-
Biomedical Optics Express Nov 2015Cell imaging using low-light techniques such as bioluminescence, radioluminescence, and low-excitation fluorescence has received increased attention, particularly due to...
Cell imaging using low-light techniques such as bioluminescence, radioluminescence, and low-excitation fluorescence has received increased attention, particularly due to broad commercialization of highly sensitive detectors. However, the dim signals are still regarded as difficult to image using conventional microscopes, where the only low-light microscope in the market is primarily optimized for bioluminescence imaging. Here, we developed a novel modular microscope that is cost-effective and suitable for imaging different low-light luminescence modes. Results show that this microscope system features excellent aberration correction capabilities and enhanced image resolution, where bioluminescence, radioluminescence and epifluorescence images were captured and compared with the commercial bioluminescence microscope.
PubMed: 26601020
DOI: 10.1364/BOE.6.004585 -
ELife Mar 2024Fluorescence microscopy is a fundamental tool in the life sciences, but the availability of sophisticated equipment required to yield high-quality, quantitative data is...
Fluorescence microscopy is a fundamental tool in the life sciences, but the availability of sophisticated equipment required to yield high-quality, quantitative data is a major bottleneck in data production in many laboratories worldwide. This problem has long been recognized and the abundancy of low-cost electronics and the simplification of fabrication through 3D-printing have led to the emergence of open-source scientific hardware as a research field. Cost effective fluorescence microscopes can be assembled from cheaply mass-produced components, but lag behind commercial solutions in image quality. On the other hand, blueprints of sophisticated microscopes such as light-sheet or super-resolution systems, custom-assembled from high quality parts, are available, but require a high level of expertise from the user. Here, we combine the UC2 microscopy toolbox with high-quality components and integrated electronics and software to assemble an automated high-resolution fluorescence microscope. Using this microscope, we demonstrate high resolution fluorescence imaging for fixed and live samples. When operated inside an incubator, long-term live-cell imaging over several days was possible. Our microscope reaches single molecule sensitivity, and we performed single particle tracking and SMLM super-resolution microscopy experiments in cells. Our setup costs a fraction of its commercially available counterparts but still provides a maximum of capabilities and image quality. We thus provide a proof of concept that high quality scientific data can be generated by lay users with a low-budget system and open-source software. Our system can be used for routine imaging in laboratories that do not have the means to acquire commercial systems and through its affordability can serve as teaching material to students.
Topics: Humans; Microscopy, Fluorescence; Biological Science Disciplines; Culture; Data Accuracy; Laboratories
PubMed: 38436658
DOI: 10.7554/eLife.89826 -
Journal of Family Medicine and Primary... May 2020In recent years, a new concept has emerged at the forefront of slide-based diagnosis and telepathology. This is the concept of whole slide imaging. This has very...
CONTEXT
In recent years, a new concept has emerged at the forefront of slide-based diagnosis and telepathology. This is the concept of whole slide imaging. This has very recently also been tried with smartphones using complex software for photo analysis and stitching. This study is aimed to evaluate and compare the use of Jenoptik photomicrograph camera and smartphone camera-based whole slide imaging (WSI) for various microscopic preparations and slides. The study also uses a commonly available IOS smartphone device, a commonly available phone to microscope attachment, and the most commonly used Adobe Photoshop software for all stitching purposes.
AIM
To evaluate and compare smartphone and photomicrography-based whole slide imaging.
SUBJECTS AND METHODS
The study was conducted at the Department of Oral Pathology and Microbiology in Bapuji Dental College and Hospital, Davangere. The samples included in this study are 10 slides of routine hematoxylin and eosin stain slides and 10 ground section slides of teeth and bone samples. Continuous photographs of the entire slide were captured using an IOS device and a Jenoptik camera attached research microscope at 10× magnification. The photographs were stitched using Adobe Photoshop 2017 software to obtain a whole slide image. Then, two observers analyzed the whole slide image for a possible diagnosis and thus compared the efficiency of both the methods.
STATISTICAL ANALYSIS USED
The statistical Chi-square test and analysis of variance (ANOVA) H test were done using Statistical Package for the Social Sciences (SPSS) 2010 Software.
RESULTS
Percentage of measure of agreement was 79.5%, 83.7% and 86.3%, 89.1% for the whole slide images taken in the smartphone and photomicrograph and analyzed by Observer 1 and Observer 2, respectively.
CONCLUSIONS
In this study, it can be seen that the WSI by research microscopes is better than that with a smartphone and a compound microscope but still both the methods are equally good and can be followed with accurate results.
