Why use confocal

This figure depicts the effect of the pinhole, or iris diaphragm, on the thickness of the optical plane that is collected. The pinhole and focal plane in the sample are at conjugate planes of focus. The small pinhole opening in the diagram on the left enables data collection from a thin optical plane within the specimen. Points that are out of the plane of focus red will have a different secondary focal plane thus, most of the data is deflected.

On the other hand, the widefield microscope carries a risk of high background. There is also the risk of channel-to-channel bleed-through when fluorescent dyes have overlapping spectral profiles. Also, excitation wavelength bands depend on the filter sets available; this can be a limitation. Using a confocal microscope , we can obtain a superior image quality and improve the signal-to-noise ratio.

Due to light scattering, image blurring can be easily removed. Magnification can be adjusted electronically, and stitching across large surfaces of the specimen is also possible.

A confocal microscope gives us a clear examination and 3D reconstruction of thick specimens due to effective Z-axis scanning. However, it can be time-consuming to use a confocal microscope depending on the scanning speed and it has a more complicated image acquisition procedure compared to a widefield microscope. Also, confocal images are only obtained digitally from the PMT detector the signal observed through the ocular lens is a widefield image.

For most uses, a widefield fluorescence microscope is sufficient and provides the best trade-off between quality, speed, ease of use, and cost. Therefore, it is a perfect tool for initial screens of protocols, and for live cell imaging applications where the speed of acquisition offers an advantage over scanning using confocal-based approaches if the signal-to-noise ratio for a particular staining is sufficient. It is generally considered good practice to verify the quality of staining of your samples for confocal microscopy using a widefield microscope first Figure 3.

For example, it is advisable to use a confocal microscope for subcellular localization or protein—protein interaction studies, colocalizations, 3D imaging of thick tissues, or larger surfaces of a specimen stitching between fields. Figure 3. Recent advances in confocal microscopy have made possible multi-dimensional views of living cells and tissues that include image information in 3D over time. This information is presented in multiple colors.

Having temporal data collected from time-lapse experiments or through real-time image acquisition is a powerful tool for cellular biology. The capabilities of confocal microscopy increase as new laser systems are being developed to limit cell damage and as computer processing speeds and storage capacities increase.

The primary advantage of laser scanning confocal microscopy is to produce thin optical sections through fluorescent specimens that have a thickness beyond 50 micrometers. Images are collected by coordinating incremental changes in the microscope fine focus mechanism using a stepper motor with sequential image acquisition at each step. Contrast and definition are greatly improved over other techniques due to reduction of background fluorescence and improved signal-to-noise.

Faster scanning rates to near video speed can be achieved using acousto-optic devices or oscillating mirrors. In contrast, multiple-beam scanning confocal microscopes are equipped with a spinning Nipkow disk containing an array of pinholes and microlenses. These instruments often use arc-discharge lamps for illumination instead of lasers to reduce specimen damage and enhance the detection of low fluorescence levels during real time image collection. Another important feature of the multiple-beam microscopes is their ability to readily capture images with an array detector, such as a charge-coupled device CCD camera system.

All laser scanning confocal microscope designs are centered around a conventional upright or inverted research-level optical microscope. However, instead of the standard tungsten-halogen or mercury arc-discharge lamp, one or more laser systems are used as a light source to excite fluorophores in the specimen.

After a series of images usually serial optical sections has been acquired and stored on digital media, analysis can be conducted utilizing numerous image processing software packages available on the host or a secondary computer. The primary advantage of laser scanning confocal microscopy is the ability to serially produce thin 0. The image series is collected by coordinating incremental changes in the microscope fine focus mechanism using a stepper motor with sequential image acquisition at each step.

Image information is restricted to a well-defined plane, rather than being complicated by signals arising from remote locations in the specimen. Contrast and definition are dramatically improved over widefield techniques due to the reduction in background fluorescence and improved signal-to-noise.

