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8. Relationship between raster size, pixel size, and " light dose. " (A) If a microscope scanning at a zoom setting of 1 is switched from a 512 ¥ 512 raster to one of 1024 ¥ 1024 pixels, the dimensions of each pixel on the specimen will drop by 50%. (B) If the same amount of signal is split between more pixels, the signal level from each one goes down (and the Poisson noise goes up), but if the beam scans more slowly or is made more intense so that the same amount of signal is still collected from each pixel, (C), then the amount of damage/pixel increases. There is no free lunch!
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Microscopical images are now almost always recorded digitally. To accomplish this, the flux of photons that forms the final
image must be divided into small geometrical subunits called pixels. The light intensity in each pixel will be stored as a
single number. Changing the objective magnification, the zoom magnification on your confocal control pa...
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Citations
... Effective image contrast can be controlled through software using detector and amplifier gain settings. Detector gain should be as low as possible so the detector is not oversaturated (Pawley, 2006c). Our microscope was purchased in 1997. ...
We report non-destructive 3-dimensional imaging and analysis techniques for material returned by the Stardust cometary collector. Our technique utilizes 3-dimensional laser scanning confocal microscopy (3D LSCM) to image whole Stardust tracks, in situ, with attainable resolutions <90 nm/pixel edge. LSCM images illustrate track morphology and fragmentation history; image segmentation techniques provide quantifiable volumetric and dynamic measurements. We present a method for multipart image acquisition and registration in 3-D. Additionally, we present a 3D deconvolution method for aerogel, using a theoretically calculated point spread function for first-order corrections of optical aberrations induced by light diffraction and refractive index mismatches. LSCM is a benchtop technique and is an excellent alternative to synchrotron x-ray computed microtomography for optically transparent media. Our technique, developed over the past 2 years, is a non-invasive, rapid technique for fine-scale imaging of high value returned samples from the Stardust mission, as well as various other samples from the geosciences.
... Per the Nyquist-Shannon sampling theorem, retaining the resolution of a continuous signal (i.e. the spatially varying distribution of photons/electrons incident on the detector) in discrete digital space (i.e. the pixels in the acquired image) requires sampling at at least double the frequency of the smallest resolvable feature 22 . When the Nyquist-Shannon theorem is applied to the two-dimensional nature of images, the theoretical pixel size for adequate sampling should in fact be~2.8 times smaller than the resolving power of the microscope 23 . This sampling should be observed if very fine structures within the sample are to be measured and quantified, as larger pixel sizes will lead to a loss of information due to undersampling. ...
Images are at the core of most modern biological experiments and are used as a major source of quantitative information. Numerous algorithms are available to process images and make them more amenable to be measured. Yet the nature of the quantitative output that is useful for a given biological experiment is uniquely dependent upon the question being investigated. Here, we discuss the 3 main types of visual information that can be extracted from microscopy data: intensity, morphology, and object counts or categorical labels. For each, we describe where they come from, how they can be measured, and what may affect the relevance of these measurements in downstream data analysis. Acknowledging that what makes a measurement "good" is ultimately down to the biological question being investigated, this review aims at providing readers with a toolkit to challenge how they quantify their own data and be critical of conclusions drawn from quantitative bioimage analysis experiments.
... Epi-illumination approaches (wide-field and confocal microscopy methodologies) illuminate the focal plane along the detection axis and, in this way, excitation light is absorbed above and below this plane. Consequently, phototoxicity is induced along the z-axis (Pawley, 2006;Verveer et al., 2007). The use of selective sample illumination microscopy techniques such as total internal reflection fluorescence (TIRF), two-photon, and light-sheet fluorescence microscopy can overcome some of these caveats. ...
Mitochondrial toxicology is a growing field of study focusing disturbances in the natural function of mitochondria, which occur as off-target effects of drugs or other agents. This is an important reason for failure of drugs in clinical trials, which is associated with significant losses of time and money. Failure to detect mitochondrial effects in pre-clinical studies can be explained by limitations in the traditional methodologies, from cell culture to microscopy or bioinformatic analyses. Some of the most popular in vitro models lack physiological relevance, as the widely used culture cell conditions favor pro-glycolytic metabolism and reduce cellular reliance on mitochondria for energy production, which can artificially lower the cells’ sensitivity to mitochondrial toxicants. Several points are also often overlooked in animal models that may lead to inadequate translation of pre-clinical trials into humans. This chapter tackles the most common problems in this area while suggesting some alternatives to overcome them.
