Laser Capture Microdissection of Vascular Endothelial Cells from Frozen Heart Tissues.

Laser capture microdissection (LCM) enables researchers to selectively evaluate gene expression profiling of a specific cell type within a tissue. Vascular endothelial cells (EC) line the inside of vessel lumen and play an essential role in new blood vessel formation. It remains a challenge to determine vascular ECs-specific genes expression in vivo. Here, we described a method to dissect vascular ECs from the frozen heart tissue by LCM. The total RNA or proteins are then extracted from the ECs for further analysis.

Laser Capture Microdissection-Liquid Vortex Capture Mass Spectrometry Metabolic Profiling of Single Onion Epidermis and Microalgae Cells.

Laser capture microdissection is a valuable technique in individually isolating single cells whether in tissue networks or deposited from a cell suspension. New developments have enabled coupling of laser capture microdissection with mass spectrometry via liquid vortex capture sampling probe. This enables online metabolic profiling of sectioned cells. Here, we describe the protocol used to deposit, isolate, and individually chemically characterize single Allium cepa and Chlamydomonas reinhardtii cells by laser capture microdissection-liquid vortex capture mass spectrometry.

Plant-Associated Microbes Alter Root Growth by Modulating Root Apical Meristem.

Rhizobacteria are known to produce a variety of signal molecules which may modify plant growth by interfering with phytohormone balance. Among the microbial signals are phytohormones, known to contribute to plant endogenous pool of phytohormones. The current chapter describes different methods to study the regulation of gene expression in root apical meristem in response to rhizobacterial inoculation. We describe protocol for the detection of in planta modulation of CKs and IAA by rhizobacteria and their impact on root growth, dissecting the underlying plant signaling pathway by RNA sequencing.

Laser Capture Microdissection on Frozen Sections for Extraction of High-Quality Nucleic Acids.

Many cancers harbor a large fraction of nonmalignant stromal cells intermixed with neoplastic tumor cells. While single-cell transcriptional profiling methods have begun to address the need to distinguish biological programs in different cell types, such methods do not enable the analysis of spatial information available through histopathological examination. Laser capture microdissection offers a means to separate cellular samples based on morphological criteria. We present here an optimized method to retrieve intact RNA from laser capture microdissected tissue samples, using pancreatic ductal adenocarcinoma as an example, in order to separately profile tumor epithelial and stromal compartments. This method may also be applied to nonmalignant tissues to isolate cellular samples from any morphologically identifiable structure.

Applications of Single-Cell Sequencing for Multiomics.

Single-cell sequencing interrogates the sequence or chromatin information from individual cells with advanced next-generation sequencing technologies. It provides a higher resolution of cellular differences and a better understanding of the underlying genetic and epigenetic mechanisms of an individual cell in the context of its survival and adaptation to microenvironment. However, it is more challenging to perform single-cell sequencing and downstream data analysis, owing to the minimal amount of starting materials, sample loss, and contamination. In addition, due to the picogram level of the amount of nucleic acids used, heavy amplification is often needed during sample preparation of single-cell sequencing, resulting in the uneven coverage, noise, and inaccurate quantification of sequencing data. All these unique properties raise challenges in and thus high demands for computational methods that specifically fit single-cell sequencing data. We here comprehensively survey the current strategies and challenges for multiple single-cell sequencing, including single-cell transcriptome, genome, and epigenome, beginning with a brief introduction to multiple sequencing techniques for single cells.

The Colorectal Cancer Microenvironment: Strategies for Studying the Role of Cancer-Associated Fibroblasts.

Colorectal cancer (CRC) is a key public health concern and the second highest cause of cancer related death in Western society. A dynamic interaction exists between CRC cells and the surrounding tumor microenvironment, which can stimulate not only the development of CRC, but its progression and metastasis, as well as the development of resistance to therapy. In this chapter, we focus on the role of fibroblasts within the CRC tumor microenvironment and describe some of the key methods for their study, as well as the evaluation of dynamic interactions within this biological ecosystem.

Quantitative Proteomic Analysis of Mass Limited Tissue Samples for Spatially Resolved Tissue Profiling.

Traditionally, proteomic studies have been carried out on whole tissues or organs enabling the profiling of thousands of proteins within a single LC-MS analysis. A disadvantage of this approach is that proteomes generated from whole tissues are an "average" that represents a blend of cell types and distinct anatomical regions which can obscure important biological phenomena. Laser capture microdissection (LCM) is an elegant method that allows tissue features of interest, as small as a single cell, to be identified and isolated for downstream analysis. Herein we describe an approach that utilizes an immobilized enzyme reactor (IMER) coupled directly to nanoLC-MS/MS for highly sensitive, automated, quantitative proteomic analysis of the microscopic tissue specimens generated by LCM.

