Brain Sample Preparation After Performing in Utero Electroporation to Proliferating and Differentiated Cells in the Developing Mouse Neocortex.

In utero electroporation (IUE) enables labeling and manipulating specific types of cells by introducing DNA plasmids with desired promoters. After the surgery, mouse brains are fixed at any stage and analyzed after staining using specific antibodies. Here, we describe the flow of the IUE experiment from the preparation to microscopic observations.

Technical note: Cryostat transmission characterization for MR linac - temporal stability, clinical impact and change implementation.

BACKGROUND: In the Unity MR linac (Elekta AB, Stockholm, Sweden), the radiation beam traverses the cryostat and the coil support structure. The resulting beam attenuation must be considered for output calibration and its variation with gantry angle must be characterized in the treatment planning system (TPS). PURPOSE: The aim of this work was to investigate the impact of a change of the cryostat transmission characterization (CTC) curve, due to the helium level modification, on clinical treatment plan dosimetry and to report on the experience with the CTC curve update. METHODS: Twenty stereotactic body radiotherapy (SBRT) treatment plans: 10 prostate and 10 oligo-metastatic cancer plans, prepared with a beam model incorporating the CTC curve acquired at installation time, were re-calculated using the model implementing CTC curve post helium top-up. To account for the CTC change as well as to align our system to the recent reference conditions recommendations, the new model was commissioned with the emphasis on the specifics associated with the treatment plan adaptation and the existence of the offline and online TPS components. RESULTS: Average CTV mean dose reduction by 0.45% in prostate cases and average GTV mean dose reduction by 0.22% in oligo-metastatic cases was observed. Updated model validation showcased good agreement between measurements and TPS calculations. CONCLUSIONS: The agreement between CTC measurements demonstrates its temporal constancy and robustness of the measurement method employed. A helium fill level change was shown to affect the CTC and led to a small but systematic dose calculation inaccuracy. Finally, model validation and end-to-end testing results presented, underscore the minimal impact of transitioning to the new beam model and new reference conditions.

Novel correction procedure for compensating thermal contraction errors in the measurement of the magnetic field of superconducting undulator coils in a liquid helium cryostat.

Superconducting undulators (SCUs) can offer a much higher on-axis undulator field than state-of-the-art cryogenic permanent-magnet undulators with the same period and vacuum gap. The development of shorter-period and high-field SCUs would allow the free-electron laser and synchrotron radiation source community to reduce both the length of undulators and the dimensions of the accelerator. Magnetic measurements are essential for characterizing the magnetic field quality of undulators for operation in a modern light source. Hall probe scanning is so far the most mature technique for local field characterization of undulators. This article focuses on the systematic error caused by thermal contraction that influences Hall probe measurements carried out in a liquid helium cryostat. A novel procedure, based on the redundant measurement of the magnetic field using multiple Hall probes at known relative distance, is introduced for the correction of such systematic error.

Dosimetric validation of the couch and coil model for high-field MR-linac treatment planning.

PURPOSE: The precision of the dose delivery in radiation therapy with high-field MR-linacs is challenging due to the substantial variation in the beam attenuation of the patient positioning system (PPS) (the couch and coils) as a function of the gantry angle. This work aimed to compare the attenuation of two PPSs located at two different MR-linac sites through measurements and calculations in the treatment planning system (TPS). METHODS: Attenuation measurements were performed at every 1° gantry angle at the two sites with a cylindrical water phantom with a Farmer chamber inserted along the rotational axis of the phantom. The phantom was positioned with the chamber reference point (CRP) at the MR-linac isocentre. A compensation strategy was applied to minimise sinusoidal measurement errors due to, e.g. air cavity or setup. A series of tests were performed to assess the sensitivity to measurement uncertainties. The dose to a model of the cylindrical water phantom with the PPS added was calculated in the TPS (Monaco v5.4 as well as in a development version Dev of an upcoming release), for the same gantry angles as for the measurements. The TPS PPS model dependency of the dose calculation voxelisation resolution was also investigated. RESULTS: A comparison of the measured attenuation of the two PPSs yielded differences of less than 0.5% for most gantry angles. The maximum deviation between the attenuation measurements for the two different PPSs exceeded ±1% at two specific gantry angles 115° and 245°, where the beam traverses the most complex PPS structures. The attenuation increases from 0% to 25% in 15° intervals around these angles. The measured and calculated attenuation, as calculated in v5.4, was generally within 1-2% with a systematic overestimation of the attenuation for gantry angles around 180°, as well as a maximum error of 4-5% for a few discrete angles in 10° gantry angle intervals around the complex PPS structures. The PPS modelling was improved compared to v5.4 in Dev, especially around 180°, and the results of those calculations were within ±1%, but with a similar 4% maximum deviation for the most complex PPS structures. CONCLUSIONS: Generally, the two tested PPS structures exhibit very similar attenuation as a function of the gantry angle, including the angles with a steep change in attenuation. Both TPS versions, v5.4 and Dev delivered clinically acceptable accuracy of the calculated dose, as the differences in the measurements were overall better than ±2%. Additionally, Dev improved the accuracy of the dose calculation to ±1% for gantry angles around 180°.