PubMed: 32754495
DOI: 10.4103/jfmpc.jfmpc_18_20 -
Optics Express Jul 2022Microscopes are vital pieces of equipment in much of biological research and medical diagnostics. However, access to a microscope can represent a bottleneck in research,...
Microscopes are vital pieces of equipment in much of biological research and medical diagnostics. However, access to a microscope can represent a bottleneck in research, especially in lower-income countries. 'Smart' computer controlled motorized microscopes, which can perform automated routines or acquire images in a range of modalities are even more expensive and inaccessible. Developing low-cost, open-source, smart microscopes enables more researchers to conceive and execute optimized or more complex experiments. Here we present the OpenFlexure Delta Stage, a 3D-printed microscope designed for researchers. Powered by the OpenFlexure software stack, it is capable of performing automated experiments. The design files and assembly instructions are freely available under an open licence. Its intuitive and modular design-along with detailed documentation-allows researchers to implement a variety of imaging modes with ease. The versatility of this microscope is demonstrated by imaging biological and non-biological samples (red blood cells with Plasmodium parasites and colloidal particles in brightfield, epi-fluorescence, darkfield, Rheinberg and differential phase contrast. We present the design strategy and choice of tools to develop devices accessible to researchers from lower-income countries, as well as the advantages of an open-source project in this context. This microscope, having been open-source since its conception, has already been built and tested by researchers around the world, promoting a community of expertise and an environment of reproducibility in science.
Topics: Microscopy; Reproducibility of Results; Software
PubMed: 36236831
DOI: 10.1364/OE.450211 -
Biomedical Optics Express Jan 2022Confocal microscopy is a standard approach for obtaining volumetric images of a sample with high axial and lateral resolution, especially when dealing with scattering...
Confocal microscopy is a standard approach for obtaining volumetric images of a sample with high axial and lateral resolution, especially when dealing with scattering samples. Unfortunately, a confocal microscope is quite expensive compared to traditional microscopes. In addition, the point scanning in confocal microscopy leads to slow imaging speed and photobleaching due to the high dose of laser energy. In this paper, we demonstrate how the advances in machine learning can be exploited to "teach" a traditional wide-field microscope, one that's available in every lab, into producing 3D volumetric images like a confocal microscope. The key idea is to obtain multiple images with different focus settings using a wide-field microscope and use a 3D generative adversarial network (GAN) based neural network to learn the mapping between the blurry low-contrast image stacks obtained using a wide-field microscope and the sharp, high-contrast image stacks obtained using a confocal microscope. After training the network with widefield-confocal stack pairs, the network can reliably and accurately reconstruct 3D volumetric images that rival confocal images in terms of its lateral resolution, z-sectioning and image contrast. Our experimental results demonstrate generalization ability to handle unseen data, stability in the reconstruction results, high spatial resolution even when imaging thick (∼40 microns) highly-scattering samples. We believe that such learning-based microscopes have the potential to bring confocal imaging quality to every lab that has a wide-field microscope.
PubMed: 35154871
DOI: 10.1364/BOE.444488 -
Journal of Microscopy Mar 2021Live observation of biological phenomena in the context of living organisms can provide important insights in the mechanisms of these phenomena. However, the spatially...
Live observation of biological phenomena in the context of living organisms can provide important insights in the mechanisms of these phenomena. However, the spatially complex and dynamic physiology of multicellular organisms can be a challenging environment to make observations with fluorescence microscopy. Due to the illumination of out-of-focus planes, confocal and particularly widefield fluorescence microscopy suffer from low signal-to-background ratio (SBR), photo toxicity and bleaching of fluorescent probes. In light-sheet microscopy (LSM), solely the focal plane of the detection objective is illuminated, minimising out-of-focus fluorescence and photobleaching, thereby enhancing SBR, allowing for low laser intensities and longer acquisition periods. Here we present a straightforward light-sheet microscope with a 1.0-NA detection objective and a fast sample-positioning stage that allows for four degrees of freedom. By imaging the sensory cilia and nervous system of living young adult C. elegans, we demonstrate that the instrument is well suited for relatively fast, volumetric imaging of larger (hundreds of micrometres cubed) living samples. These experiments demonstrate that such an instrument provides a valuable addition to commonly used widefield and confocal fluorescence microscopes. LAY DESCRIPTION: In fluorescence microscopy, sharp images can only be obtained when the light obtained from the section of the image that is in focus is not overwhelmed by light emerging from elsewhere. In this paper, we present a light-sheet fluorescence microscope, based on the OpenSPIM initiative, with a magnification of 90× and a sensitive sample positioning stage that allows for fast controlled linear movement and rotation. In a light-sheet microscope (LSM), the sample is illuminated from the side, compared to the direction of detection, limiting illumination only to the part of the sample that is imaged in the focal plane (general resources: Wikipedia or MicroscopyU). This does not only limit background noise, but also reduces damage to the sample due to phototoxicity. This makes a LSM particularly suitable for imaging living samples at high resolution, in three dimensions, over long periods of time. Our instrument was specifically designed for imaging adult C. elegans nematodes. We show here how the instrument compares to a standard epifluorescence microscope, imaging neuronal structures in the animals. The instrument proved well suited for fast volumetric imaging of larger cellular structures such as C. elegans neuronal cell bodies. Our experiments show that the instrument provides a valuable addition to widefield and confocal fluorescence microscopes commonly used to image adult C. elegans.
