging, iterative phase recovery, and color imaging. These approaches share a reliance on computational techniques, which are typically necessary to reconstruct meaningful images from the raw data captured by digital image sensors. When these approaches are combined with physical innovations in sample preparation and fabrication, lensless imaging can be used to image and sense cells, viruses, nanoparticles, and biomolecules. We conclude by discussing several ways in which lensless imaging and sensing might develop in the near future.

Improved quantitative phase imaging in lensless microscopy by single-shot multi-wavelength illumination using a fast convergence algorithm.

We report on a novel algorithm for high-resolution quantitative phase imaging in a new concept of lensless holographic microscope based on single-shot multi-wavelength illumination. This new microscope layout, reported by Noom et al. along the past year and named by us as MISHELF (initials incoming from Multi-Illumination Single-Holographic-Exposure Lensless Fresnel) microscopy, rises from the simultaneous illumination and recording of multiple diffraction patterns in the Fresnel domain. In combination with a novel and fast iterative phase retrieval algorithm, MISHELF microscopy is capable of high-resolution (micron range) phase-retrieved (twin image elimination) biological imaging of dynamic events. In this contribution, MISHELF microscopy is demonstrated through qualitative concept description, algorithm implementation, and experimental validation using both a synthetic object (resolution test target) and a biological sample (swine sperm sample) for the case of three (RGB) illumination wavelengths. The proposed method becomes in an alternative instrument improving the capabilities of existing lensless microscopes.

Pixel size adjustment in coherent diffractive imaging within the Rayleigh-Sommerfeld regime.

The reconstruction of the smallest resolvable object detail in digital holography and coherent diffractive imaging when the detector is mounted close to the object of interest is restricted by the sensor's pixel size. Very high resolution information is intrinsically encoded in the data because the effective numerical aperture (NA) of the detector (its solid angular size as subtended at the object plane) is very high. The correct physical propagation model to use in the reconstruction process for this setup should be based on the Rayleigh-Sommerfeld diffraction integral, which is commonly implemented via a convolution operation. However, the convolution operation has the drawback that the pixel size of the propagation calculation is preserved between the object and the detector, and so the maximum resolution of the reconstruction is limited by the detector pixel size, not its effective NA. Here we show that this problem can be overcome via the introduction of a numerical spherical lens with adjustable magnification. This approach enables the reconstruction of object details smaller than the detector pixel size or of objects that extend beyond the size of the detector. It will have applications in all forms of near-field lensless microscopy.

Multimode fibres for micro-endoscopy

AbstractThere has been a tremendous effort in modern microscopy towards miniaturisation and fibre-based technology, driven by the need to access hostile or difficult environments in situ and in vivo. Most of these rely on reducing the size of endoscopes based on fibre-optic bundles, and systems incorporating microfabricated lenses. Recently, the use of standard multimode optical fibres for lensless microscopy has become possible mainly due to advances in holographic beam shaping. This article reviews the methods and techniques behind this progress paving theway towards minimally invasive in vivo imaging as well as other applications of multimode waveguides including on-chip integration of optical micro-manipulation and numerous other biophotonics techniques.

Holography and coherent diffraction with low-energy electrons: A route towards structural biology at the single molecule level

The current state of the art in structural biology is led by NMR, X-ray crystallography and TEM investigations. These powerful tools however all rely on averaging over a large ensemble of molecules. Here, we present an alternative concept aiming at structural analysis at the single molecule level. We show that by combining electron holography and coherent diffraction imaging estimations concerning the phase of the scattered wave become needless as the phase information is extracted from the data directly and unambiguously. Performed with low-energy electrons the resolution of this lens-less microscope is just limited by the De Broglie wavelength of the electron wave and the numerical aperture, given by detector geometry. In imaging freestanding graphene, a resolution of 2Å has been achieved revealing the 660.000 unit cells of the graphene sheet from a single data set. Once applied to individual biomolecules the method shall ultimately allow for non-destructive imaging and imports the potential to distinguish between different conformations of proteins with atomic resolution.