Laser Capture Microdissection of Tissue Sections for High-Throughput RNA Analysis.

The heterogeneous nature of most human organs and tissues represents a common challenge when analyzing specific structures or cells. Laser capture microdissection (LCM) enables isolation of pure cells from a mixed population of cells or tissue samples via usage of laser energy. Combined with high-throughput gene or protein techniques, compartment specific analysis elucidating the role of specialized cell types in physiological or pathophysiological activity can be performed. This chapter describes the crucial steps that have to be taken into consideration when designing and conducting a LCM project. Detailed protocols describing the workflow from project planning to high-throughput analysis of LCM material used in our laboratory are provided. Routinely occurring challenges and appropriate solutions, e.g., when working with fibrotic tissue are described.

Using Laser Capture Microdissection to Isolate Cortical Laminae in Nonhuman Primate Brain.

Laser capture microdissection (LCM) is a technique that allows procurement of an enriched cell population from a heterogeneous tissue sample under direct microscopic visualization. Fundamentally, laser capture microdissection consists of three main steps: (1) visualizing the desired cell population by microscopy, (2) melting a thermolabile polymer onto the desired cell populations using infrared laser energy to form a polymer-cell composite (capture method) or photovolatizing a region of tissue using ultraviolet laser energy (cutting method), and (3) removing the desired cell population from the heterogeneous tissue. In this chapter, we discuss the infrared capture method only. LCM technology is compatible with a wide range of downstream applications such as mass spectrometry, DNA genotyping and RNA transcript profiling, cDNA library generation, proteomics discovery, and signal pathway mapping. This chapter profiles the ArcturusXT™ laser capture microdissection instrument, using isolation of specific cortical lamina from nonhuman primate brain regions, and sample preparation methods for downstream proteomic applications.

Laser microdissection: A promising tool for exploring microorganisms and their interactions with hosts.

Laser microdissection is a method that allows for the isolation of homogenous cell populations from their native niches in tissues for downstream molecular assays. This method is widely used for genomic analysis, gene expression profiling and proteomic and metabolite assays in various fields of biology, but it remains an uncommon approach in microbiological research. In spite of the limited number of publications, laser microdissection was shown to be an extremely useful method for studying host-microorganism interactions in animals and plants, investigating bacteria within biofilms, identifying uncultivated bacteria and performing single prokaryotic cell analysis. The current paper describes the methodological aspects of commercially available laser microdissection instruments and representative examples that demonstrate the advantages of this method for resolving a variety of issues in microbiology.

Laser dissection sampling modes for direct mass spectral analysis.

RATIONALE: Laser microdissection coupled directly with mass spectrometry provides the capability of on-line analysis of substrates with high spatial resolution, high collection efficiency, and freedom on shape and size of the sampling area. Establishing the merits and capabilities of the different sampling modes that the system provides is necessary in order to select the best sampling mode for characterizing analytically challenging samples. METHODS: The capabilities of laser ablation spot sampling, laser ablation raster sampling, and laser 'cut and drop' sampling modes of a hybrid optical microscopy/laser ablation liquid vortex capture electrospray ionization mass spectrometry system were compared for the analysis of single cells and tissue. RESULTS: Single Chlamydomonas reinhardtii cells were monitored for their monogalactosyldiacylglycerol (MGDG) and diacylglyceryltrimethylhomo-Ser (DGTS) lipid content using the laser spot sampling mode, which was capable of ablating individual cells (~4-15 µm) even when agglomerated together. Turbid Allium Cepa cells (~150 µm) having unique shapes difficult to precisely measure using the other sampling modes could be ablated in their entirety using laser raster sampling. Intact microdissections of specific regions of a cocaine-dosed mouse brain tissue were compared using laser 'cut and drop' sampling. Since in laser 'cut and drop' sampling whole and otherwise unmodified sections are captured into the probe, 100% collection efficiencies were achieved. Laser ablation spot sampling has the highest spatial resolution of any sampling mode, while laser ablation raster sampling has the highest sampling area adaptability of the sampling modes. CONCLUSIONS: Laser ablation spot sampling has the highest spatial resolution of any sampling mode, useful in this case for the analysis of single cells. Laser ablation raster sampling was best for sampling regions with unique shapes that are difficult to measure using other sampling modes. Laser 'cut and drop' sampling can be used for cases where the highest sensitivity is needed, for example, monitoring drugs present in trace amounts in tissue.

Tissue Microdissection.

