Bright-field Signals Research Articles

Coaxial Bright and Dark Field Optical Coherence Tomography.

To improve the signal collection efficiency of Optical Coherence Tomography (OCT) for biomedical applications. A novel coaxial optical design was implemented, utilizing a wavefront-division beam splitter in the sample arm with a 45-degree rod mirror. This design allowed for the simultaneous collection of bright and dark field signals. The bright field signal was detected within its circular aperture in a manner similar to standard OCT, while the dark field signal passed through an annular-shaped aperture and was collected by the same spectrometer via a fiber array. This new configuration improved the signal collection efficiency by ∼3 dB for typical biological tissues. Dark-field OCT images were found to provide higher resolution, contrast and distinct information compared to standard bright-field OCT. By compounding bright and dark field images, speckle noise was suppressed by ∼ √2 . These advantages were validated using Teflon phantoms, chicken breast ex vivo, and human skin in vivo. This new OCT configuration significantly enhances signal collection efficiency and image quality, offering great potential for improving OCT technology with better depth, contrast, resolution, speckles, and signal-to-noise ratio. We believe that the bright and dark field signals will enable more comprehensive tissue characterization with the angled scattered light. This advancement will greatly promote the OCT technology in various clinical and biomedical research applications.

Resolution of Virtual Depth Sectioning from Four-Dimensional Scanning Transmission Electron Microscopy.

One approach to three-dimensional structure determination using the wealth of scattering data in four-dimensional (4D) scanning transmission electron microscopy (STEM) is the parallax method proposed by Ophus et al. (2019. Advanced phase reconstruction methods enabled by 4D scanning transmission electron microscopy, Microsc Microanal25, 10-11), which determines the scattering matrix and uses it to synthesize a virtual depth-sectioning reconstruction of the sample structure. Drawing on an equivalence with a hypothetical confocal imaging mode, we derive contrast transfer and point spread functions for this parallax method applied to weakly scattering objects, showing them identical to earlier depth-sectioning STEM modes when only bright field signal is used, but that improved depth resolution is possible if dark field signal can be used. Through a simulation-based study of doped Si, we show that this depth resolution is preserved for thicker samples, explore the impact of shot noise on the parallax reconstructions, discuss challenges to making use of dark field signal, and identify cases where the interpretation of the parallax reconstruction breaks down.

Label-Free Analyses of Minimal Residual Disease in ALL Using Deep Learning and Imaging Flow Cytometry

MD/MC and PR/JI contributed equallyMinimal residual disease is an important response biomarker for risk stratification in acute lymphoblastic leukaemia (ALL) and is assessed by two methodologies, molecular analyses of patient-specific, clonal antigen receptor rearrangements or flow cytometric analyses of aberrant leukaemia-associated immunophenotypes. Some clinical regimens use both methodologies and sequential samples of bone marrow and/or peripheral blood are assessed during, at the end of induction therapy and at time points beyond. Both MRD methods are highly specialised, laborious and for resource poor countries, are not affordable. In addition, a proportion of patients are not evaluable by either method.Technological advances in cytometry have led to imaging flow cytometers (IFC) which deliver all standard flow cytometric parameters, as well as a high quality image of each cell. We have previously shown that machine learning of IFC data can classify cell cycle phases within a cell, using only data from bright field (BF) and dark field (DF)1 and we hypothesised that this might be possible in the MRD setting. Thus, paired diagnostic and follow up bone marrow aspirates taken during remission induction from children with ALL (n=23, 6 at day 8 or day 15 or day 28 following the start of treatment) were stained with CD19 APC, CD10PE, CD34Texas Red, CD45APCH7 and DAPI and ran on an imaging flow cytometer (Amnis, Imagestream X Mark II) at x40 magnification. A standard sequential gating technique identified ALL cells and levels (range 90-0.01%) correlated with that assessed by traditional flow cytometry. Next, 200 morphological features were extracted from BF and DF images of cells including pixel intensities, size, shapes, textures and subcellular components and both supervised machine and deep learning algorithms were developed to correctly classify ALL cells in clinical samples. The classifiers were trained initially using all fluorescent and BF and DF parameters but then with a sequential leave-one-parameter-out. In parallel, iteration of the training-testing sets on 23 datasets, with a leave-one-sample-out approach was performed, to ensure prediction accuracy across samples. Correct cell classifiers peaked at 98.21% when all parameters were considered but even without antibody-conjugated signals, relying only in DAPI, DF and BF signals, correct cell classification averaged 85.36%. Finally, DF/BF in combination and BF alone still gave rise to correct cell call rates of 81.62% and 71.26% respectively.Thus, we report a new MRD detection method that is label free, using only the morphological features extracted from BF and DF cell images. The method is cheap, quick, and highly applicable and with a technology that could be simplified to be laser-free and allow point of care testing. Transferability of this unique methodological approach to other types of leukaemia is also feasible and may be further developed to give functional information e.g. cell cycle status.