A Cryostat Applicable to Long-Wavelength Light-Driven Scanning Probe Microscopy.

Recently, there has been growing interest in using lightwave-driven scanning probe microscopy (LD-SPM) to break through the Abbe diffraction limit of focusing, yielding insight into various energy couplings and conversion processes and revealing the internal information of matter. We describe a compact and efficient optical cryostat designed for LD-SPM testing under magnetic fields. The exceptional multilayer radiation shielding insert (MRSI) forms an excellent temperature gradient when filled with heat conducting gas, which removes the requirement to install an optical window in the liquid helium cooling shell. This not only critically avoids the vibration and thermal drift caused by solid heat conduction but also minimizes light transmission loss. The application of gate valves and bellows allows a simpler and more effective replacement of the sample and working cell in the test cavity. ANSYS software is used for steady-state thermal analysis of the MRSI to obtain the temperature distribution and heat transfer rate, and the necessity of the flexible copper shielding strips is illustrated by the simulations. The topography and magnetic domain images of 45 nm-thick La0.67Ca0.33MnO3 thin films on NdGaO3(001) substrates under a magnetic field were obtained by a self-made lightwave-driven magnetic force microscope in this cryostat. The resolution and noise spectra during imaging reveal temperature stability and low vibration throughout the cryostat. The experience acquired during the development of this cryostat will help to establish cryostats of similar types for a variety of optic applications requiring the use of cryogenic temperatures.

Dependence of crystallographic atomic displacement parameters on temperature (25-150†K) for complexes of horse liver alcohol dehydrogenase.

Enzymes catalyze reactions by binding and orienting substrates with dynamic interactions. Horse liver alcohol dehydrogenase catalyzes hydrogen transfer with quantum-mechanical tunneling that involves fast motions in the active site. The structures and B factors of ternary complexes of the enzyme with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or NAD+ and 2,2,2-trifluoroethanol were determined to 1.1-1.3 Šresolution below the `glassy transition' in order to extract information about the temperature-dependent harmonic motions, which are reflected in the crystallographic B factors. The refinement statistics and structures are essentially the same for each structure at all temperatures. The B factors were corrected for a small amount of radiation decay. The overall B factors for the complexes are similar (13-16 Å2) over the range 25-100 K, but increase somewhat at 150 K. Applying TLS refinement to remove the contribution of pseudo-rigid-body displacements of coenzyme binding and catalytic domains provided residual B factors of 7-10 Å2 for the overall complexes and of 5-10 Å2 for C4N of NAD+ and the methylene carbon of the alcohols. These residual B factors have a very small dependence on temperature and include local harmonic motions and apparently contributions from other sources. Structures at 100 K show complexes that are poised for hydrogen transfer, which involves atomic displacements of ∼0.3 Šand is compatible with the motions estimated from the residual B factors and molecular-dynamics simulations. At 298 K local conformational changes are also involved in catalysis, as enzymes with substitutions of amino acids in the substrate-binding site have similar positions of NAD+ and pentafluorobenzyl alcohol and similar residual B factors, but differ by tenfold in the rate constants for hydride transfer.

Optimized Immunostaining of Embryonic and Early Postnatal Mouse Brain Sections.

