Table of Contents
What type of image does a confocal microscope produce?
In laser scanning confocal microscopy, the image of an extended specimen is generated by scanning the focused beam across a defined area in a raster pattern controlled by two high-speed oscillating mirrors driven by galvanometer motors.
How can I make a good confocal image?
I’ve put together some top tips to get your confocal images looking the best they can.
- Selecting Your X/Y Resolution.
- Selecting Your Z-step Resolution.
- Avoiding Crosstalk in Confocal Images.
- Ensuring Two Signals are Really Colocalized in Your Confocal Images.
- Controlling For Vibration.
- Coping with Condensation.
What is confocal imaging used for?
Confocal microscopy is widely used for fluorescence imaging in the life sciences. The last decade has seen advances in illumination sources, detectors, fluorescent probes, optics, and sample preparation techniques, which provide improvements in different combinations of speed, depth, and resolution.
What is the difference between epifluorescence and fluorescence?
Epifluorescence illumination or epi-illumination In comparison to other forms of fluorescence microscopy, epifluorescence illumination has the advantage of only requiring a small amount of emitted light to be blocked.
How do confocal microscopes produce 3D images?
By scanning several thin sections through the sample, it’s possible to create a clean 3D image formation of the sample. Using confocal microscopy, it’s possible to see visual sections of small structures that would be nearly impossible to section and build 3D structures from the resulting images.
Why are my confocal images grainy?
However, at lower probe concentrations, light intensity declines rapidly and the resulting confocal images appear more grainy due to the lowered signal level (Figure 1(b)).
What are the applications of confocal microscope?
Applications of Confocal Microscopy
- Stem cell research.
- Photobleaching studies.
- Fluorescence resonance energy transfer (FRET)
- Fluorescence recovery after photo-bleaching.
- Fluorescence in-situ hybridization.
- Lifetime imaging.
- Multiphoton microscopy.
- Total internal reflection fluorescence microscopy (TIRFM)
How does confocal microscopy differs from epifluorescence microscopy?
The key difference between fluorescence microscopy and confocal microscopy is that in fluorescence microscopy, the entire specimen is flooded evenly in light from a light source, whereas in confocal microscopy, only some points of the specimen are exposed to light from a light source.
What is epifluorescence microscopy used for?
Epifluorescence microscopy is widely used in cell biology as the illumination beam penetrates the full depth of the sample, allowing easy imaging of intense signals and co-localization studies with multi-colored labeling on the same sample.
What is the resolution of confocal microscope?
When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches.
How do I reduce background in confocal microscopy?
The practical strategy for dealing with excessive background noise in the confocal fluorescence microscope configuration is to reduce the size of the detector aperture in order to exclude more of the background noise, thereby increasing both the signal-to-background and signal-to-noise ratios.
How does confocal microscopy improve resolution?
The resolution of confocal microscopy relies on the pinhole diameter, that is, higher resolution comes from the smaller sized pinhole filter. Such a small pinhole rejects the unwanted out‐of‐focus light, while parts of the desired in‐focus emission are filtered out simultaneously.
How deep can confocal images be?
0-1000 µm
Confocal imaging: 0-1000 µm beneath specimen surface For every specimen, there is a depth at which scattering, photobleaching and noise occur at such high levels that traditional fluorescence techniques are no longer effective.