Automation Highlights from the Literature

Automation Highlights from the Literature

High-speed multi-wavelength Fresnel diffraction imaging.

We demonstrate a compact lensless microscope which can capture video-rate phase contrast images of moving objects and allows numerical scanning of the focal distance after recording. Using only an RGB-detector and illumination from a single mode fiber, diffraction patterns at three wavelengths are recorded simultaneously, enabling high-speed data collection and reconstruction of phase and amplitude. The technique is used for imaging of a moving test target, beads in a flow cell, and imaging of Caenorhabditis elegans moving in a droplet of liquid.

Growth phenotype screening of Schizosaccharomyces pombe using a Lensless microscope

The Lensless microscope has a large field of view and allows the capture of the diffraction pattern from a large number of cells simultaneously. A simple algorithm to measure intensity changes in the Airy Disc First Fringe (ADFF) has been derived to follow the growth characteristics of the unicellular yeast Schizosaccharomyces pombe. The performance of the algorithm is calibrated using comparison between optical image and ADFF analysis of polystyrene microspheres with known dimensions and has an accuracy of 5% over all lengths above the diffraction-limited measurements. We have observed the growth characteristics of S. pombe for N=100 cells to determine the growth phenotype distributions of Length (L(t=0)) and width (W(t=0)) on arrival at the surface, lag phase adjustment to the new growth conditions (B), the length at birth, LB, and cell cycle length, tcell. The observed cell width distribution has a median width of 3.9 (±0.1)µm, as expected, but a non-normal distribution. Similarly, all growth parameters studied, L(t=0), LB and cell cycle time are phenotypes with non-normal distributions but with medians consistent with the literature values.

X-ray lensless microscopy from undersampled diffraction intensities

X-ray coherent diffraction imaging including ptychography provides the nanoscale resolved three-dimensional description of matter. The combination of these approaches to the Bragg geometry case arouses a strong interest for its capability to provide information about strain state in crystals. Among the existing approaches, ptychography is particularly appealing because it allows the investigation of extended or weakly scattering samples. Coherent diffraction imaging approaches, based on redundancy in the collected diffraction intensity data set, are highly time consuming and rely on state-of-the-art mechanical setups, both being strong limitations for a general application. We show here that these can be overcome by regularization-based inversion algorithms introducing a priori structural knowledge. This method, which can be generalized to other wavelengths or beam sources, opens new possibilities for the imaging of radiation-sensitive specimens or very large samples.

Speckle-based configuration for simultaneousin vitroinspection of mechanical contractions of cardiac myocyte cells

An optical lensless configuration for a remote noncontact measuring of mechanical contractions of a vast number of cardiac myocytes is proposed. All the myocytes were taken from rats, and the measurements were done in an in vitro mode. The optical method is based on temporal analysis of secondary reflected speckle patterns generated in lensless microscope configuration. The processing involves analyzing the movement and the change in the statistics of the secondary speckle patterns that are created on top of the cell culture when it is illuminated by a spot of laser beam. The main advantage of the proposed system is the ability to measure many cells simultaneously (∼1000 cells) and to extract the statistical data of their movement at once. The presented experimental results also include investigation of the effect of isoproteranol on cell contraction process.

Oversampling smoothness: an effective algorithm for phase retrieval of noisy diffraction intensities.

Coherent diffraction imaging (CDI) is high-resolution lensless microscopy that has been applied to image a wide range of specimens using synchrotron radiation, X-ray free-electron lasers, high harmonic generation, soft X-ray lasers and electrons. Despite recent rapid advances, it remains a challenge to reconstruct fine features in weakly scattering objects such as biological specimens from noisy data. Here an effective iterative algorithm, termed oversampling smoothness (OSS), for phase retrieval of noisy diffraction intensities is presented. OSS exploits the correlation information among the pixels or voxels in the region outside of a support in real space. By properly applying spatial frequency filters to the pixels or voxels outside the support at different stages of the iterative process (i.e. a smoothness constraint), OSS finds a balance between the hybrid input-output (HIO) and error reduction (ER) algorithms to search for a global minimum in solution space, while reducing the oscillations in the reconstruction. Both numerical simulations with Poisson noise and experimental data from a biological cell indicate that OSS consistently outperforms the HIO, ER-HIO and noise robust (NR)-HIO algorithms at all noise levels in terms of accuracy and consistency of the reconstructions. It is expected that OSS will find application in the rapidly growing CDI field, as well as other disciplines where phase retrieval from noisy Fourier magnitudes is needed. The MATLAB (The MathWorks Inc., Natick, MA, USA) source code of the OSS algorithm is freely available from http://www.physics.ucla.edu/research/imaging.