Furthermore, optical sectioning eliminates artifacts that occur during physical sectioning and fluorescent staining of tissue specimens for traditional forms of microscopy. The non-invasive confocal optical sectioning technique enables the examination of both living and fixed specimens under a variety of conditions with enhanced clarity. With most confocal microscopy software packages, optical sections are not restricted to the perpendicular lateral x - y plane, but can also be collected and displayed in transverse planes.

Vertical sections in the x - z and y - z planes parallel to the microscope optical axis can be readily generated by most confocal software programs. Thus, the specimen appears as if it had been sectioned in a plane that is perpendicular to the lateral axis. In practice, vertical sections are obtained by combining a series of x - y scans taken along the z axis with the software, and then projecting a view of fluorescence intensity as it would appear should the microscope hardware have been capable of physically performing a vertical section.

A typical stack of optical sections often termed a z-series through a sunflower pollen grain revealing internal variations in autofluorescence emission wavelengths is illustrated in Figure 6.

Optical sections were gathered in 0. Pollen grains of from this species range between 20 and 40 micrometers in diameter and yield blurred images in widefield fluorescence microscopy see Figure 1 c , which lack information about internal structural details.

Although only 12 of the over 48 images collected through this series are presented in the figure, they represent individual focal planes separated by a distance of approximately 3 micrometers and provide a good indication of the internal grain structure. In specimens more complex than a pollen grain, complex interconnected structural elements can be difficult to discern from a large series of optical sections sequentially acquired through the volume of a specimen with a laser scanning confocal microscope.

However, once an adequate series of optical sections has been gathered, it can be further processed into a three-dimensional representation of the specimen using volume-rendering computational techniques. This approach is now in common use to help elucidate the numerous interrelationships between structure and function of cells and tissues in biological investigations.

In order to ensure that adequate data is collected to produce a representative volume image, the optical sections should be recorded at the appropriate axial z -step intervals so that the actual depth of the specimen is reflected in the image. Most of the software packages accompanying commercial confocal instruments are capable of generating composite and multi-dimensional views of optical section data acquired from z-series image stacks.

The three-dimensional software packages can be employed to create either a single three-dimensional representation of the specimen Figure 7 or a video movie sequence compiled from different views of the specimen volume. These sequences often mimic the effect of rotation or similar spatial transformation that enhances the appreciation of the specimen's three-dimensional character. In addition, many software packages enable investigators to conduct measurements of length, volume, and depth, and specific parameters of the images, such as opacity, can be interactively altered to reveal internal structures of interest at differing levels within the specimen.

Typical three-dimensional representations of several specimens examined by serial optical sectioning are presented in Figure 7. The pollen grain optical sections illustrated in Figures 1 and 6 were combined to produce a realistic view of the exterior surface Figure 7 a as it might appear if being examined by a scanning electron microscope.

The algorithm utilized to construct the three-dimensional model enables the user to rotate the pollen grain through degrees for examination.

The tissue culture cells in Figure 7 b are derived from the Chinese hamster ovary CHO line and were transfected with a chimeric plasmid vector containing the green fluorescent protein and a human immunodeficiency virus HIV protein that is expressed in the nucleus thus, labeling the nuclear region.

Thick tissue sections are also easily viewed in three-dimensions constructed from optical sections. The mouse intestine section illustrated in Figure 7 c was labeled with several fluorophores and created from a stack of 45 optical sections.

In many cases, a composite or projection view produced from a series of optical sections provides important information about a three-dimensional specimen than a multi-dimensional view.

For example, a fluorescently labeled neuron having numerous thin, extended processes in a tissue section is difficult if not impossible to image using widefield techniques due to out-of-focus blur.

Confocal thin sections of the same neuron each reveal portions of several extensions, but these usually appear as fragmented streaks and dots and lack continuity.

Composite views created by flattening a series of optical sections from the neuron will reveal all of the extended processes in sharp focus with well-defined continuity.