Simultaneous Polychromatic Immunofluorescent Staining of Tissue Sections and Consecutive Imaging of up to Seven Parameters by Standard Confocal Microscopy.

Confocal microscopy has been an important imaging tool for life scientists for over 20 years. Early techniques focused on indirect staining processes that involved staining with an unconjugated primary antibody, followed by incubation with a secondary fluorescent antibody that would reveal and amplify the signal of the primary antibody. With more and more directly conjugated fluorescent primary antibodies becoming commercially available, staining with multiple fluorescent primary antibodies is now more frequent. To date, staining with up to three primary antibodies and a nuclear dye is widely practiced. Here, we describe an important improvement to the standard polychromatic immunofluorescent staining protocol that allows the simultaneous detection of seven fluorescent parameters using a standard confocal laser scanning microscope with four laser lines and four photomultiplier tubes. By incorporating recently available tandem dyes that emit in the blue and violet regions of the visible light spectrum (Brilliant Blue and Brilliant Violet), we were able to differentiate several additional fluorochromes simultaneously. Due to the added complexity of 7-color immunofluorescent imaging, we developed a clear methodology to optimize antibody concentrations and simple guidelines on how to identify and correct non-specific signals. These are detailed in the following protocol. © 2019 by John Wiley & Sons, Inc. Basic Protocol: 7-Color immunofluorescent staining protocol using directly conjugated antibodies Support Protocol 1: Antibody titration protocol Support Protocol 2: Spillover optimization protocol.

Immediate Label-Free Ex Vivo Evaluation of Human Brain Tumor Biopsies With Confocal Reflectance Microscopy.

Confocal microscopy utilizing fluorescent dyes is widely gaining use in the clinical setting as a diagnostic tool. Reflectance confocal microscopy is a method of visualizing tissue specimens without fluorescent dyes while relying on the natural refractile properties of cellular and subcellular structures. We prospectively evaluated 76 CNS lesions with confocal reflectance microscopy (CRM) to determine cellularity, architecture, and morphological characteristics. A neuropathologist found that all cases showed similar histopathological features when compared to matched hematoxylin and eosin-stained sections. RNA isolated from 7 tissues following CRM imaging retained high RNA integrity, suggesting that CRM does not alter tissue properties for molecular studies. A neuropathologist and surgical pathologist masked to the imaging results independently evaluated a subset of CRM images. In these evaluations, 100% of images reviewed by the neuropathologist and 95.7% of images reviewed by the surgical pathologist were correctly diagnosed as lesional or nonlesional. Furthermore, 97.9% and 91.5% of cases were correctly diagnosed as tumor or not tumor by the neuropathologist and surgical pathologist, respectively, while 95.8% and 85.1% were identified with the correct diagnosis. Our data indicate that CRM is a useful tool for rapidly screening patient biopsies for diagnostic adequacy, molecular studies, and biobanking.

Gastroenteropancreatic neuroendocrine tumors: indications for and pitfalls of frozen section examination.

Gastroenteropancreatic neuroendocrine tumors constitute a heterogeneous group of neoplasms. Surgical resection remains the only curative treatment. Frozen section examination is requested by the surgeon in a large variety of surgical situations, but its use differs greatly according to the location of the tumor and the type of surgery performed. The objective of this review is to describe the main indications for and pitfalls of frozen section examination of gastroenteropancreatic neuroendocrine tumors.

Fiducial-less alignment of cryo-sections.

Cryo-electron tomography of vitreous sections is currently the most promising technique for visualizing arbitrary regions of eukaryotic cells or tissue at molecular resolution. Despite significant progress in the sample preparation techniques over the past few years, the three dimensional reconstruction using electron tomography is not as simple as in plunge frozen samples for various reasons, but mainly due to the effects of irradiation on the sections and the resulting poor alignment. Here, we present a new algorithm, which can provide a useful three-dimensional marker model after investigation of hundreds to thousands of observations calculated using local cross-correlation throughout the tilt series. The observations are chosen according to their coherence to a particular model and assigned to virtual markers. Through this type of measurement a merit figure can be calculated, precisely estimating the quality of the reconstruction. The merit figures of this alignment method are comparable to those obtained with plunge frozen samples using fiducial gold markers. An additional advantage of the algorithm is the implicit detection of areas in the sections that behave as rigid bodies and can thus be properly reconstructed.

Can you trust your cryostat? Reproducibility of cryostat section thickness.

Reproducibility of cryostat section thickness is required for valid quantitative microscopy. This is generally pursued by motorized sectioning using a low but constant speed. The purpose of our study was to compare variation in section thickness between motorized and manual cryostat sectioning. Serial sections were cut from a frozen block of homogenized tissue on different days. Lactate dehydrogenase activity was histochemically detected and calibrated absorbance measurements were taken. The coefficients of variation of measurements was 9.7% for motorized sectioning and 3.3% for manual sectioning. In conclusion, section thickness is similarly reproducible after manual sectioning compared with motorized sectioning, if not better.

Visible Korean human: improved serially sectioned images of the entire body.

