IMSI—Guidelines for Sperm Quality Assessment

Intracytoplasmic sperm injection (ICSI) is a widely used and accepted treatment of choice for oocyte fertilization. However, the quality of sperm selection depends on the accurate visualization of the morphology, which can be achieved with a high image resolution. We aim to correct the conviction, shown in a myriad of publications, that an ultra-high magnification in the range of 6000×–10,000× can be achieved with an optical microscope. The goal of observing sperm under the microscope is not to simply get a larger image, but rather to obtain more detail—therefore, we indicate that the optical system’s resolution is what should be primarily considered. We provide specific microscope system setup recommendations sufficient for most clinical cases that are based on our experience showing that the optical resolution of 0.5 μm allows appropriate visualization of sperm defects. Last but not least, we suggest that mixed research results regarding the clinical value of IMSI, comparing to ICSI, can stem from a lack of standardization of microscopy techniques used for both ICSI and IMSI.

Photoacoustic imaging of 3D-printed vascular networks

Thrombosis in the circulation system can lead to major myocardial infarction and cardiovascular deaths. Understanding thrombosis formation is necessary for developing safe and effective treatments. In this work, using digital light processing (DLP)-based 3D printing, we fabricated sophisticated in vitro models of blood vessels with internal microchannels that can be used for thrombosis studies. In this regard, photoacoustic microscopy (PAM) offers a unique advantage for label-free visualization of the 3D-printed vessel models, with large penetration depth and functional sensitivity. We compared the imaging performances of two PAM implementations: optical-resolution PAM and acoustic-resolution PAM, and investigated 3D printed- vessel structures with different patterns of microchannels. Our results show that PAM can provide clear microchannel structures at depths up to 3.6 mm. We further quantified the blood oxygenation in the 3D-printed vascular models, showing that thrombi had lower oxygenation than the normal blood. We expect that PAM can find broad applications in 3D printing and bioprinting for in vitro studies of various vascular and other diseases.

Колебательно-вращательный спектр высокого разрешения в районе полос 3ν-=SUB=-4-=/SUB=-, ν-=SUB=-2-=/SUB=-+2ν-=SUB=-4-=/SUB=- и 2ν-=SUB=-2-=/SUB=-+ν-=SUB=-4-=/SUB=- молекулы -=SUP=-72-=/SUP=-GeH-=SUB=-4-=/SUB=-

The high-resolution spectrum of the 72GeH4 molecule was recorded on a Bruker IFS 125HR Fourier spectrometer with an optical resolution of 0.003 cm-1. The line positions were analyzed for ten interacting vibrational-rotational bands 3ν4 (1F2, F1, 2F2), v2+ 2ν4 (1E, F1, F2, 2E) and 2ν2+v4 (1F2, F1, 2F2) in the range 2350-2750 cm-1. As a result of the analysis, 1726 experimental lines were identified with the maximum value of the quantum number Jmax = 17; then used in the fitting procedure with parameters of the effective Hamiltonian. The resulting set of 35 spectroscopic parameters describes the vibrational-rotational structure of the spectrum with drms = 7.5 · 10-4 cm-1.

Fluorescent Probes for STED Optical Nanoscopy

Progress in developing fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, is inseparable from the advancement in optical fluorescence microscopy. Super-resolution microscopy, or optical nanoscopy, overcame the far-field optical resolution limit, known as Abbe’s diffraction limit, by taking advantage of the photophysical properties of fluorescent probes. Therefore, fluorescent probes for super-resolution microscopy should meet the new requirements in the probes’ photophysical and photochemical properties. STED optical nanoscopy achieves super-resolution by depleting excited fluorophores at the periphery of an excitation laser beam using a depletion beam with a hollow core. An ideal fluorescent probe for STED nanoscopy must meet specific photophysical and photochemical properties, including high photostability, depletability at the depletion wavelength, low adverse excitability, and biocompatibility. This review introduces the requirements of fluorescent probes for STED nanoscopy and discusses the recent progress in the development of fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, for the STED nanoscopy. The strengths and the limitations of the fluorescent probes are analyzed in detail.

Physics-based machine learning for subcellular segmentation in living cells

Segmenting subcellular structures in living cells from fluorescence microscope images is a ground truth (GT)-deficient problem. The microscopes’ three-dimensional blurring function, finite optical resolution due to light diffraction, finite pixel resolution and the complex morphological manifestations of the structures all contribute to GT-hardness. Unsupervised segmentation approaches are quite inaccurate. Therefore, manual segmentation relying on heuristics and experience remains the preferred approach. However, this process is tedious, given the countless structures present inside a single cell, and generating analytics across a large population of cells or performing advanced artificial intelligence tasks such as tracking are greatly limited. Here we bring modelling and deep learning to a nexus for solving this GT-hard problem, improving both the accuracy and speed of subcellular segmentation. We introduce a simulation-supervision approach empowered by physics-based GT, which presents two advantages. First, the physics-based GT resolves the GT-hardness. Second, computational modelling of all the relevant physical aspects assists the deep learning models in learning to compensate, to a great extent, for the limitations of physics and the instrument. We show extensive results on the segmentation of small vesicles and mitochondria in diverse and independent living- and fixed-cell datasets. We demonstrate the adaptability of the approach across diverse microscopes through transfer learning, and illustrate biologically relevant applications of automated analytics and motion analysis.

Awake mouse brain photoacoustic and optical imaging through a transparent ultrasound cranial window

Optical resolution photoacoustic microscopy (OR-PAM) can map the cerebral vasculature at capillary level resolution. However, the OR-PAM setup’s bulky imaging head makes awake mouse brain imaging challenging and inhibits its integration with other optical neuroimaging modalities. Moreover, the glass cranial windows used for optical microscopy are unsuitable for OR-PAM due to the acoustic impedance mismatch between the glass plate and the tissue. To overcome these challenges, we propose a lithium niobate based transparent ultrasound trans-ducer (TUT) as a cranial window on a thinned mouse skull. The TUT cranial window simplifies the imaging head considerably due to its dual functionality as an optical window and ultrasound transducer. The window remains stable for six weeks, with no noticeable inflammation and minimal bone regrowth. The TUT window’s potential is demonstrated by imaging the awake mouse cerebral vasculature using OR-PAM, intrinsic optical signal imaging and two-photon microscopy. The TUT cranial window can potentially also be used for ultrasound stimulation and simultaneous multimodal imaging of the awake mouse brain.

VAMP2 and synaptotagmin mobility in chromaffin granule membranes: implications for regulated exocytosis

Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may influence docking, and membrane fusion. We measured the mobility of vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field produced by total internal reflection (TIR) was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, enabling the measurement of diffusion coefficients. We found that VAMP2-EGFP and Syt7-EGFP are mobile with a diffusion coefficient of approximately 3 × 10-10 cm2/s. Syt1-EGFP mobility was below the detection limit. Utilizing these diffusion parameters, we estimated the time required for these proteins to arrive at docking and nascent fusion sites to be many tens of milliseconds. Our analyses raise the possibility that the diffusion characteristics of VAMP2 and Syt proteins could be a factor that influences the rate of exocytosis.