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Dark-field microscopy is known to offer both high resolution and direct visualization of thin samples. However, its performance and optimization on thick samples is under-explored and so far, only meso-scale information from whole organisms has been demonstrated. In this work, we carefully investigate the difference between trans- and epi-illumination configurations. Our findings suggest that the epi-illumination configuration is superior in both contrast and fidelity compared to trans-illumination, while having the added advantage of experimental simplicity and an "open top" for experimental intervention. Guided by the theoretical analysis, we constructed an epi-illumination dark-field microscope with measured lateral and axial resolutions of 260 nm and 520 nm, respectively. Subcellular structures in whole organisms were directly visualized without the need for image reconstruction, and further confirmed via simultaneous fluorescence imaging. With an imaging speed of 20 to 50 fps, we visualize fast dynamic processes such as cell division and pharyngeal pumping in Caenorhabditis elegans., (© 2021 Wiley-VCH GmbH.)

Recent developments in phase contrast microscopy have enabled the label-free visualization of certain organelles due to their distinct morphological features, making this method an attractive alternative in the study of cellular dynamics. However tubular structures such as endoplasmic reticulum (ER) networks and complex dynamics such as the fusion and fission of mitochondria, due to their low phase contrast, still need fluorescent labeling to be adequately imaged. In this article, we report a quantitative phase microscope with ultra-oblique illumination that enables us to see those structures and their dynamics with high contrast for the first time without labeling. The imaging capability was validated through comparison to the fluorescence images with the same field-of-view. The high image resolution (~270 nm) was validated using both beads and cellular structures. Furthermore, we were able to record the vibration of ER networks at a frame rate of 250 Hz. We additionally show complex cellular processes such as remodeling of the mitochondria networks through fusion and fission and vesicle transportation along the ER without labels. Our high spatial and temporal resolution allowed us to observe mitochondria "spinning", which has not been reported before, further demonstrating the advantages of the proposed method., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Manual hand counting of parasites in fecal samples requires costly components and substantial expertise, limiting its use in resource-constrained settings and encouraging overuse of prophylactic medication. To address this issue, a cost-effective, automated parasite diagnostic system that does not require special sample preparation or a trained user was developed. It is composed of an inexpensive (~US$350), portable, robotic microscope that can scan over the size of an entire McMaster chamber (100 mm 2 ) and capture high-resolution (~1 μm lateral resolution) bright field images without need for user intervention. Fecal samples prepared using the McMaster flotation method were imaged, with the imaging region comprising the entire McMaster chamber. These images are then automatically segmented and analyzed using a trained convolution neural network (CNN) to robustly separate eggs from background debris. Simple postprocessing of the CNN output yields both egg species and egg counts. The system was validated by comparing accuracy with hand-counts by a trained operator, with excellent performance. As a further demonstration of utility, the system was used to conveniently quantify drug response over time in a single animal, showing residual disease due to Anthelmintic resistance after 2 weeks., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this paper we report the development of a cost-effective, modular, open source, and fully automated slide-scanning microscope, composed entirely of easily available off-the-shelf parts, and capable of bright field and fluorescence modes. The automated X-Y stage is composed of two low-cost micrometer stages coupled to stepper motors operated in open-loop mode. The microscope is composed of a low-cost CMOS sensor and low-cost board lenses placed in a 4f configuration. The system has approximately 1 micron resolution, limited by the f/# of available board lenses. The microscope is compact, measuring just 25×25×30 cm, and has an absolute positioning accuracy of ±1 μm in the X and Y directions. A Z-stage enables autofocusing and imaging over large fields of view even on non-planar samples, and custom software enables automatic determination of sample boundaries and image mosaicking. We demonstrate the utility of our device through imaging of fluorescent- and transmission-dye stained blood and fecal smears containing human and animal parasites, as well as several prepared tissue samples. These results demonstrate image quality comparable to high-end commercial microscopes at a cost of less than US$400 for a bright-field system, with an extra US$100 needed for the fluorescence module.

Raman scattering is the inelastic scattering of light by chemical bonds, and can therefore show molecular specificity. It can be used both in pure spectroscopy mode, and in imaging mode. While many applications of Raman spectroscopy and imaging in the biomedical field have been so far demonstrated, the use of this technology for pathology applications is still in its early stages. In this paper we review some of the most important recent developments in this field, including a description of relevant technologies, applications to molecular sensing, characterization of cells and tissues of interest, and disease detection via Raman scattering.