Noise models for low counting rate coherent diffraction imaging

Coherent diffraction imaging (CDI) is a lens-less microscopy method that extracts the complex-valued exit field from intensity measurements alone. It is of particular importance for microscopy imaging with diffraction set-ups where high quality lenses are not available. The inversion scheme allowing the phase retrieval is based on the use of an iterative algorithm. In this work, we address the question of the choice of the iterative process in the case of data corrupted by photon or electron shot noise. Several noise models are presented and further used within two inversion strategies, the ordered subset and the scaled gradient. Based on analytical and numerical analysis together with Monte-Carlo studies, we show that any physical interpretations drawn from a CDI iterative technique require a detailed understanding of the relationship between the noise model and the used inversion method. We observe that iterative algorithms often assume implicitly a noise model. For low counting rates, each noise model behaves differently. Moreover, the used optimization strategy introduces its own artefacts. Based on this analysis, we develop a hybrid strategy which works efficiently in the absence of an informed initial guess. Our work emphasises issues which should be considered carefully when inverting experimental data.

A Lensless, Automated Microscope for Disease Diagnostics

Optical microscopy is widely accepted as one of the gold standards in disease diagnosis. However, factors such as cost and the need for a trained eye limit the prevalence of such equipment, particularly in resource-limited areas such as rural clinics. Lensless microscopy, which is based on principles of digital holography, has illustrated the possibility of using simple and cheap optical components combined with software algorithms to implement microscope platforms. We present a digital in-line holographic microscope (DIHM) platform to be used with image processing and classification algorithms to provide a low cost, portable and automated microscope. Initial results show that the images obtained using the DIHM platform are similar to those obtained using a conventional bright field microscope. Applications of this work are targeted towards the implementation of a full blood count, which could provide resource-limited areas with improved healthcare facilities and diagnosis times.

Micro camera based Lensless Microscope for a miniaturized diagnostic platform

The fast paced development in life sciences in recent years resulted in the integration of laboratory equipment into miniaturized mobile point-of-care diagnostic units as well as biotechnological microfluidic systems. Similar to the evolution of microelectronics, the “Micro System Technology” is the driver for the integration and miniaturization on the system level. For PoC, this clearly is reflected in the increased functionality, the number of analysis performed per devices and the reduced amount of sample liquid required to run a high-quality analysis. Due to the physical constraints, however, this evolution is not easily adopted to the optical analytics, like microscopy. As neither the lightpath nor the lens sizes can be modified without a complete re-engineering of the system, the evolution has stalled here. Therefore, the presented work targets the development and implementation of a lens-less microscope combining optics and microfluidics specifically for cell and particle analysis. Conceptually designed without any movable elements or lenses, the images obtained can be processed digitally for analysis and visualization. This is achieved by combining modern image sensors, holography principles and image deconvolution to enable novel approaches to microscopic observations, be it bacteria, cell clusters or micro particles. We show in this work the path to realization for the lens less microscope from the initial concept, the implementation strategy to demonstrating the capabilities of the device. The innovation is compared to the capabilities of conventional microscopy, using both live organisms and particles as well as simulation models to holography imaging as well as photonic wave theory. The microscope platform is demonstrated as a 3D rapid prototype, offering a versatile system for easy implementation in a laboratory and research setting. Microscopic sampling is used to offer correlation of the actual achievements of the system and derive future optimization criteria. Also, the viability w.r.t. cytometrics and live observation in microfluidic bioreactors id demonstrated. A synopsis on the achievements as well as the outlook w.r..t. fluorescence or tomography analysis is given. Biomed Tech 2012; 57 (Suppl. 1) © 2012 by Walter de Gruyter · Berlin · Boston. DOI 10.1515/bmt-2012-4008 447 Unauthenticated Download Date | 5/27/16 6:33 AM