The mammalian neocortex, the outer layer of the cerebrum and most recently evolved brain region, is characterized by its unique areal and laminar organization. Distinct cortical layers and areas can be identified by the protein expression of graded transcription factors and molecular determinants that define the identity of different projection neurons. Thus, specific detection and visualization of protein expression is crucial for assessing the identity of neocortical neurons and, more broadly, for understanding early and late developmental mechanisms and function of this complex system. Several immunostaining/immunofluorescence methods exist to detect protein expression. Published protocols vary with regard to subtle details, which may impact the final outcome of the immunofluorescence. Here, we provide a detailed protocol, suitable for both thin cryostat sections and thick vibratome sections, which has successfully worked for a wide range of antibodies directed against key molecular players of neocortical development. Ranging from early technical steps of brains collection down to image analysis and statistics, we include every detail concerning sample inclusion and sectioning, slide storage and optimal antibody dilutions aimed at reducing non-specific background. Routinely used in the lab, our background-optimized immunostaining protocol allows efficient detection of area- and layer- specific molecular determinants of distinct neocortical projection neurons. Graphic abstract: Workflow chart for the optimized immunostaining protocol of mouse brain sections. A. A flow chart for different steps of the optimized immunostaining protocol on both thin cryostat and thick vibratome sections. B. Example for immunostaining against Satb2 and Ctip2 on a thin coronal section (20 µm) at the level of the somatosensory cortex. The first column to the left shows the binning system where 6 bins can be overlaid on the image. On the bottom, an example of counting analysis showing the percentage of marker-positive cells normalized to the total number of DAPI or Hoechst-positive cells. C. Example for immunostaining against Satb2 and Ctip2 on a GFP+ thick vibratome section (200 µm). Images are taken at low magnification (10x, left) and high magnification (40x, right). The graph shows a counting of the percentage of Ctip2-positive neurons normalized to the total number of GFP-electroporated neurons on high-magnification images. Images on B and C are modified from Harb et al. (2016).

A multisample 7 T dynamic nuclear polarization polarizer for preclinical hyperpolarized MR.

Dynamic nuclear polarization (DNP) provides the opportunity to boost liquid state magnetic resonance (MR) signals from selected nuclear spins by several orders of magnitude. A cryostat running at a temperature of ~ 1 K and a superconducting magnet set to between 3 and 10 T are required to efficiently hyperpolarize nuclear spins. Several DNP polarizers have been implemented for the purpose of hyperpolarized MR and recent systems have been designed to avoid the need for user input of liquid cryogens. We herein present a zero boil-off DNP polarizer that operates at 1.35 ± 0.01 K and 7 T, and which can polarize two samples in parallel. The samples are cooled by a static helium bath thermally connected to a 1 K closed-cycle 4 He refrigerator. Using a modified version of the commercial fluid path developed for the SPINlab polarizer, we demonstrate that, within a 12-minute interval, the system can produce two separate hyperpolarized 13 C solutions. The 13 C liquid-state polarization of [1-13 C]pyruvate measured 26 seconds after dissolution was 36%, which can be extrapolated to a 55% solid state polarization. The system is well adapted for in vitro and in vivo preclinical hyperpolarized MR experiments and it can be modified to polarize up to four samples in parallel.

A helium mini-cryostat for the nanoprobe beamline ID16B at ESRF: characteristics and performance.

A helium mini-cryostat has been developed for the hard X-ray nanoprobe ID16B of the European Synchrotron to collect X-ray excited optical luminescence and X-ray fluorescence at low temperature (<10 K). The mini-cryostat has been specifically designed to fit within the strong space restrictions and high-demanding mechanical constraints imposed by the beamline to provide vibration-free operation and maximal thermal stability. This paper reports the detailed design, architecture and technical requirements of the mini-cryostat, and presents the first experimental data measured using the cryogenic equipment. The resulting cryo-system features ultimate thermal stability, fast cool-down and ultra-low vibrations. The simultaneous X-ray fluorescence and X-ray excited optical luminescence data acquired from bulk GaN and core/shell InGaN/GaN multi-quantum wells validated the excellent performance of the cryostat with ultimate resolution, stability and sensitivity.

