Table of Contents
Can a live specimen be used in a laser scanning confocal microscope?
Although LSCM accomplishes the best axial resolution relative to other modes of confocal microscopy, it is not optimal for rapid z imaging of live embryos, as the intense laser beam and long scan times negatively affect live samples.
What is the specimen placed on in a confocal microscope?
Specimens that have three-dimensional structure that is to be studied with the confocal microscope, have to be mounted in such a way as to preserve the structure. Some sort of spacer, such as fishing line or a piece of coverslip, is commonly placed between the slide and the coverslip to avoid deforming the specimen.
How do you prepare slides for confocal microscopy?
Pour off the media, wash each chamber twice quickly with 0.5 mL PBS, and then fix the cells by adding 0.5 mL of 4% PFA to each chamber and incubating for 15 min. 4. Wash the cells three times in 0.5 mL PBT (per chamber), 5 min each, and then incubate the cells for 30 min. in 0.5 mL of 1% BSA.
What is the main principle of confocal microscopy?
The basic principle of confocal microscopy is that the illumination and detection optics are focused on the same diffraction-limited spot, which is moved over the sample to build the complete image on the detector.
What would you use a confocal microscope for?
As a distinctive feature, confocal microscopy enables the creation of sharp images of the exact plane of focus, without any disturbing fluorescent light from the background or other regions of the specimen. Therefore, structures within thicker objects can be conveniently visualized using confocal microscopy.
Why the specimen should be prepared properly before microscopic examination?
Proper preparation of metallographic specimens is a key step to determine the accurate microstructure of a material and often requires sectioning, mounting, course grinding, fine grinding, polishing, etching and microscopic examination.
What is confocal microscope and its uses?
Confocal microscopy is a versatile imaging method, through which viewing cells and tissues is made possible. It is a major improvement from conventional light microscopy, as it also helps create images in 3D. The term ‘confocal’ is used when two lenses are arranged to focus on the same point.
How do you focus a confocal microscope?
Use the control pad and joystick to the left of the microscope to move the slide and select the area of interest. Focus the microscope by turning the focus knob, on the right of the microscope.
What are the steps of specimen preparation?
There are 5 steps for the preparation of samples:
- Fixation. Fixation is carried out immediately after the removal of the sample to be observed.
- Embedding. Embedding is the step that follows fixation in a fixative solution.
- Sectioning. Sectioning is performed using microtomy or cryotomy.
- Staining and immunolabeling.
- Mounting.
How do you prepare a specimen for a scanning electron microscope?
SEM sample preparation techniques
- Step 1: Primary fixation with aldehydes (proteins)
- Step 2: Secondary fixation with osmium tetroxide (lipids)
- Step 3: Dehydration series with solvent (ethanol or acetone)
- Step 4: Drying.
- Step 5: Mounting on a stub.
- Step 6: Sputter coating with conductve material.
What are the specimen preparation techniques?
Specimen Preparation
- Focused Ion Beam.
- Transmission Electron Microscopy.
- Atom Probe Tomography.
- Electron Microscope.
What is a modified confocal laser scanning microscope for intracellular activity imaging?
A modified confocal laser scanning microscope allows fast ultraviolet ratio imaging of intracellular Ca2+ activity using Fura 2, Euro. J. Physiologie, Vol. 433: pp. 653-663, (1997) 10. Oldenbourg,\bR.\bet\bal.,\b Image sharpness and contrast transfer in coherent confocal microscopy, Journal of Microscopy Vol. 172, pp. 31-39, (10/1993)
What is the purpose of a laser scanning confocal microscope?
Abstract 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.
What is the best approach in developing a new confocal microscope protocol?
The best approach in developing a new protocol for a specimen to be imaged with the confocal microscope is to begin with one known to be appropriate for conventional microscopy, and to modify it as necessary.
What are the basic principles of confocal microscopy?
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 are introduced including single optical sections, multiple wavelength images, three-dimensional reconstruc- tions, and living cell and tissue sequences.