Exploiting multimode waveguides for pure fibre-based imaging

There has been an immense drive in modern microscopy towards miniaturization and fibre-based technology. This has been necessitated by the need to access hostile or difficult environments in situ and in vivo. Strategies to date have included the use of specialist fibres and miniaturized scanning systems accompanied by ingenious microfabricated lenses. Here we present a novel approach for this field by utilizing disordered light within a standard multimode optical fibre for lensless microscopy and optical mode conversion. We demonstrate the modalities of bright- and dark-field imaging and scanning fluorescence microscopy at acquisition rates that allow observation of dynamic processes such as Brownian motion of mesoscopic particles. Furthermore, we show how such control can realize a new form of mode converter and generate various types of advanced light fields such as propagation-invariant beams and optical vortices. These may be useful for future fibre-based implementations of super-resolution or light-sheet microscopy.

Imaging of highly inhomogeneous strain field in nanocrystals using x-ray Bragg ptychography: A numerical study

X-ray ptychography is a lensless microscopy method able to provide extended field of view with spatial resolution above the diffraction limit. A series of intensity coherent diffraction patterns measured in the far field is used to obtain the numerical deconvolution between the sample scattering contrast and the illumination function. The measurements are performed with a finite-size beam spot scanned across the sample. The scan step, smaller than the beam size, ensures a high redundancy in the data set, which allows for the convergence of the iterative inversion algorithm. This work explores the possibility to use ptychography for the investigation of strained crystals by means of coherent x-ray Bragg diffraction, taking advantage of the high sensitivity to the atomic displacement fields. The Bragg diffraction scattering contrast is described by an effective complex-valued electron density, where the phase holds the information on the displacement field. The detailed two-dimensional numerical study of Bragg ptychography is presented, both for the known and unknown illumination cases. It demonstrates the high robustness of the ptychographical iterative engine for highly nonhomogeneous strain fields. In particular, the local information is extracted from the individual diffraction patterns to calculate the modulus and phase estimates of the electron density, which are further used to constrain the newly derived algorithm. From this work, it is foreseen that Bragg ptychography when experimentally feasible, will open the way to the nondestructive imaging of strain fields at the nanoscale.

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.

Three-dimensional high-resolution quantitative microscopy of extended crystals

Hard X-ray lens-less microscopy raises hopes for a non-invasive quantitative imaging, capable of achieving the extreme resolving power demands of nanoscience. However, a limit imposed by the partial coherence of third generation synchrotron sources restricts the sample size to the micrometer range. Recently, X-ray ptychography has been demonstrated as a solution for arbitrarily extending the field of view without degrading the resolution. Here we show that ptychography, applied in the Bragg geometry, opens new perspectives for crystalline imaging. The spatial dependence of the three-dimensional Bragg peak intensity is mapped and the entire data subsequently inverted with a Bragg-adapted phase retrieval ptychographical algorithm. We report on the image obtained from an extended crystalline sample, nanostructured from a silicon-on-insulator substrate. The possibility to retrieve, without transverse size restriction, the highly resolved three-dimensional density and displacement field will allow for the unprecedented investigation of a wide variety of crystalline materials, ranging from life science to microelectronics.

The self-imaging effect in lensless holographic microscopy and its application in characterising periodic structures

A lensless holographic microscope was used to record a hologram of self-organised monolayers of micron-sized polystyrene spheres. By reconstructing the hologram digitally using a computer it was found that the original periodicity of the object produces planes of identical images, as well as planes that show reversed contrast images at separations predicted by the first-order approximation to the Talbot theory of the self-imaging effect. The performance of a compound light microscope is compared and contrasted with that of the holographic system, specifically examining the ability of the different systems to recover the distribution at the Talbot planes and to characterise properties of periodic structures. A theoretical model is developed and experimental results are presented.