Basic Neuroanatomical Methods.

This unit covers some basic procedures that are common to a wide range of neuroanatomical protocols for brain tissue. Procedures are provided for preparation of unfixed fresh brain tissue as well as for perfusion fixation of animals to obtain fixed neural tissue. A variety of methods for sectioning are described, including frozen sectioning using a cryostat or microtome and sectioning with a vibratome. The choice of sectioning method depends on how the brain has been prepared and what histochemical method is to be used. A fluorescent immunohistochemical method to localize endogenous molecules as well as induced markers such as green fluorescent protein and red fluorescent protein is also provided. Additionally, three post-sectioning procedures are described: defatting of slide-mounted sections, fluorescent Nissl staining, and thionin staining of sections. Finally, support protocols are provided, describing a method for maintaining the correct order of cut tissue, whether rostral to caudal or lateral to medial; a procedure for subbing slides with gelatin, which is necessary in some protocols in order for sections to adhere to slides; and preparation of custom 3D-printed 10- or 20-well tissue plates and trays for subsequent immunostaining. Published 2019. U.S. Government. Basic Protocol 1: Preparation of unfixed fresh-frozen brain tissue Basic Protocol 2: Perfusion fixation Basic Protocol 3: Cryostat sectioning of frozen brain tissue Basic Protocol 4: Sliding-microtome sectioning of fixed brain tissue Basic Protocol 5: Vibratome and Compresstome sectioning Support Protocol 1: Tissue collection in a 1-in-10 series Support Protocol 2: Preparation of gelatin-subbed microscope slides Support Protocol 3: Custom 3D-printed 10- and 20-well tissue plates Basic Protocol 6: Post-sectioning procedures I: Fluorescent immunohistochemical localization Basic Protocol 7: Post-sectioning procedures II: Defatting Basic Protocol 8: Post-sectioning procedures III: Nissl staining Basic Protocol 9: Post-sectioning procedures IV: Thionin staining.

Sticker method for preparation of frozen section using adhesive film.

BACKGROUND: In basic research, especially animal experiments, tissue histology with preserved organ morphology is essential, and the reliability of experiments depends on the quality of tissue sections. Frozen sections adequately maintain the antigenicity of tissues and are suitable for immunohistochemistry. However, thin frozen sections are often difficult to prepare from specimens. Therefore, a simple and fast method with a high success rate of specimen preparation is desired. NEW METHOD: In this study, we propose the "Sticker method," for preparing frozen sections using adhesive film, which is easy and maintains the whole organ morphology even in frozen section. RESULTS: This method requires a simple adhesive film, and other components of general tissue fixation method with embedding medium. The present sticker method showed a higher success rate than the conventional method in preparing frozen sections. COMPARISON WITH EXISTING METHOD: Fragile frozen sections can be prepared with intact whole organ morphology without wrinkles. The advantage of the present method is that tissues can be embedded using only a common embedding medium and besides the adhesive film, no other special equipment is required. Furthermore, this method can be easily used by virtually every lab performing cryosections. CONCLUSIONS: The sticker method using adhesive films is characterized by a shortened preparation time and increased success rate of frozen section compared with conventional method.

Methods for In Situ Protein Visualization in Dental Mineralized Tissues.

Immunohistochemistry (IHC) is a technique based on the specificity of antibody-antigen principle used commonly to detect antigens in tissue sections. The immune labeling can be performed in paraffin sections, cryostat sections, and ultrathin sections and can be observed in light confocal and transmission electron microscopy. However, the use of immunohistochemical techniques for the study of mineralized tissues has been a challenge for decades (Berdal et al., Arch Oral Biol 36:715-725, 1991; Nanci et al., Eur J Histochem 52:201-214, 2008). Specific procedures are necessary when compared with soft tissue immunohistochemistry. This chapter describes methods for IHC on Tissue-Tek O.C.T. compound and paraffin-embedded sections to detect antigens in the dental mineralized tissues.

Ultra-stable super-resolution fluorescence cryo-microscopy for correlative light and electron cryo-microscopy.