Topics: Animals; Caenorhabditis elegans; Fluorescent Dyes; Lasers; Microscopy, Fluorescence; Photobleaching
PubMed: 32949409
DOI: 10.1111/jmi.12964 -
Cureus Jun 2022Access to microneurosurgical care in low- and middle-income countries remains limited mainly due to a lack of equipment. High purchasing and maintenance costs hinder the...
Access to microneurosurgical care in low- and middle-income countries remains limited mainly due to a lack of equipment. High purchasing and maintenance costs hinder the use of operating microscopes in low-resource facilities. The authors present an improved version of their previously introduced low-cost exoscope to achieve high magnification and illumination in low-resource environments. The setup included a 48-megapixel two-dimensional digital microscope camera, a wide field C-mount lens, ring light, and a two-link cantilever with a screw terminal. The surgical field was projected to a portable 17.3-inch 2K resolution monitor. Ten patients underwent exoscope-assisted transforaminal lumbar interbody fusion via the Wiltse paraspinal approach. The simple construction allowed a fast and intuitive preoperative setup. The in-plane switching type display provided a clear and bright image regardless of the viewing angle. The two-link arm of the cantilever allowed smooth positioning of the camera, overcoming the cumbersome up and down movements needed to zoom in and out with the previous prototype. Industrial microscope cameras are effective low-budget alternatives to conventional operating microscopes in lumbar microdiscectomy. The improved system is superior compared to the authors' previous prototype with regard to affordability, image quality, and adjustability of position and angle.
PubMed: 35836461
DOI: 10.7759/cureus.25858 -
Frontiers in Neuroscience 2020Animal behavior is regulated by environmental stimuli and is shaped by the activity of neural networks, underscoring the importance of assessing the morpho-functional... (Review)
Review
Animal behavior is regulated by environmental stimuli and is shaped by the activity of neural networks, underscoring the importance of assessing the morpho-functional properties of different populations of cells in freely behaving animals. In recent years, a number of optical tools have been developed to monitor and modulate neuronal and glial activity at the protein, cellular, or network level and have opened up new avenues for studying brain function in freely behaving animals. Tools such as genetically encoded sensors and actuators are now commonly used for studying brain activity and function through their expression in different neuronal ensembles. In parallel, microscopy has also made major progress over the last decades. The advent of miniature microscopes (mini-microscopes also called mini-endoscopes) has become a method of choice for studying brain activity at the cellular and network levels in different brain regions of freely behaving mice. This technique also allows for longitudinal investigations while animals carrying the microscope on their head are performing behavioral tasks. In this review, we will discuss mini-endoscopic imaging and the advantages that these devices offer to research. We will also discuss current limitations of and potential future improvements in mini-endoscopic imaging.
PubMed: 32848576
DOI: 10.3389/fnins.2020.00819 -
Micromachines May 2022Bright field microscopes are particularly useful tools for biologists for cell and tissue observation, phenotyping, cell counting, and so on. Direct cell observation...
Bright field microscopes are particularly useful tools for biologists for cell and tissue observation, phenotyping, cell counting, and so on. Direct cell observation provides a wealth of information on cells' nature and physiological condition. Microscopic analyses are, however, time-consuming and usually not easy to parallelize. We describe the fabrication of a stand-alone microscope able to automatically collect samples with 3D printed pumps, and capture images at up to 50× optical magnification with a digital camera at a good throughput (up to 24 different samples can be collected and scanned in less than 10 min). Furthermore, the proposed device can store and analyze pictures using computer vision algorithms running on a low power integrated single board computer. Our device can perform a large set of tasks, with minimal human intervention, that no single commercially available machine can perform. The proposed open-hardware device has a modular design and can be freely reproduced at a very competitive price with the use of widely documented and user-friendly components such as Arduino, Raspberry pi, and 3D printers.
PubMed: 35744447
DOI: 10.3390/mi13060833