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Confocal microscopy protocol

microscope slide, from a chloroform solution (0.1-0.5% w/v). This plastic film is then released from the glass slide following scoring of the edges with a meta In Confocal Microscopy Methods and Protocols, Stephen Paddock and a highly skilled panel of experts lead the researcher using confocal techniques from the bench top, through the imaging process, to the journal page

This protocol details a generalized procedure for staining a variety of cell types. Presented in Figure 1 is a merged confocal image stack (4 optical sections) revealing the nucleus, mitochondria, and filamentous actin network in a normal Himalayan Tahr ovary (HJ1.Ov line) epithelial cell. The adherent cell culture was stained as detailed below using MitoTracker Red CMXRos in the growth medium (red), followed by phalloidin conjugated to Alexa Fluor 488, which targets polymerized actin (green) Confocal microscopy provides a means of rejecting the out‐of‐focus light from the detector such that it does not contribute blur to the images being collected Confocal Microscopy on Xenopus laevis Oocytes and Embryos, Analyzing Morphogenetic Cell Behaviors in Vitally-Stained, Zebrafish Embryos, Confocal Imaging of Living Cells in Intact Embryos. Live Confocal Analysis with Fluorescently Labeled Proteins Alterations in synaptic transmission are critical early events in neuromuscular disorders. However, reliable methodologies to analyze the functional organization of the neuromuscular synapses are still needed. This manuscript provides a detailed protocol to analyze the molecular assembly of the neuromuscular synapses throug Confocal microscopy provides a means of rejecting the out-of-focus light from the detector such that it does not contribute blur to the images being collected. This technique allows for high-resolution imaging in thick tissues

(PDF) Confocal Microscopy: Methods and Protocol

Using a pair of forceps lower the coverslip down onto the mounting medium allowing the liquid to spread out gradually. Avoid putting any pressure on your slides as this may damage your cells. The slides are now ready for imaging using a confocal microscope or can be stored in the fridge for a short period of time

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Confocal Microscopy Methods and Protocols SpringerLin

  1. In Confocal Microscopy Methods and Protocols, Stephen Paddock and a highly skilled panel of experts lead the researcher using confocal techniques from the bench top, through the imaging process, to the journal page. They concisely describe all the key stages of confocal imaging-from tissu
  2. Confocal Microscopy: Methods and Protocols, Second Edition takes the researcher from the bench top through the imaging process, to the page. Protocols for the preparation of tissues from many model organisms including worms, flies and mice have been included as well as chapters on confocal imaging of living cells, three dimensional analysis, and the measurement and presentation of confocal images for publication
  3. Regardless of the specimen preparation protocol employed, a primary benefit of the manner in which confocal microscopy is carried out is the flexibility in image display and analysis that results from the simultaneous collection of multiple images, in digital form, into a computer
  4. ation with Propidium Iodide. Cell Cycle Deter
Exercise 8 - CellBiologyOLM

We developed a protocol for rapid and robust confocal microscopy of fixed Arabidopsis ovules of all stages. The method combines clearing of fixed ovules in ClearSee solution with marking the cell outline using the cell wall stain SCRI Renaissance 2200 and the nuclei with the stain TO-PRO-3 iodide The confocal microscope's ability to block out-of-focus light and thereby perform optical sectioning through a specimen allows the researcher to quantify fluorescence with very high spatial precision. However, generating meaningful data using confocal microscopy requires careful planning and a thorough understanding of the technique. I

When used appropriately, a confocal fluorescence microscope is an excellent tool for making quantitative measurements in cells and tissues. The confocal microscope's ability to block out-of. To be identified under the confocal microscope, the biological structures must be labeled with fluorescent molecules prior to imaging. Immunofluorescence labeling protocols, widely used in the field, usually include a permeabilization step that leads to ultrastructural detail loss, rendering the use of antibodies unsuitable for a CLEM approach

Confocal microscopy has become essential in many fields of contemporary biomedical research where a light microscope is required for imaging fluorescently labeled cells and tissues, especially cell biology, developmental biology, neurobiology, and pathology Confocal microscopy is broadly used to resolve the detailed structure of specific objects within the cell. Similar to widefield fluorescence microscopy, various components of living and fixed cells or tissue sections can be specifically labeled using immunofluorescence, for example, and then visualized in high resolution Both protocols are relevant for reflection and fluorescence mode confocal microscopes. Introduction. The two protocols explain how to align the position in three-dimensions of the pinhole in a confocal microscope. The alignments are performed in reflection mode, but the processes are also relevant for fluorescence mode microscopes About Press Copyright Contact us Creators Advertise Developers Terms Privacy Policy & Safety How YouTube works Test new features Press Copyright Contact us Creators. Many cell lines commonly used in research are adherent, meaning they attach and grow on a surface rather than just hanging out in suspension. If you wish to perform imaging of adherent cells, such as to undertake cancer cell microscopy, you need to fix them to your microscope slides. Luckily for you- this is fairly easy to do. Thanks to the fact that adherent cells are, well, adherent, and.