... The magnification, M , and pixelation, p (p = M a/d pix ), can be varied independently of this. In previous studies, the minimum number of pixels required to resolve atomic positions on a lattice without loss of information is reasoned to be p = 2.5 [27,37], based on linear-optics calculations (Shannon-Nyquist sampling). ...
Quantum-gas microscopes are used to study ultracold atoms in optical lattices at the single particle level. In these system atoms are localised on lattice sites with separations close to or below the diffraction limit. To determine the lattice occupation with high fidelity, a deconvolution of the images is often required. We compare three different techniques, a local iterative deconvolution algorithm, Wiener deconvolution and the Lucy-Richardson algorithm, using simulated microscope images. We investigate how the reconstruction fidelity scales with varying signal-to-noise ratio, lattice filling fraction, varying fluorescence levels per atom, and imaging resolution. The results of this study identify the limits of singe-atom detection and provide quantitative fidelities which are applicable for different atomic species and quantum-gas microscope setups.
... Based on the Nyquist criterion, sampling with at least more than two times the resolution is required. 16,20 Thus, in our example, the pixel size of the camera should be close to 9 lm. However, oversampling the image with much smaller pixels will not bring additional resolution while it can decrease the sensitivity of the detection. ...
Live-cell microscopy is a powerful tool that can reveal cellular behavior as well as the underlying molecular processes. A key advantage of microscopy is that by visualizing biological processes, it can provide direct insights. Nevertheless, live-cell imaging can be technically challenging and prone to artifacts. For a successful experiment, many careful decisions are required at all steps from hardware selection to downstream image analysis. Facing these questions can be particularly intimidating due to the requirement for expertise in multiple disciplines, ranging from optics, biophysics, and programming to cell biology. In this review, we aim to summarize the key points that need to be considered when setting up and analyzing a live-cell imaging experiment. While we put a particular focus on yeast, many of the concepts discussed are applicable also to other organisms. In addition, we discuss reporting and data sharing strategies that we think are critical to improve reproducibility in the field.
... According to the Nyquist theorem, it is required to have the pixel size of a digital image to be at least 2.3 times smaller than the optical resolution of a microscope to prevent from losing the information of a resolved image by an objective (Pawley 2006). We can use the following equations (Murphy & Davidson 2013) to determine proper combinations of magnification and the physical size of a sensor of a camera that satisfy the Nyquist theorem. ...
Wood identification is a crucial step to verify claims of the legality of wood and wood-derived products for compliance with the laws against illegal logging. For accurate determination of wood species, transmitted light microscopy has been utilized to identify microscopic features of wood from microscopic slides with thin sections from the transverse, radial, and tangential planes. When there are many woods in trading for identification, producing microscopic or permanent slides can be problematic because the production of the slides is time-consuming and slows down wood identification. However, the slides are not required for alternative microscopy such as epi-illumination light microscopy. In this study, we suggest the utilization of epi-illumination light microscopy as an alternative method to conventional transmitted light microscopy with microscopic slides for wood identification. We investigated the performance of selected epi-illumination light microscopic techniques: brightfield reflected light microscopy with a polarizer (RLBF), darkfield reflected light microscopy (RLDF), and fluorescence light microscopy by observing intervessel pits and vessel-ray pits. Among the selected epi-illumination light microscopy, brightfield reflected light
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... We scanned 200×200 steps in the lateral direction to collect enough data to provide overlap while stitching images. The minimum scanning step size criteria to fulfill the maximum optical resolution can be calculated using the Nyquist-Shannon sampling theorem [39]. The maximum spatial frequency that can be measured by the current system is group 8 element 6 (i.e., 1.096 µm). ...
Multifocal illumination can improve image acquisition time compared to single point scanning in confocal microscopy. However, due to an increase in the system complexity, obtaining uniform multifocal illumination throughout the field of view with conventional methods is challenging. Here, we propose a volume holographic lenslet array illuminator (VHLAI) for multifocal confocal microscopy. To obtain uniform array illumination, a super Gaussian (SG) beam has been incorporated through VHLAI with an efficiency of 43%, and implemented in a confocal microscope. The design method for a photo-polymer based volume holographic beam shaper is presented and its advantages are thoroughly addressed. The proposed system can significantly improve image acquisition time without sacrificing the quality of the image. The performance of the proposed multifocal confocal microscopy was compared with wide-field images and also evaluated by measuring optically sectioned microscopic images of fluorescence beads, florescence pollen grains, and biological samples. The proposed multifocal confocal system generates images faster without any changes in scanning devices. The present method may find important applications in high-speed multifocal microscopy platforms.
