Annular Dark Field Imaging with Variable Angle for Improving STEM Tomography of Biological Samples
Year: 2023
Type: article
Abstract: Three-dimension (3D) chromatin structure visualization is key to answering the mysteries of gene expression process and regulations [1]. This requires high resolution tomographic techniques down to nm range, which is challenging to achieve in biological samples. Advances in light microscopy such as stochastic optical reconstruction microscopy (STORM) and structured illumination microscopy (SIM) allowed super resolution imaging beyond diffraction limits [2, 3], but 3D imaging capability of these techniques is limited. The recent development of ChromEMT uses Click-EM labeling and electron tomography techniques and is one of the closest to revealing the 3D chromatin structure in nucleus [4]. However, since the contrast variation in transmission electron microscopy (TEM) cannot be directly interpreted, binarization is required for analysis which makes chromatin concentration analysis not reliable. ChromSTEM is another approach following ChromEMT that applies high-angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) for electron tomography on chromatin samples [5]. HAADF scattering intensity is proportional to mass of the sample, therefore ChromSTEM probes the mass distribution of chromatin in the sample by the continuous intensity distribution of each pixel without binarization. However, STEM suffers from statistical noises especially in case of dose-limited condition in bio-imaging applications to reduce beam damages [6]. It is, therefore, important to optimize the signal-to-noise ratio to improve the tomogram quality without increasing beam dosage. One of such methods is based on varying the detection angle of HAADF-STEM imaging. The method is useful in material science research and is widely applied to chemical analysis in materials composed of two or more elements [7, 8]. By varying the detection angle, the contrast specific to an element can be enhanced or suppressed. This can be used to enhance osmium-stained biological structure in HAADF imaging. This is also very important to biological STEM tomography when fiducial markers such as gold nanoparticles are added to help to align images in a tilt-series. Due to the high contrast of gold nanoparticles, together with the unavoidable “missing cone” effect in electron tomography, it greatly impacts the tomogram quality of the volume around the nanoparticles. By choosing an appropriate detection angle, the intensity of the fiducial markers can also be suppressed and reduce its impact on the resultant tomogram. In this work, HCT116 cells were prepared following ChromEMT protocol [4]. The samples were then sectioned into 100nm thick samples using Leica EM UC7 ultramicrotome. HAADF STEM images were collected in Hitachi HD2300 STEM with 200kV. To vary the collection angle of the HAADF detector, the objective lens current was adjusted. At higher detection angle (increased the objective lens current), the contrast from osmium improved and it shows clearer nuclear membrane as well as chromatin clusters inside the nucleus in Figure 1. Meanwhile, comparing the intensity of gold nanoparticles and osmium, gold nanoparticles have a decreased contrast. This can potentially help to prevent the gold nanoparticles signals from overshadowing osmium-stained features in a tomogram. Although there is a trend that the osmium intensity will continue to increase relative to the gold intensity at higher objective lens current, the higher objective lens current is not preferred in these images because the off-axis electrons are deflected differently and it creates difference in brightness in the image periphery compared to the image center, as shown in Figure 2. Therefore, higher objective lens current requires higher magnification images where field of view is limited. In summary, adjusting detection angle in HAADF-STEM can help to adjust the contrast of specific elements. It is useful in biological electron microscopy tomography to suppress fiducial marker intensity and highlight the interested structures without increasing beam dosage and beam damage. It may also be applied to other biological samples labeled with multiple elements [9]. Osmium-stained structures have increased contrast at (b) higher objective lens current than at (a) lower objective lens. (c) The gold nanoparticles and osmium-stained membrane of each image were segmented, and their intensities were compared in (d). Left: Image of whole nucleus with lower objective lens current. Right: Image of whole nucleus with higher objective lens current.
Source: Microscopy and Microanalysis
Language: English
Subfield: Structural Biology
Domain: Life Sciences
Open Access status: bronze
Funder: National Cancer Institute
OpenAlex ID: W4385141964