Remarkable progress in correlative light and electron cryo-microscopy (cryo-CLEM) has been made in the past decade. A crucial component for cryo-CLEM is a dedicated cryo-fluorescence microscope (cryo-FM). Here, we describe an ultra-stable super-resolution cryo-FM that exhibits excellent thermal and mechanical stability. The temperature fluctuations in 10 h are less than 0.06 K, and the mechanical drift over 5 h is less than 200 nm in three dimensions. We have demonstrated the super-resolution imaging capability of this system (average single molecule localization accuracy of ∼13.0 nm). The results suggest that our system is particularly suitable for long-term observations, such as single molecule localization microscopy (SMLM) and cryogenic super-resolution correlative light and electron microscopy (csCLEM).

Imaging the Lymph Node Stroma.

Lymph node (LN) stromal cells are being recognized as key organizers of the immune system. They assemble in complex 3D networks and hence, need to be studied in situ to fully understand their exact functions. Here, we describe two distinct but complementary procedures that allow analyzing LN stromal cells at high resolution by confocal imaging.

Large Angle Optical Access in a Sub-Kelvin Cryostat.

The development of lens-antenna-coupled aluminum-based microwave kinetic inductance detectors (MKIDs) and on-chip spectrometers needs a dedicated cryogenic setup to measure the beam patterns of the lens-antenna system over a large angular throughput and broad frequency range. This requires a careful design since the MKID has to be cooled to temperatures below 300 mK to operate effectively. We developed such a cryostat with a large opening angle θ = ± 37 . 8 ∘ and an optical access with a low-pass edge at 950 GHz . The system is based upon a commercial pulse tube cooled 3 K system with a 4 He - 3 He sorption cooler to allow base temperatures below 300 mK . A careful study of the spectral and geometric throughput was performed to minimize thermal loading on the cold stage, allowing a base temperature of 265 mK . Radio-transparent multi-layer-insulation was employed as a recent development in filter technology to efficiently block near-infrared radiation.

Enzymatic Assays and Enzyme Histochemistry ofTuta absoluta Feeding on Tomato Leaves.

Enzymes play a key role in insect-plant relationships. For a better understanding of these interactions, we analyzed Tuta absoluta digestive enzymes. Here, we describe a detailed protocol for the detection of trypsin and papain-like enzymes in Tuta absoluta larvae by enzyme histochemistry. This assay uses frozen and unfixed samples to avoid the loss of enzymatic activity. We also describe a protocol for the quantification of trypsin and papain-like enzymes in the larvae of Tuta absoluta at different developmental instars.

Evaluation of critical parameters for in vitro skin permeation and penetration studies using animal skin models.

In vitro skin permeation/penetration studies may be affected by many sources of variation. Herein, we aimed to investigate the major critical procedures of in vitro skin delivery studies. These experiments were performed with model drugs according to official guidelines. The influence of skin source on penetration studies was studied as well as the use of a cryopreservation agent on skin freezing evaluated by transepidermal water loss, electrical resistance, permeation/penetration profiles and histological changes of the skin. The best condition for tape stripping procedure was validated through the evaluation of the distribution of corneocytes, mass of stratum corneum (SC) removed and amount of protein removed using finger pressure, a 2kg weight and a roller. The interchangeability of the tape stripping procedures followed by the epidermis and dermis homogenate and the micrometric horizontal cryostat skin sectioning methods were also investigated, besides the effect of different formulations. Noteworthy, different skin sources were able to ensure reliable interchangeability for in vitro permeation studies. Furthermore, an increased penetration was obtained for stored frozen skin compared to fresh skin, even with the addition of a cryoprotectant agent. The best method for tape stripping was the finger pressure followed by the addition of a propylene glycol solvent leading to better SC removal. Finally, no significant difference was found in skin penetration studies performed by different methods suggesting their possible interchangeability.

Intra-operative Diagnosis of Breast Lesions by Imprint Cytology and Frozen Section with Histopathological Correlation.