The data from the Visible Human Project (VHP) and the Chinese Visible Human (CVH), which are the serially sectioned images of the entire cadaver, are being used to produce three-dimensional (3-D) images and software. The purpose of our research, the Visible Korean Human (VKH), is to produce an enhanced version of the serially sectioned images of an entire cadaver that can be used to upgrade the 3-D images and software. These improvements are achieved without drastically changing the methods developed for the VHP and CVH; thus, a complementary solution was found. A Korean male cadaver was chosen without anything perfused into the cadaver; the entire body was magnetic resonance (MR) and computed tomography (CT) scanned at 1.0-mm intervals to produce MR and CT images. After scanning, entire body of the cadaver was embedded and serially sectioned at 0.2-mm intervals; each sectioned surface was inputted into a personal computer to produce anatomical images (pixel size: 0.2 mm) without any missing images. Eleven anatomical organs in the anatomical images were segmented to produce segmented images. The anatomical and segmented images were stacked and reconstructed to produce 3-D images. The VKH is an ongoing research; we will produce a female version of the VKH and provide more detailed segmented images. The data from the VHP, CVH, and VKH will provide valuable resources to the medical image library of 3-D images and software in the field of medical education and clinical trials.

Reference sample method reduces the error caused by variable cryosection thickness in Fourier transform infrared imaging.

Fourier transform infrared imaging (FT-IRI) is a novel technique for characterization of the biochemical composition of biological tissues, e.g., articular cartilage. The use of cryosections is preferred in FT-IRI. Unfortunately, significant variation in section thickness often impairs the suitability of cryosections for quantitative FT-IRI analysis. The present study introduces an inexpensive reference sample method for quantitative analysis. In this technique, specimen absorption is normalized with that of nitrocellulose membrane embedded and cryosectioned with the sample. Mean variation of the infrared absorption in cartilage specimens was 11.5%, 12.1%, and 20.6% for 5 microm, 10 microm, and 14 microm thick sections, respectively, without normalization. Normalization reduced the variation to 5.2%, 4.0%, and 4.6% for the same sections, respectively. The normalization method enables usage of cryosections for quantitative work and significantly reduces the cost and time needed for FT-IRI analysis.

Cryoelectron microscopy of refrozen cryosections.

Cryoelectron microscopy makes it possible to record high-resolution detail from large and complex structures. However, its application to understanding cellular structure is limited by the requirement that samples should be no thicker than approximately 0.5-1 microm. Therefore it is important to develop the ability to section biological material so that it can be imaged in its native frozen state. Here we have adapted standard methods of preparing cryosections so that they can be imaged by cryoelectron microscopy. As used for immunolabeling, cryosections of chemically fixed, cryoprotected frozen rat cardiac muscle were thawed, applied to carbon-coated grids, and rinsed on a drop of buffer. The special step here is that the cryosections were then refrozen by being plunged into liquid ethane and imaged at approximately -180 degrees C in a 200-kV field-emission gun electron microscope. The unstained cryosections have good contrast, allowing the identification of optimum regions of the sample. Considerable fine detail is observed within the substructure of the sarcomere A-band and I-band. Fourier transform analysis of the micrographs shows that this method preserves high structural order, hence these sections are well-suited to 3D reconstruction. We conclude that this approach has considerable potential for obtaining intermediate- and high-resolution structural detail from bulk tissue.

Use of touch preps for diagnosis and evaluation of surgical margins in breast cancer.

BACKGROUND: The best cosmetic results with conservative breast surgery are obtained at the time of initial excisional biopsy. The usefulness of the touch prep (TP) technique was evaluated for accuracy in diagnosis as well as in evaluation of margins at the time of original breast biopsy. METHODS: Four hundred twenty-eight consecutive patients with breast masses seen from January 1993 to December 1994 were evaluated prospectively using TP. RESULTS: Three hundred forty-five benign and 83 malignant tumors were evaluated. Tumors ranged in size from microscopic to 8 cm. Pathologic diagnosis was correct as compared to permanent section in 99.3%. The three carcinomas missed on TP were focal and in situ. Sensitivity was 96.39%, and specificity was 100%. Positive predictive value was 100%, and negative predictive value was 99.3%. For margin evaluation, the sensitivity and specificity were both estimated to be 100%. CONCLUSIONS: TP has the advantage of being a simple, quick (2 to 3 minutes), safe (no loss of diagnostic material), and accurate method for diagnosis and estimation of tumor margins at the time of the original surgery.

Application of cryotechniques in cartilage tissue preservation and immunoelectron microscopy: potentials and problems.

Cryotechnical processing of cartilage has the potential to solve many of the tissue-specific problems associated with various routine chemical fixation protocols. This is particularly the case with respect to extracellular matrix architecture, the distortion or destruction of which (caused by extraction and/or precipitation of proteoglycan molecules) may be prevented. Adoption of such techniques also permits high-sensitivity immunoelectron-microscopy of the extracellular matrix space (carbohydrate epitopes). However, a number of difficulties still remain to be resolved, particularly that of matrix-cell interface separation occurring during freeze substitution and low temperature embedding. These problems are briefly addressed and possible solutions outlined.