Stretch synchronizes sarcomere shortening in isolated cardiomyocytes

According to the Frank-Starling mechanism, stretching cardiac muscle augments contractile force. However, it is unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres and their local Ca2+ signals. To investigate this issue, we simultaneously monitored cytosolic Ca2+ and sarcomere striations in isolated rat cardiomyocytes by recording Fluo-4 fluorescence (515–560 nm) and brightfield signals respectively, using a high-speed sCMOS camera. Cells were affixed to glass rods (CellTester, World Precision Instruments) to allow control of cell length and measurement of force production during electrical stimulation at 1 Hz. At resting sarcomere length (SL), differential sarcomere deformation was observed despite uniform Ca2+ transients corresponding to individual sarcomeres. Specifically, while most sarcomeres contracted during the stimulus, a minority of sarcomeres (~13%) were observed to be stretched or were stationary. Sarcomeres which exhibited stretch during the Ca2+ transient tended to be shorter than those that contracted, and were significantly shorter than their neighbours. Step-wise stretch of the cell generated an expected increase in force development, but also revealed that more sarcomeres exhibited contraction, reaching nearly 100% of sarcomeres at 20% stretch. During cell lengthening, resting SL increased in all sarcomeres, suggesting that the shortest sarcomeres surpassed a critical length which enabled sufficient force generation and shortening. SL variability in unstretched cells was not caused by regional differences in Ca2+, as it remained apparent during permeabilization in Ca2+-free conditions. Rather, electron microscopy analysis traced differences in SL to the I-band, where dimensions are regulated by titin. We postulate, therefore, that slackened titin in unstretched cells allows greater variability in I-band length, cross-bridge formation, and active force generation between sarcomeres. When the cell is lengthened, sarcomere shortening is synchronized, which may contribute to augmented force production defined by the Frank-Starling mechanism.

An Inexpensive Approach for Bright-Field and Dark-Field Imaging by Scanning Transmission Electron Microscopy in Scanning Electron Microscopy

Scanning transmission electron microscopy in scanning electron microscopy (STEM-in-SEM) is a convenient technique for soft materials characterization. Various specimen-holder geometries and detector arrangements have been used for bright-field (BF) STEM-in-SEM imaging. In this study, to further the characterization potential of STEM-IN-SEM, a new specimen holder has been developed to facilitate direct detection of BF signals and indirect detection of dark-field (DF) signals without the need for substantial instrument modification. DF imaging is conducted with the use of a gold (Au)-coated copper (Cu) plate attached to the specimen holder which directs highly scattered transmitted electrons to an off-axis yttrium-aluminum-garnet (YAG) detector. A hole in the copper plate allows for BF imaging with a transmission electron (TE) detector. The inclusion of an Au-coated Cu plate enhanced DF signal intensity. Experiments validating the acquisition of true DF signals revealed that atomic number (Z) contrast may be achieved for materials with large lattice spacing. However, materials with small lattice spacing still exhibit diffraction contrast effects in this approach. The calculated theoretical fine probe size is 1.8 nm. At 30 kV, in this indirect approach, DF spatial resolution is limited to 3.2 nm as confirmed experimentally.

Determining On-Axis Crystal Thickness with Quantitative Position-Averaged Incoherent Bright-Field Signal in an Aberration-Corrected STEM

An accurate determination of specimen thickness is essential for quantitative analytical electron microscopy. Here we demonstrate that a position-averaged incoherent bright-field signal recorded on an absolute scale can be used to determine the thickness of on-axis crystals with a precision of ±1.6 nm. This method measures both the crystalline and the noncrystalline parts (surface amorphous layers) of the sample. However, it avoids the systematic error resulting from surface plasmon contributions to the inelastic mean-free-path thickness estimated by electron energy loss spectroscopy.

Understanding imaging modes in the helium ion microscope

Recent investigations are gaining us a better understanding of the nature of the beam-sample interactions in the helium ion microscope and what they mean for the image information provided. In secondary electron (SE) imaging, for example, the surface sensitivity is attributed to the low SE-II fraction. Voltage contrast imaging shows the ability to see both buried structures and to probe the conductance to ground of surface contacts. It is found, however, that the prominence of these two types of contrast varies oppositely with beam energy, yielding information about the nature of the interactions that gives rise to them. Transmission ion imaging can yield information about material density, atomic number, grain structure, and electronic structure. It is possible to capture the top-side SE signal, bright field signal, and dark field signal from a given sample simultaneously. The detection of diffraction contrast is under investigation.

