Confocal Microscopy

Confocal microscopy can eliminate common fluorescence microscopy issues that arise from out of focus illumination and allow thick specimens (up to roughly 200 nm) to be viewed with high resolution. In confocal microscopy, only one diffraction-limited region of the specimen is illuminated at a time, and a single signal is accepted from that specific region. This illumination technique overcomes issues present in wide field (WF) microscopy where much of the emitted light may appear out of focus.

In design, the confocal microscope is composed of one or more electronic detectors, a multi-laser system, a beam scanning assembly, and an adapted computer and software for sample imaging, analysis, and data storage. Spatial filtering techniques remove blurred components and glares, common elements in images from thicker specimens.

Some advantages of confocal microscopy lie in the ability to finely, optically, section samples, where unwanted light can be removed above and/or below the focal plane. Confocal microscopy can provide better contrast and allow 3D reconstruction by combining the image data from a stack of images using appropriate software. This technique also gives the user the ability to control the depth of field, and to remove or reduce background noise. For these reasons it has become the premier choice of microscopy in the field of immunofluorescence.

 

Limitations of Confocal Microscopy

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Confocal microscopy, however, does not come without limitations. Even though techniques offer less signal degradation, photobleaching is still a significant issue, especially for samples with weaker fluorescent signals. Resolution of images is less than other techniques, and the cost may be 2-7 times more than WF microscopy. The best resolution possible remains around 0.2 μm on the x-axis (worse than WF microscopy) and 0.6um on the y-axis. High resolution of the Z-axis can only be achieved by sacrificing the level of light that reaches the detector.

Similar to WF microscopy, confocal microscopy is also limited by diffraction effects. Image capture is, as well, determined by a number of variables including the technology of the imaging system, image brightness, and the speed of the laser raster that scans the specimen. Because of this, traditional confocal microscopy can only capture 1-10 images per second where dwell time, or the time that excitation light remains in one location in the specimen, may increase considerably.

 

Subcategories of Confocal Microscopy

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Laser Scanning Confocal Microscopy (LSCM)

In LSCM, a focused laser beam controlled by two high-speed oscillating mirrors scans across a sample region in a defined area via a raster pattern. Next, the laser light is scattered and reflected and any fluorescent light from the illuminated spot is received by an objective lens. From here, a device known as a beam splitter separates a portion of the received light and sends it through a filter thereby selectively allowing emission, but not excitation, light to pass. The filtered light passes on to a photodetection device, which then transforms the light into electrical signals, recorded by a computer.

As LSCM sequentially scans excitation at specified points, fluorescence intensities across the specimen can be collected and whole images can be sequentially generated. LSCM has a number of advantages, in that the technology offers programmable sampling densities at very high resolutions. Out-of-focus light is blocked, which increases image resolution and prevents non-target elements from appearing in the background.