The new opportunities of modern assays of molecular biology can only be exploited fully if the results can be accurately correlated to the tissue phenotype under investigation. This is a general problem of non-in situ techniques, whereas results from in situ techniques are often difficult to quantify. The use of bulk tissue, which is not precisely characterized in terms of histology, has long been the basis for molecular analysis. It has, however, become apparent, that this simple approach is not sufficient for a detailed analysis of molecular alterations, which might be restricted to a specific tissue phenotype (e.g., tumor or normal tissue, stromal or epithelial cells). Microdissection is a method to provide minute amounts of histologically characterized tissues for molecular analysis with non-in situ techniques and has become an indispensable research tool. If tissue diversity is moderate and negligible, manual microdissection can be an easy and cost-efficient method of choice. In contrast, the advantage of laser microdissection is a very exact selection down to the level of a single cell, but often with a considerable time exposure to get enough material for the following analyses. The latter issue and the method of tissue preparation needed for laser microdissection are the main problems to solve if RNA, highly sensitive to degradation, shall be analyzed. This chapter focuses on optimized procedures for manual microdissection and laser microdissection to analyze RNA of malignant and nonmalignant prostate tissue.

Single-Cell Isolation and Gene Analysis: Pitfalls and Possibilities.

During the last two decades single-cell analysis (SCA) has revealed extensive phenotypic differences within homogenous cell populations. These phenotypic differences are reflected in the stochastic nature of gene regulation, which is often masked by qualitatively and quantitatively averaging in whole tissue analyses. The ability to isolate transcripts and investigate how genes are regulated at the single cell level requires highly sensitive and refined methods. This paper reviews different strategies currently used for SCA, including harvesting, reverse transcription, and amplification of the RNA, followed by methods for transcript quantification. The review provides the historical background to SCA, discusses limitations, and current and future possibilities in this exciting field of research.

A microchannel device tailored to laser axotomy and long-term microelectrode array electrophysiology of functional regeneration.

We designed a miniaturized and thin polydimethylsiloxane (PDMS) microchannel device compatible with commercial microelectrode array (MEA) chips. It was optimized for selective axonal ablation by laser microdissection (LMD) to investigate the electrophysiological and morphological responses to a focal injury in distinct network compartments over 45 days in vitro (45 DIV). Low-density cortical or hippocampal networks (<3500 neurons per device) were cultured in quasi-closed somal chambers. Their axons were selectively filtered through neurite cavities and guided into the PDMS microchannels aligned over the recording electrodes. The device geometries amplified extracellularly recorded signals in the somal reservoir and the axonal microchannels to detectable levels. Locally extended areas along the microchannel, so-called working stations, forced axonal bundles to branch out and thereby allowed for their repeatable and controllable local, partial or complete dissections. Proximal and distal changes in the activity and morphology of the dissected axons were monitored and compared to those of their parent networks and of intact axons in the control microchannels. Microscopy images confirmed progressive anterograde degeneration of distal axonal segments over four weeks after surgery. Dissection on cortical and hippocampal axons revealed different cell type- and age-dependent network responses. At 17 DIV, network activity increased in both the somal and proximal microchannel compartments of the dissected hippocampal or cortical axons. At later days (24 DIV), the hippocampal networks were more susceptible to axonal injury. While their activity decreased, that in the cortical cultures actually increased. Subsequent partial dissections of the same axonal bundles led to a stepwise activity reduction in the distal hippocampal or cortical axonal fragments. We anticipate that the MEA-PDMS microchannel device for the combined morphological and electrophysiological study of axonal de- and regeneration can be easily merged with other experimental paradigms like molecular or pharmacological screening studies.

Application of tissue mesodissection to molecular cancer diagnostics.

AIMS: To demonstrate clinical application of a mesodissection platform that was developed to combine advantages of laser-based instrumentation with the speed/ease of manual dissection for automated dissection of tissue off standard glass slides. METHODS: Genomic analysis for KRAS gene mutation was performed on formalin fixed paraffin embedded (FFPE) cancer patient tissue that was dissected using the mesodissection platform. Selected reaction monitoring proteomic analysis for quantitative Her2 protein expression was performed on FFPE patient tumour tissue dissected by a laser-based instrument and the MilliSect instrument. RESULTS: Genomic analysis demonstrates highly confident detection of KRAS mutation specifically in lung cancer cells and not the surrounding benign, non-tumour tissue. Proteomic analysis demonstrates Her2 quantitative protein expression in breast cancer cells dissected manually, by laser-based instrumentation and by MilliSect instrumentation (mesodissection). CONCLUSIONS: Slide-mounted tissue dissection is commonly performed using laser-based instruments or manually scraping tissue by scalpel. Here we demonstrate that the mesodissection platform as performed by the MilliSect instrument for tissue dissection is cost-effective; it functions comparably to laser-based dissection and which can be adopted into a clinical diagnostic workflow.