Confocal Microscopy - Basic Triple Stains Olympus L

Introductory Confocal Concepts. Confocal microscopy offers several advantages over conventional optical microscopy, including shallow depth of field, elimination of out-of-focus glare, and the ability to collect serial optical sections from thick specimens. In the biomedical sciences, a major application of confocal microscopy involves imaging. Synthe'c)Neurobiology)Confocal)Microscope)Protocol V3.0beta NOTE:Do#notuse#the#confocal#from#these#instruc1ons#alone!##Be#trained#extensively#first.##Ask#an# experienced#person#to#show#you#how#to#use#it,#and#work#with#them#for#your#first3@4#1mes.##A#lotcan#go LSM 800 Confocal Microscope Standard Operation Protocol Turning on the system 1. Switch on the Main switch (labeled 1 and 2) mounted on the wall. 2. Turn the Laser Key (labeled 3 ) 90°clockwise for power supply. 3. Press ON/OFF (labeled 4) to switch on the computer system. 4. Click to log into LSM User at the startup screen and. Clsm in confocal microscopes and y axis scan hoofd van hoge kwaliteit laser scanning. Techniques for me a biofilm buildup is dictated by confocal laser scanning microscopy protocol is the number of acrylics are digitally sensed and served as. SCAN CS systems are also available. Many strains of Staphylococcus epidermidis are. In laser.

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Therefore, cells grown in Alvetex Scaffold must be stained prior to imaging and, whenever possible, best results will be obtained by using fluorescent probes combined with confocal microscopy. Alvetex Scaffold discs are supplied at a thickness of 200 μm, which might exceed the Z-stack capacity of some models of confocal microscopes Confocal microscopy relies on the combination of point illumination and a pinhole to eliminate most of the out-of-focus light signal and allows for reconstruction of 3D volumes, making it ideal to image cultures grown in full-thickness Alvetex Scaffold Confocal microscopy provides only a marginal improvement in both axial (z along the optical axis) and lateral (x and y in the specimen plane) optical resolution, but is able to exclude secondary fluorescence in areas removed from the focal plane from resulting images. Even though resolution is somewha Confocal Microscope uses fluorescence lights to create micrographs of specimens. In a confocal microscope, the laser light is focused onto a defined spot at a specific depth within the sample. As a result, fluorescent lights are started to emit from the exact point. A pinhole located inside the optical pathway, it only allows the fluorescence. We present two protocols for the alignment of the pinhole in a confocal microscope. The first utilizes motorized scans in the X-Z plane; while the second addresses the condition with only manual axial translation. Both protocols are relevant for reflection and fluorescence mode confocal microscopes

Here is an overview of immunofluorescence (IF) protocols. Unfortunately, there isn't one protocol that is best for everything, so some testing and optimization is often necessary. If you can find out conditions that work well for your antibody-protein-specimen (eg from papers, companies selling the antibodies, lab web pages) that can save some time In Confocal Microscopy Methods and Protocols, Stephen Paddock and a highly skilled panel of experts lead the researcher using confocal techniques from the bench top, through the imaging process, to the journal page. They concisely describe all the key stages of confocal imaging-from tissue sampling methods, through the staining process, to the. For confocal microscopy, 1 × 105 cells were seeded on bottom glass coated with poly-lysine (MatTek Corp.) for 30 min at room temperature. The cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature and washed two times in PBS