Electron tomography of Pt nanocatalyst particles and their carbon support

Industrial nanocatalysts usually comprise crystalline particles of high atomic number that have sizes of between 1 and 20 nm and are supported or embedded in a lower atomic number matrix. The physical characterisation of the three-dimensional shapes and sizes of such particles can now be carried out using high-angle annular darkfield electron tomography. The spatial distribution of the particles with respect to their matrix is an issue of paramount importance for their performance as catalysts. Here, we show experimental electron tomography results from platinum particles dispersed in a carbon support. We show that both the high and the low atomic number regions of the same region of a sample can be characterised by using a combination of high and low angle annular dark field and bright field signals.

Theory of bright-field scanning transmission electron microscopy for tomography

Radiation transport theory is applied to electron microscopy of samples composed of one or more materials. The theory, originally due to Goudsmit and Saunderson, assumes only elastic scattering and an amorphous medium dominated by atomic interactions. For samples composed of a single material, the theory yields reasonable parameter-free agreement with experimental data taken from the literature for the multiple scattering of 300-keV electrons through aluminum foils up to 25μm thick. For thin films, the theory gives a validity condition for Beer’s law. For thick films, a variant of Molière’s theory [V. G. Molière, Z. Naturforschg. 3a, 78 (1948)] of multiple scattering leads to a form for the bright-field signal for foils in the multiple-scattering regime. The signal varies as [tln(e1−2γt∕τ)]−1 where t is the path length of the beam, τ is the mean free path for elastic scattering, and γ is Euler’s constant. The Goudsmit–Saunderson solution interpolates numerically between these two limits. For samples with multiple materials, elemental sensitivity is developed through the angular dependence of the scattering. From the elastic scattering cross sections of the first 92 elements, a singular-value decomposition of a vector space spanned by the elastic scattering cross sections minus a delta function shows that there is a dominant common mode, with composition-dependent corrections of about 2%. A mathematically correct reconstruction procedure beyond 2% accuracy requires the acquisition of the bright-field signal as a function of the scattering angle. Tomographic reconstructions are carried out for three singular vectors of a sample problem with four elements Cr, Cu, Zr, and Te. The three reconstructions are presented jointly as a color image; all four elements are clearly identifiable throughout the image.

Imaging of 30nm gold spheres by dark-field scanning transmission x-ray microscopy

In a scanning transmission x-ray microscope (STXM), such as that operated at the beamline X-lA at the National Synchrotron Light Source, it is possible to obtain dark-field images by blocking the undeviated transmitted photons so that only x rays scattered by the specimen are detected. Although the signal that is detected in dark-field is much weaker than the conventional bright-field signal the method offers much higher contrast of small features and the possibility of detecting small features with higher signal to noise for the same incident x-ray flux. Also, the signal depends on both the amplitude and phase of the specimen, rather than just the amplitude. One important application of dark-field x-ray microscopy is the imaging of gold-labelled biological specimens. This unites the advantages of x-ray microscopy (the ability of imaging thick biological objects, low radiation dose) with those of gold labelling (specific-site or specific-protien labelling, well documented protocols).The x-ray microscope consists of the scanning transmission x-ray microscope, in which a zone-plate is used to focus monochromatic x rays to a probe, through which the sample is scanned.

Electron holography of ferroelectric materials

Electron holography gives to the electron microscope the ability the visualize, and measure, electric and magnetic fields at high spatial resolution . The principle of the method is shown in figure (1). Some fraction of a coherent electron beam, typically produced from a cold field emission electron gun, is incident on the specimen while the rest passes through vacuum, or through some other region of the sample, to form a reference wave. An electrostatic bi-prism converges these two components to form an interferogram or ‘hologram’ at the viewing screen. The hologram consists of a fringe pattern, with a spacing determined by the potential on the bi-prism, modulated by the intensity variations of the transmitted bright-field signal. Variations in the phase of the transmitted signal are stored as shifts in the fringe pattern. The hologram can then be reconstructed, either optically or by using a computer and appropriate software, to yield two images, one - the 'Amplitude Image' - containing normal intensity contrast (bright field) information, and a second- the 'Phase Image'- which displays variations in the relative phase of the signal as changes in grey level. The phase of the transmitted beam will be shifted by an amount proportional to the mean inner potential of the specimen multiplied by the thickness, and also by the potential that the electron encounters as it travels through the sample.

