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Bring It In Focus

February 1, 2005
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Getting a high-quality image using confocal technology lies in the details.

Just as the telescope allows one to catch a broad glimpse of the universe, the microscope enables one to narrow the focus down to the smallest of worlds: molecules and atoms. Although conventional light and fluorescence microscopy allow for the examination of both fixed and small living samples, one challenge is that it tends to blur the details of key structures of interest because of the excitation of light-or out-of-focus

fluorescence.

Out-of-focus information often can obscure thicker, intact fluorescent samples. In a conventional fluorescence microscope setup, not only is the plane of focus illuminated, much of the sample above and below this point is simultaneously illuminated and imaged. That's where a "confocal" solution can be used.



The basics of confocal

Unlike conventional fluorescence microscopy, confocal microscopes are among the most sophisticated microscope equipment in the world. They have a shallow depth of field, allowing information to be collected from a well-defined optical section, or single plane, rather than from most of the sample depth. The result is the virtual elimination of out-of-focus fluorescence, which increases clarity and contrast within the image.

The principal function and ray pathway of confocal laser scanning microscopy relies on a light source, beamsplitter and excitation filter wavelengths. The light source is a laser that produces a high-intensity, clear light of a defined wavelength. Between the laser and beamsplitter, a light aperture-referred to as a pinhole-excites fluorescence. Although other techniques are used, laser serves as an ideal light source for radiating photon waves that form perfectly coherent wavelengths.

The pinhole, by providing for optical sectioning of the observed material, produces a graphic recreation of the sample as a three-dimensional image. The laser-scanning microscope (LSM) scans the sample sequentially, point by point and line by line, and assembles the pixel information. Optical slices of the sample are imaged with high contrast and high resolution in the X, Y and Z planes. By moving the focus plane, single images-or optical slices-are arranged into a three-dimensional stack that is digitally processed via an electronic detector and viewed as one image on a computer. The displayed image represents the entire sample.



More detailed solutions

p> Three steps can be taken for obtaining a confocal image. First, view the sample in visible mode. Focus the specimen in epi-fluorescence mode using the binocular and center the part of interest; select fluorescence filter cube according to application via software, and match the field of view; and change to the appropriate objective magnification. The use of a correct immersion medium also might be considered.

Second, load an LSM configuration by going to the LSM mode. Then, opening up the appropriate menus in the software, select a predefined configuration from the list, either single track or multi-track. This will set up the system with laser lines, attenuation, filters, beamsplitters, pinhole diameter and detector settings. Third, scan the image. This, too, can be accomplished using software. A system will automatically open the image window, optimize detector settings and scan the image.

To enhance the image quality, change to longer pixel dwell times by reducing scanning speed and use the "average" method, which is a calculation of sum or mean value of pixels of consecutive line or frame scans. Also, increase bandwidth of the emission filter by using long pass instead of band pass. Enlarge the pinhole diameter, though note that optical slice thickness will increase accordingly. Finally, increase excitation energy, or the laser power. In taking these steps, one must pay attention to bleaching, saturation and photoxic effects.

Further refinement of the image quality may include using an objective with higher numerical aperture, increasing the image frame size, optimizing scan zoom and increasing the dynamic range. More reliability may be achieved through multi-tracking, as this provides for fast switching of excitation wavelengths and prevents crosstalk between signals on different channels.

To accomplish multi-tracking, software often supplies predefined configurations. Using region of interest (ROI), excited areas of the sample are significantly reduced and the acquisition rate is increased at a constant signal-to-noise ratio. Several ROIs of any shape desired can be defined and used.



Popular but cautious

Recent years have seen an increase in the use of confocal microscopy because of the relative ease with which high-quality images can be obtained from samples prepared for conventional optical microscopy. However, for all of its benefits, safe laser operation is a key issue in effective use of LSM. The two critical concerns are exposure to the beam and the electrical hazards associated with high voltages in the laser and its power supply.

Safety precautions should be clearly accessible and operators should be trained in correct usage, avoiding, for instance, looking directly in the beam and avoiding exposure to the radiation. Observation of diffuse reflection usually is safe. However, because the laser beam never freely propagates in the room where the system is installed, there is no need to install a door interlock device. NDT



Quality magazine and NDT would like to thank Carl Zeiss MicroImaging (Thornwood, NY) for its contributions to this article.

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