Laser scanning confocal microscopes employ a pair of pinhole apertures to limit the specimen focal plane to a confined volume approximately a micron in size. Relatively thick specimens can be imaged in successive volumes by acquiring a series of sections along the optical (z) axis of the microscope.

This tutorial explores imaging of specimens with a Nikon PCM 2000 laser scanning confocal microscope by creating virtual control systems that simulate how the actual microscope operates. All specimens contained in this tutorial were imaged with the Nikon instrument and are presented as successive z-axis optical sections obtained from the original data.

The tutorial initializes with a randomly selected specimen appearing in the image windows. The left window (entitled Widefield Image) reveals how the specimen appears when viewed through widefield illumination in a classical fluorescence microscope, while the right window (entitled Confocal Image) presents a thin optical section from the confocal microscope at the same focal plane on the microscope z-axis. Photomultiplier gain is set to approximately 25 percent for both the red and green channels, and the initial scan speed is set to the medium scan rate.

The confocal Z-Axis Position and widefield Focus sliders are locked together at the same focal plane. They can be uncoupled with the Focus Lock checkbox for observation of a single focal plane in one window while the other is translated through successive view fields along the microscope optical axis. Use either the Z-Axis Position or Focus sliders to scan through successive focal planes, which will produce thin optical sections of the specimen in the confocal image window, and a succession of blurred images in the widefield image window.

To increase the photomultiplier gain in the confocal portion of the tutorial, translate the PMT Red Gain and PMT Green Gain sliders by moving one or both bars with the mouse cursor. Moving the sliders to the right will increase photomultiplier gain, while moving the sliders to the left decreases the gain. A similar Brightness slider for the widefield microscope portion of the applet allows the visitor to change image brightness as seen in a standard fluorescence microscope. The Scan Line Speed slider can be employed to adjust the speed of the virtual confocal scanning galvanometer module. As the scan speed is increased to higher values, a corresponding increase in the amount of background noise captured by the photomultipliers is observed in the confocal images. Adjust the scan speed slider to achieve the fastest scan rate with a minimum amount of signal noise.

Pinhole size can be varied using the Large, Medium, and Small radio buttons to toggle between the available Pinhole Aperture Sizes. Small pinholes afford the greatest resolution with the confocal microscope, while successively larger pinholes permit more of the fluorescence background noise to appear in the image. After examining a specimen, the Choose A Specimen pull-down menu can be accessed to select a new specimen for observation.

In a conventional widefield microscope, thick specimens will produce an image that represents the sum of sharp image details from the in-focus region combined with blurred images from all of the regions that are out of focus. This effect does not significantly deteriorate images at low magnification (10x and below) where the depth of field is large. However, high magnification objectives often feature correspondingly high numerical apertures that produce a limited depth of field, which is defined as the distance between the upper and lower planes of the in-focus region. The area where sharp specimen focus is observed can be a micron or less at the highest numerical apertures. The result is that specimens having a thickness greater than three to five microns will produce images in which most of the light is contributed by regions that are not in exact focus. Contrast will be reduced because of the contribution from a blurred background that is superimposed over a weaker in-focus image.

Several methods have been developed to overcome the poor contrast inherent in imaging thick specimens with a conventional microscope. The easiest solution is to modify the specimen by slicing it into very thin sections, which requires fixation, dehydration, embedding, and staining. This approach is useful for specimens that are obtained from larger sections of tissue, but it will not work for living cells or tissue sections in culture. Another approach is to modify the microscopy techniques utilized in collection of images. Confocal microscopy, multiphoton excitation, and deconvolution techniques enable observation of the details within thick specimens by a process known as optical sectioning, without the artifacts that accompany specimen preparation by physical sectioning.

Specimens having a moderate degree of thickness (5 to 15 microns) will produce dramatically improved images with confocal, multiphoton, or deconvolution techniques. The thickest specimens (20 microns and above) will suffer from a tremendous amount of extraneous light originating in out-of-focus regions, and are probably best-imaged using confocal or multiphoton techniques. In order to judge whether a particular specimen should be imaged with conventional, deconvolution, multiphoton excitation, or confocal microscopy, first view the specimen with widefield illumination. Specimens that produce blurred images, but still contain regions allowing the observer to set focus and see some level of detail, can benefit from either confocal or deconvolution imaging techniques. However, if the view through a conventional microscope is virtually featureless, providing no landmarks for choosing the appropriate area for imaging or for setting focus, then confocal microscopy should be employed for detailed analysis.

Living specimens require special consideration, because they are often sensitive to fluorophores and photobleaching cannot be prevented by addition of suppression reagents. In addition, necessary low concentrations of fluorescent labels usually produce a weak signal having poor contrast. Another concern is that long exposure to intense low-wavelength illumination often limits cell and tissue viability and, consequently, the length of experiments. A good solution may be to employ high quantum efficiency CCD cameras (commonly utilized in widefield microscopy) instead of the photomultiplier and avalanche diode detectors used for confocal and multiphoton methods. In this case, deconvolution techniques can offer the most satisfactory imaging solution.

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