Dark-field imaging with the scanning transmission x-ray microscope

The King’s College London scanning transmission x-ray microscope in use on beam line 5U2 at the SRS, SERC Daresbury Laboratory, has been modified to allow dark-field images to be formed using only the x rays scattered by the specimen. Experiments have been performed with a number of different detector geometries, and this has confirmed that the strongest scattering arises from edges or thickness gradients in the specimen. Although the dark-field signal is only a small fraction of the normal transmitted bright-field signal, features can be revealed with high contrast, and it has proved possible to detect the presence of features that are below the resolution limit of the microscope.

Quantitative Energy-Filtered CRYO-EM of Hydrated Chromosome Fibers, Viruses, and Heavy Atoms

We have calculated and experimentally measured the extent to which energy filtration improves the S/N in phase- and scattering-contrast images of frozen-hydrated molecules. Multiple scattering calculations based on PW (partial wave) complex scattering amplitudes agree quantitatively with 80 KV image intensities of 50-500nm-thick ice. For an objective aperture of 2.5 nm-1 the PW elastic mean free path is 280 nm. From the ratio of filtered to unfiltered image intensities from 50-500 nm of ice at 80 KV, we calculate an empirical inelastic mean free path of 180 nm. The PW calculations show that Beer's Law should be obeyed for thicknesses up to five mean free paths, whether or not energy filtration is used. Thus the bright-field signal can be used to determine the absolute mass distribution of molecules embedded in thick ice layers.Energy filtration removes background intensity due to inelastic scattering (Fig.1a), and increases the scattering signal from molecules (becoming the sum of both elastic electrons excluded by the objective aperture and inelastic electrons excluded by the energy filter). As the result, the calculated contrast is 2-15 times greater in the Zeiss EM902 than in a TEM without energy filtration (Fig.1b). 80 KeV energy-filtered images of frozen-hydrated molecules should have a higher S/N than unfiltered images taken at intermediate or high voltage (Fig. 1c,d). TMV contrast and S/N is improved by filtration and agrees with calculations (Fig.1b,2). The advantages of energy-filtration are important to the microscopy of chromatin fibers, which are excluded from ice layers ≤200 nm and viruses, which are usually excluded from layers ≤100 nm. Radial density reconstructions from near-focus energy-filtered images of frozen-hydrated chromatin from Thyone sperm show that the centers are not hollow, but very dense, probably containing DNA (Fig.3). Energy-filtered images of icosahedral viruses in even the thinnest possible ice have a much improved S/N ratio, decreasing the dose required for analysis (Fig.2a,b).

Detection and Imaging of Supported Catalyst Particles

ABSTRACTIn this short review we contrast and compare the relative merits of three imaging techniques in the CTEM and STEM, for detecting subnanometer size catalyst clusters. It is shown that incoherent dark field signals, such as those obtained by hollow-cone illumination in the CTEM, and the annular detector in the STEM, are significantly more sensitive to subnanometer size clusters than the bright field signal. Sensitivity to clusters of high atomic number increases as the illumination (or detector) semi-angle increases, although at the expense of decreased signal-to-noise. The STEM annular detector signal offers advantages over the CTEM hollow-cone signal in this respect since it has a higher electron collection efficiency. A further advantage of the STEM is that image contrasts may be enhanced by electronically combining the annular detector signal with the axial energy loss signal. This ratio method, or Z contrast technique, has been used with success to detect single Pt atoms on thin low atomic number supports.

Observations of Pt-Ir on Al2O3 Catalysts Using a Fast Digital System Controlled STEM

The familiar Scanning Transmission Electron Microscope (STEM) technique of Z-contrast, used to image heavy atoms on amorphous supports, can be applied to the study of metal catalysts with only partial success. The contrast is reduced when crystalline systems are studied since a Bragg contribution is introduced in the standard annular dark-field collector. At large angles, the Bragg reflections diminish due to thermal vibrations; and the scattering cross section is approximately proportional to Z2. Thus scattering in this region is predominately Rutherford-like. The atomic number dependence of this high angle detectoro (HAD) signal makes it particularly suited for identifying small (20-50Å diameter) catalyst particles suspended in polycrystal line alumina.The experimental configuration for this imaging mode suggests concurrent acquisition of the HAD signal and the corresponding bright field signal , as on-line arithmetic of the two is necessary to optimize image contrast. In the conventional STEM it is impossible to detect the HAD signal because it is cut off by the specimen cartridge. Treacy, et al. have developed a sophisticated method for obtaining these signals.