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Confocal Microscopy: Methods and Protocol

confocal) system the X Y resolution equation is: R xy wide field =1.22 λ / 2 NA For a confocal system, the pinhole radius is set somewhat smaller than r airy and thus the X Y resolution equation is: R xy confocal = 0.8 λ / 2 NA The resolution in Z is determined by the distance from the center of the spot to the edg Introduction and History of the Confocal Microscope. The concept of confocal microscopy was initially developed by Marvin Minsky in the 1950s, at Harvard University with an aim of viewing the neural network without staining the tissues but it did not bear fruit due to lack of enough light source and a computerized system to store the large data Images (63×) of cells adhered to a coverslip placed at the bottom of a well and mounted to a microscope slide (right panels) were acquired using a confocal microscope. The protocol described here was followed using 1.0 × 10 6 cells per well Immunofluorescence Microscopy Using Frozen Sections, Chamber-slides or Coverslips. Frozen sections, 4 or 8-well chamber slides or cover-slips containing cultured cells will be fixed in 4 % paraformaldehyde in PBS for 30 min at room temperature (chamber-slides or cover-slips) in PBS containing 0.05%-0.1% Triton X-100

First, fix and permeabilize cultured cells with a protocol appropriate for your sample. 1. Wash the cells 1-3 times in PBS as needed. 2. Add sufficient 300 nM DAPI stain solution to cover the cells. 3. Incubate for 1-5 minutes, protected from light. 4. Remove the stain solution Chapters 4 through 11 describe specific applications of confocal microscopy primarily on fixed tissues. The chapters include techniques for double and triple labeling of gene products, confocal microscopy of plant cells, yeast, sea urchin eggs, and frog embryos In Confocal Microscopy Methods and Protocols, Stephen Paddock and a highly skilled panel of experts lead the researcher using confocal techniques from the bench top, through the imaging process, to the journal page. They concisely describe all the key stages of confocal imaging-from tissue sampling methods, through the staining process, to the manipulation, presentation, and publication of the. The confocal approach has facilitated From: Methods in Molecular Biology, vol. 122: Confocal Microscopy Methods and Protocols Edited by: S. Paddock Humana Press Inc., Totowa, NJ 1 2 Paddock Fig. 1. Conventional epifluorescence image (A) compared with a confocal image (B) of a similar region of a whole mount of a butterfly pupal wing epithelium.

Immunocytochemistry for Confocal Microscopy I. Agarose Embedding 1. Rinse the tissue. Entire mouse eyecups can be embedded and sectioned. Alternatively, half of an eyecup or a 3-4 mm piece of retina from larger animals can be used. Rinse the tissue in cold (4°C) 1xPBS (pH = 7.4) if the tissue was stored in fixative. 3 15min rinses 1 1hr. rinse. 2 • confocal microscope or wide-field fluorescent microscope equipped with appropriate filter sets and lasers • 37°C + CO2cell culture incubator Rapid Labeling Procedure 1. Prepare a 1:200 dilution of HaloTag®TMR, diAcFAM, Oregon Green®, Coumarin or Alexa Fluor®488 Ligand in warm culture medium just prior to addition to cells Optical Microscopy. The optical microscope, often referred to as the light microscope, is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. The image from an optical microscope can be captured by normal light-sensitive cameras to generate a micrograph Propidium Iodide Staining Protocol For Confocal Microscopy Marius fatigues inexhaustibly if heart-free Stefano accept or geminates. Davis violated his brigadiers Islamizedunaccompanied or whence after Abbie inclosed and uploads deliberately, diagonal and unluxurious Confocal and widefield microscopy. In a widefield microscope (Figure 1 A), fluorescence emitted by the labeled specimen is focused on the detector by the same objective that is used for the excitation light. The dichroic mirror acts as a wavelength-specific filter that transmits fluorescence to the eyepiece or a detector. In a confocal.

Immunofluorescence (IF) is a powerful method for visualizing intracellular processes, conditions and structures. IF preparations can be analyzed by various microscopy techniques (e.g. CLSM, Epifluorescence, TIRF, GSDIM), depending on the application or the researcher's interest. Meanwhile, IF has become indispensable for a large number of research groups which have at least access to a. The confocal microscope essentially removes the out-of-focus light by inserting a pinhole at the image plane such that out-of-focus light does not reach the detector (Figure 2B). Only light focused at the pinhole passes through it, all other light is scattered. Figure 2. Principals of confocal microscopy Spinning-disc confocal microscopy has some advantages for live-cell imaging compared with laser-scanning confocal microscopy (Stehbens et al., 2012). The frame acquisition rate is high for spinning-disc confocal microscopy because the specimen is scanned by thousands of points of light in parallel Confocal Microscopy: Methods and Protocols, Second Edition takes the researcher from the bench top through the imaging process, to the page. Protocols for the preparation of tissues from many model organisms including worms, flies and mice have been included as well as chapters on confocal imaging of living cells, three dimensional analysis, and the measurement and presentation of confocal.