Application of laser microdissection ICP-MS for high resolution elemental mapping in mouse brain tissue: a comparative study with laser ablation ICP-MS.

Mapping of elements in biological tissue by laser induced mass spectrometry is a fast growing analytical methodology in life sciences. This method provides a multitude of useful information of metal, nonmetal, metalloid and isotopic distribution at major, minor and trace concentration ranges, usually with a lateral resolution of 12-160 µm. Selected applications in medical research require an improved lateral resolution of laser induced mass spectrometric technique at the low micrometre scale and below. The present work demonstrates the applicability of a recently developed analytical methodology - laser microdissection associated to inductively coupled plasma mass spectrometry (LMD ICP-MS) - to obtain elemental images of different solid biological samples at high lateral resolution. LMD ICP-MS images of mouse brain tissue samples stained with uranium and native are shown, and a direct comparison of LMD and laser ablation (LA) ICP-MS imaging methodologies, in terms of elemental quantification, is performed.

SIVQ-LCM protocol for the ArcturusXT instrument.

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process. It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

Identification of a microscopically selected microorganism in milk samples.

Identification of unwanted microbial contaminants microscopically observed in food products is challenging due to their low abundance in a complex matrix, quite often containing other microorganisms. Therefore, a selective identification method was developed using laser capture microdissection in combination with direct-captured cell PCR. This procedure was validated with Geobacillus stearothermophilus and further used to identify microbial contaminants present in some industrial milk samples. The microscopically observed contaminants were identified as mainly Methylobacterium species.

Optimizing Frozen Sample Preparation for Laser Microdissection: Assessment of CryoJane Tape-Transfer System®.

Laser microdissection is an invaluable tool in medical research that facilitates collecting specific cell populations for molecular analysis. Diversity of research targets (e.g., cancerous and precancerous lesions in clinical and animal research, cell pellets, rodent embryos, etc.) and varied scientific objectives, however, present challenges toward establishing standard laser microdissection protocols. Sample preparation is crucial for quality RNA, DNA and protein retrieval, where it often determines the feasibility of a laser microdissection project. The majority of microdissection studies in clinical and animal model research are conducted on frozen tissues containing native nucleic acids, unmodified by fixation. However, the variable morphological quality of frozen sections from tissues containing fat, collagen or delicate cell structures can limit or prevent successful harvest of the desired cell population via laser dissection. The CryoJane Tape-Transfer System®, a commercial device that improves cryosectioning outcomes on glass slides has been reported superior for slide preparation and isolation of high quality osteocyte RNA (frozen bone) during laser dissection. Considering the reported advantages of CryoJane for laser dissection on glass slides, we asked whether the system could also work with the plastic membrane slides used by UV laser based microdissection instruments, as these are better suited for collection of larger target areas. In an attempt to optimize laser microdissection slide preparation for tissues of different RNA stability and cryosectioning difficulty, we evaluated the CryoJane system for use with both glass (laser capture microdissection) and membrane (laser cutting microdissection) slides. We have established a sample preparation protocol for glass and membrane slides including manual coating of membrane slides with CryoJane solutions, cryosectioning, slide staining and dissection procedure, lysis and RNA extraction that facilitated efficient dissection and high quality RNA retrieval from CryoJane preparations. CryoJane technology therefore has the potential to facilitate standardization of laser microdissection slide preparation from frozen tissues.

Laser capture microdissection: should an ultraviolet or infrared laser be used?

Laser capture microdissection (LCM) is a well-established cell separation technique. It combines microscopy with laser beam technology and allows targeting of specific cells or tissue regions that need to be separated from others. Consequently, this biological material can be used for genome or transcriptome analyses. Appropriate methods of sample preparation, however, are crucial for the success of downstream molecular analysis. The aim of this study was to objectively compare the two main LCM systems, one based on an ultraviolet (UV) laser and the other based on an infrared (IR) laser, on different criteria ranging from user-friendliness to sample quality. The comparison was performed on two types of samples: peripheral blood mononuclear cells and blastocysts. The UV laser LCM system had several advantages over the IR laser LCM system. Not only does the UV system allow faster and more precise sample collection, but also the obtained samples-even single cell samples-can be used for DNA extraction and downstream polymerase chain reaction (PCR) applications. RNA-based applications are more challenging for both LCM systems. Although sufficient RNA can be extracted from as few as 10 cells for reverse transcription quantitative PCR (RT-qPCR) analysis, the low RNA quality should be taken into account when designing the RT-qPCR assays.