INTRODUCTION: The most common malignant tumour among women is breast carcinoma. Early lesions of the breast including carcinoma are potentially curable if treated properly in the era of breast conservative therapy. For this purpose different diagnostic strategies are in practice. Intra operative Imprint Cytology (IC) and Frozen Section (FS) have a role in spite of the intense recognition of aspiration cytology in the following settings: inconclusive cytological/core biopsy findings; for evaluation of lumpectomy margins and intra operative nodal status. Both IC and FS are dependable intra operative diagnostic consultation modalities and provide accurate results in minutes thereby making appropriate therapeutic decision. AIM: To study the IC and FS features of breast lesions with correlation of final Histopathologic (HP) diagnosis and to determine its diagnostic accuracy. MATERIALS AND METHODS: This was a descriptive cross-sectional study conducted in the Department of Pathology, JSS Medical College, JSS University, Mysuru over a period of two years (October 2011-September 2013). A total of 62 cases of surgically resected breast specimens were evaluated for features of IC, FS and correlation with HP diagnosis. After describing gross features, representative tissue fragment was taken, three imprint smears were made and same tissue fragment was subjected to FS and later for routine HP processing. Slides prepared by IC, FS were interpreted and later correlated with HP diagnosis. Statistical analysis was done with SPSS for windows (version 16.0) by applying appropriate tests. RESULTS: Out of 62 cases, 33(53.23%) were malignant and 29(46.77%) were benign. Sensitivity, specificity and accuracy of IC were 100%, 96.43% and 98.36% and for FS were 100%, 96.55% and 98.39% respectively. p-value for both were <0.001 indicating significant correlation with HP study. CONCLUSION: IC is a simple, accurate, rapid and cost effective diagnostic tool intra operatively where services for FS are not available. It preserves crisp cellular and nuclear detail. FS tissue architecture strongly approximates permanent HP sections but is frequently hampered by freezing artifact. But FS is able to differentiate carcinoma in situ and infiltrative lesions from benign breast lesions. Thus, IC and FS together offer a more reliable diagnosis; hence, it is always useful to prepare both the slides intra operatively. HP study still remains the gold standard in final diagnosis of any breast lesion. In surgical pathology, the correlation of intra operative IC and FS diagnosis with the final HP diagnosis form an essential part of quality assurance activity.

Optimization and validation of cryostat temperature conditions for trans-reflectance mode FTIR microspectroscopic imaging of biological tissues.

In Fourier transform infrared (FTIR) microspectrocopy, the tissue preparation method is crucial, especially how the tissue is cryo-sectioned prior to the imaging requires special consideration. Having a temperature difference between the cutting blade and the specimen holder of the cryostat greatly affects the quality of the sections. Therefore, we have developed an optimal protocol for cryo-sectioning of biological tissues by varying the temperature of both the cutting blade and the specimen holder. Using this protocol, we successfully cryo-sectioned four different difficult-to-section tissues including white adipose tissue (WAT), brown adipose tissue (BAT), lung, and liver. The optimal temperatures that required to be maintained at the cutting blade and the specimen holder for the cryo-sectioning of WAT, BAT, lung, and liver are (-25, -20 °C), (-25, -20 °C), (-17, -13 °C) and (-15, -5 °C), respectively. The optimized protocol developed in this study produced high quality cryo-sections with sample thickness of 8-10 µm, as well as high quality trans-reflectance mode FTIR microspectroscopic images for the tissue sections. •Use of cryostat technique to make thin sections of biological samples for FTIR microspectroscopy imaging.•Optimized cryostat temperature conditions by varying the temperatures at the cutting blade and specimen holder to obtain high quality sections of difficult-to-handle tissues.•FTIR imaging is used to obtain chemical information from cryo-sectioned samples with no interference of the conventional paraffin-embedding agent and chemicals.

STED Imaging in Drosophila Brain Slices.

Super-resolution microscopy is a very powerful tool to investigate fine cellular structures and molecular arrangements in biological systems. For instance, stimulated emission depletion (STED) microscopy has been successfully used in recent years to investigate the arrangement and colocalization of different protein species in cells in culture and on the surface of specimens. However, because of its extreme sensitivity to light scattering, super-resolution imaging deep inside tissues remains a challenge. Here, we describe the preparation of thin slices from the fruit fly (Drosophila melanogaster) brain, subsequent immunolabeling and imaging with STED microscopy. This protocol allowed us to image small dendritic branches from neurons located deep in the fly brain with improved resolution compared with conventional light microscopy.