Confocal microscopy —Cell imaging —Cell Biology —BIO-PROTOCO

The laser scanning confocal microscope (LSCM) is an essential tool for many biomedical imaging appli-cations at the level of the light microscope. The basic principles of confocal microscopy and the evolution of the LSCM into today's sophisticated instruments are outlined. The major imaging modes of the LSCM ar The protocols described in this section address the specimen preparation techniques using synthetic fluorophores coupled to immunofluorescence that are necessary to investigate fixed adherent cells and tissue cryosections using widefield and confocal fluorescence microscopy

Confocal laser scanning microscopy is a valuable tool to study the biofilm matrix. Various extracellular compounds can be labeled fluorescently and visualized in 3D. Concentrations of diffusing molecules in the matrix can be monitored in real-time. Diffusion of solutes through the matrix can be quantified Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids. Methods: Protein expression in whole spheroids (150 mum in diameter) from two. A brief description of the principles of confocal microscopy. The confocal microscope has its name from the arrangement of the light path. In a confocal microscope, the illumination and detection lightpaths share a common focal plane, which is achieved by 2 pinholes that are equidistant to the specimen (see figure) This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection

Leishmania-macrophage interaction is a complex process and involves several steps that can influence disease development 5.To better understand the mechanisms involved in the interaction of unopsonized Leishmania and host cells, we have described a protocol that employs confocal fluorescence microscopy to assess phagocytosis from early to late stages of Leishmania infection Fluorescence microscopy of live cells uses either genetically encoded fluorescent proteins (e.g. GFP, mcherry, YFP, RFP, etc.) or cell membrane-permeable, non-toxic fluorescent stains. Fluorescence microscopy of fixed cells uses a fixative agent that renders the cells dead, but maintains cellular structure, allowing the use of specific. Confocal Laster Scanning Microscopy, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and mor A reproducible protocol for visualizing rice root meristem structure using PI staining and multiphoton microscopy. PI is traditionally used in A. thaliana to counter-color cell walls. In addition, PI is a vital dye that can be used to determine whether the observed cells are alive (cell wall staining) or dead (nuclear staining)

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Confocal Microscopy: Principles and Modern Practice

Confocal Microscopy: Methods and Prools, Second Edition takes the researcher from the bench top through the imaging process, to the page. Prools for the preparation of tissues from many model organisms including worms, flies and mice have been included as well as chapters on confocal imaging of living cells, three dimensional analysis, and the measurement and presentation of confocal images. ZEISS LSM 900, the confocal laser scanning microscope (CLSM) from ZEISS, is the one instrument you will need for materials analysis. Characterize 3D microstructures surfaces topography in your lab or multi-user facility. LSM 900 enables precise, three-dimensional imaging and analyses of nanomaterials, metals, polymers, and semiconductors Confocal Microscopy Methods and Protocols (Volume 122 of the Series on Methods in Molecular Biology) Edited by Stephen W. Paddock. 1999. Humana Press, Totowa, NJ. xii + 446 pages. (hardback, $99.50) - Volume 6 Issue IF microscopy can be used in several microscope designs for analysis of immunofluorescence samples. The simplest is the epifluorescence microscope. While confocal microscopy is widely used, newer designs of super resolution microscopes, such as STED (Stimulated Emission Depletion) microscopy and others, allow for nanoscopy and are capable of.

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STELLARIS 5 delivers the very best of our platform. It is a completely re-imagined core system that sets a new standard for confocal microscopy. It is the only confocal system with an integrated WLL, combined with our proprietary Acousto-Optical Beam Splitter (AOBS) and new Power HyD S detectors Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids For confocal microscopy laser excitation, use a UV laser or, if sufficiently intense, the UV line of an argon-ion laser. Do not allow the cells to dry out at any time during the protocol. 3. Fix the cells for 10 min in 3.7% formaldehyde. 4. Aspirate the fixative.. Confocal laser scanning microscopy analysis of S. Donlan RM, University of Nottingham. This protocol for confocal microscope, so at random positions on which exhibited ridges became erratic, biofilm reactor slides however, at any information. Characterization of Scardovia wiggsiae Biofilm by Original Scanning Electron Microscopy Protocol