... In the context of any digital systems involving digitization of an analog signal, the Nyquist-Shannon criterion (Nyquist, 1928;Shannon, 1949) enforces the sampling frequency to be at least twice the maximum frequency of the analog signal to avoid the phenomenon of aliasing. In a digital microscopy/imaging system, undersampling-induced aliasing might convert the optics-limited high spatial frequencies of the objects into low spatial frequencies in the final image by the Moiré effect (Pawley, 2006;Heintzmann and Sheppard, 2007). An adequate digitization capability thus becomes an imperative factor to digitally retrieve a submicron optical resolution over a large millimeter-scale FOV. ...
... Theoretical considerations before you start Timing: 30 min 1. The Nyquist-Shannon sampling theorem (Nyquist, 1928;Shannon, 1949) in the context of a digital microscopy/imaging system (Pawley, 2006;Heintzmann and Sheppard, 2007) demands the l exc , n, and NA stand for excitation wavelength, order of the multiphoton process, and numerical aperture of the objective lens, respectively. (1), for a fast-axis field of view of FOV max , the minimum required pixel number P n can be given as ...
A resonant-scanning multiphoton optical microscope (MPM) with a millimeter-scale field-of-view (FOV) often encounters a poor Nyquist figure-of-merit (NFOM), leading to an aliasing effect owing to limited effective voxel-sampling rate. In this protocol, we provide a design guideline to enable high-NFOM MPM imaging while simultaneously securing a large FOV/digital-resolution ratio and a fast resonant raster-scanning speed. We further provide a free version of our custom acquisition software to assist with a smooth and easy construction process.
For complete details on the use and execution of this protocol, please refer to Borah et al. (2021).
... Advancing computer technology further allows for the movement of mirrors and lenses to collect and display the best image possible [6]. With the development and aid of computer technology, modern LSCM is able to project images using quantified microscopy; that is, in a given pixel, the software calculates the light intensity in comparison to the other pixels [6][7][8]. ...
This research demonstrates the value of laser scanning confocal microscopy (LSCM) as a research tool in osteological studies, and diagenetic studies in particular. LSCM combines properties of light and scanning electron microscopy using laser light to excite fluorophores throughout the z‐axis, developing a 3‐D image. Using differential staining and selecting for specific wavelengths of light, one can image targeted materials. This research is divided into two parts: visualizing bone structures such as proteins and their decompositional products and visualizing diagenesis. Part one of this study utilized pig bones as a means of testing the overall ability of LSCM to fluoresce bone. Twenty‐three samples were imaged, including 13 samples from a decompositional study conducted 5 years previous, and 10 “fresh” samples collected from a commercial butcher. This part of the study determined that protein and organic components of the bone could be fluoresced and diagenetic alteration could be imaged. The second part of the study used human samples as a means of imaging and mapping diagenetic alterations. The second part of the study used 13 samples, including 4 clinical, 7 ancient, and 2 modern controls. The pig study used Basic Fuchsin and SlowFade Gold stains, while the human study used toluidine blue. Images were also taken with unstained elements. The results of the non‐human study found that a fresh bone fluoresced differently than that of a 5‐year subset, while the results of the human study confirmed these findings and determined that the bone diagenesis can be mapped using LSCM.
... On the other hand, according to Nyquist criteria [49] for digital resolution, the smallest resolved objects should have approximately 2.3 pixels. The confocal images of HEWL samples are presented in Fig. 10. ...
In the present work we studied the effect of 2D WS2 nanoparticles on the conformational changes in lysozyme protein at different pH values (2.0–11.5). The contributions of various structural conformations (α-helix, β-sheets parallel and antiparallel, unordered structure and side groups) were determined by decomposition of Amid I absorbance bands. The 2D WS2 were shown to have different impact on secondary structure depending on pH of the solution and protein concentration.
The amyloid fibril presence was confirmed with confocal microscopy enhanced by gold support, and fluorescent spectroscopy with amyloid-sensitive dye Thioflavin T. Our data show that WS2 can both inhibit and stimulate amyloid formation. Additionally, we have also reported an unusual spectroscopic behavior displayed by lysozyme, indicated by narrowing of Amide I and Amide II bands at pH 2.5 and 3.5 when incubated with 2D WS2 nanoparticles.
