Microparticle characterization via confocal microscopy  (Page 3/4)

The light is then reflects across a pair of mirrors or crystals, one each for the x and y directions, which enable the beam to scan across the sample ( [link] ). The speed of the scan is usually the limiting factor in the speed of image acquisition. Most confocal microscopes can create an image in 0.1 - 1 second. Usually the sample is raster scanned quickly in the x -direction and slowly in the y direction (like reading a paragraph left to right, [link] ).

The rastering is controlled by galvanometers that move the mirrors back and forth in a sawtooth motion. The disadvantage to scanning with the light beam is that the angle of light hitting the sample changes. Fortunately, this change is small. Interestingly, Minsky's original design moved the stage instead of the beam, as it was difficult to maintain alignment of the sensitive optics. Despite the obvious disadvantages of moving a bulky specimen, there are some advantages of moving the stage and keeping the optics stationary:

• The light illuminates the specimen axially everywhere circumventing optical aberrations, and
• The field of view can be made much larger by controlling the amplitude of the stage movements.

An alternative to light-reflecting mirrors is the acousto-optic deflector (AOD). The AOD allows for fast x -direction scans by creating a diffraction grating from high-frequency standing sound (pressure) waves which locally change the refractive index of a crystal. The disadvantage to AODs is that the amount of deflection depends on the wavelength, so the emission light cannot be descanned (travel back through the same path as the excitation light). The solution to this is to descan only in the y direction controlled by the slow galvanometer and collect the light in a slit instead of a pinhole. This results in reduced optical sectioning and slight distortion due to the loss of radial symmetry, but good images can still be formed. Keep in mind this is not a problem for reflected light microscopy which has the same wavelength for incident and reflected light!

Another alternative is the Nipkow disk, which has a spiral array of pinholes that create the simultaneous sampling of many points in the sample. A single rotation covers the entire specimen several times over (at 40 revolutions per second, that's over 600 frames per second). This allows descanning, but only about 1% of the excitation light passes through. This is okay for reflected light microscopy, but the signal is relatively weak and signal-to-noise ratio is low. The pinholes could be made bigger to increase light transmission but then the optical sectioning is less effective (remember depth of field is dependent on the diameter of the pinhole) and xy resolution is poorer. Highly responsive, efficient fluorophores are needed with this method.

Returning to the confocal microscope ( [link] ), light then passes through the objective which acts as a well-corrected condenser and objective combination. The illuminated fluorophores fluoresce and emitted light travels up the objective back to the dichromatic mirror. This is known as epifluorescence when the incident light has the same path as detected light. Since the emitted light now has a lower wavelength than the incident, it cannot pass through the dichromatic mirror and is reflected to the detector. When using reflected light, a beamsplitter is used instead of a dichromatic mirror. Fluorescence microscopy when used properly can be more sensitive than reflected light microscopy.