Integrated Raman and angular-scattering microscopy (IRAM) is a multimodal platform capable of noninvasively probing both the chemistry and morphology of a single cell without prior labeling. Using this system, we are able to detect activation-dependent changes in the Raman and elastic-scattering signals from CD8+ T cells stimulated with either Staphylococcal enterotoxin B (SEB) or phorbol myristate acetate (PMA). In both cases, results obtained from the IRAM instrument correlate well with results obtained from traditional fluorescence-based flow cytometry for paired samples. SEB-mediated activation was distinguished from resting state in CD8+ T cells by an increase in the number and mean size of small ( approximately 500-nm) elastic scatterers as well as a decrease in Raman bands, indicating changes in nuclear content. PMA-mediated activation induced a different profile in CD8+ T cells from SEB, showing a similar increase in small elastic scatterers but a different Raman change, with elevation of cellular protein and lipid bands. These results suggest the potential of this multimodal, label-free optical technique for studying processes in single cells.

We report on the construction of a multimodal microscope platform capable of gathering both elastically and inelastically scattered light from a 38 mum(2) region in both epi- and transillumination geometries. Simultaneous monitoring of elastic and inelastic scattering from a microscopic region allows noninvasive characterization of the chemistry and morphology of a living sample without the need for exogenous dyes or labels, thus allowing measurements to be made longitudinally in time on the same sample as it evolves naturally. A sample is illuminated either from above or below with a focused 785 nm TEM(00) mode laser beam, with elastic and inelastic scattering collected by two separate measurement arms. The measurements may be made either simultaneously, if identical illumination geometries are used, or sequentially, if the two modalities utilize opposing illumination paths. In the inelastic arm, Stokes-shifted light is dispersed by a spectrograph onto a charge-coupled device (CCD) array. In the elastic scattering collection arm, a relay system images the microscope's back aperture onto a CCD array. Postprocessing of the inelastic scattering to remove fluorescence signals yields high quality Raman spectra that report on the sample's chemical makeup. Comparison of the elastically scattered pupil images to generalized Lorenz-Mie theory yields estimated size distributions of scatterers within the sample.

A microscopy system has been constructed that is capable of simultaneously acquiring both Raman spectra and angle-resolved elastic-scattering patterns in either epi- or transillumination modes with a 7 mum spot size. The benefits and drawbacks of the epi- and transillumination modalities are discussed. Validation studies have been performed on single beads of a few micrometers in size, as well as on ensembles of submicrometer particles. In addition, transilluminated Raman and elastic-scattering spectra were obtained from single granulocytes and peripheral blood monocytes. Both the Raman- and the elastic-scattering channels show clear differences between the two types of immune cells.

Clinical applications in the developing world, such as malaria and anemia diagnosis, demand a change in the medical paradigm of expensive care given in central locations by highly trained professionals. There has been a recent explosion in optical technologies entering the consumer market through the widespread adoption of smartphones and LEDs. This technology commoditization has enabled the development of small, portable optical imaging systems at an unprecedentedly low cost. Here, we review the state-of-the-field of the application of these systems for low-cost blood imaging with an emphasis on cellular imaging systems. In addition to some promising results addressing specific clinical issues, an overview of the technology landscape is provided. We also discuss several key issues that need to be addressed before these technologies can be commercialized. [ABSTRACT FROM AUTHOR]

In this paper we report the development of two attachments to a commercial cell phone that transform the phone's integrated lens and image sensor into a 350× microscope and visible-light spectrometer. The microscope is capable of transmission and polarized microscopy modes and is shown to have 1.5 micron resolution and a usable field-of-view of ∼150×150 ηm with no image processing, and approximately 350×350 ηm when post-processing is applied. The spectrometer has a 300 nm bandwidth with a limiting spectral resolution of close to 5 nm. We show applications of the devices to medically relevant problems. In the case of the microscope, we image both stained and unstained blood-smears showing the ability to acquire images of similar quality to commercial microscope platforms, thus allowing diagnosis of clinical pathologies. With the spectrometer we demonstrate acquisition of a white-light transmission spectrum through diffuse tissue as well as the acquisition of a fluorescence spectrum. We also envision the devices to have immediate relevance in the educational field. [ABSTRACT FROM AUTHOR]