Page 243 - Fundamentals of Light Microscopy and Electronic Imaging

226 CONFOCAL LASER SCANNING MICROSCOPY

rule of thumb is to scan slowly enough that the image stands out above the
background by a small amount.
• Acquire the final image. Use frame averaging or frame accumulation. The number
of frames to be averaged depends on the noise in the image, but numbers ranging
from 4 to 16 are typical. Remember that the factor improvement in S/N is the square
root of the number of averaged frames or the square root of the factor by which the
exposure time has been increased.
• Adjust the step size for a z-series. For z-series, the step size of the stepper motor
controlling the stage or objective is adjusted to give a sample period of 2 steps per
diffraction disk radius in the z-dimension (more than this if the sample is thick
and/or high spatial resolution is not required).
• Review the following general points:
1. Use the highest numerical aperture lens available (NA 1.3–1.4) to reduce focal
plane thickness and increase image brightness.
2. Room lights must be off to see gray levels on the monitor properly.
3. For bright images, use an ND filter so that offset and gain can be used at their
midrange settings.
4. Excitation of the specimen at high laser power maximizes fluorescence emis-
sion, but causes rapid bleaching and reduces image quality.
5. ND filters are usually employed even if the resulting image is somewhat noisy
to reduce the rate of photobleaching. Noise in dimmer images can be removed
by frame averaging or frame accumulation. Noise in the PMT output, made
worse by use of a high gain setting, can be significant even in a single scan of a
bright image. Thus, frame averaging (Kalman averaging) of a moderate-quality
image nearly always produces a better result.
6. Remember that electronic zoom is related to the sampling size and affects spa-
tial resolution.
7. A freshly acquired (raw) image has up to 10–12 bit intensity resolution in com-
puter RAM; however, saving an image may reduce its resolution to 8 bits, and
the original dynamic range will be permanently lost; therefore, images must be
optimized for brightness/contrast before saving. It is often good practice to his-
togram stretch an image before saving it as byte so that image data are distrib-
uted over the maximum number (256) of gray levels.
8. Laser alignment is critical. If the laser spot does not fill the back aperture of the
objective, the scan spot in the object plane is wider and image resolution is
reduced.

TWO-PHOTON AND MULTIPHOTON
LASER SCANNING MICROSCOPY

A fluorescent molecule can be induced to fluoresce by the absorption of a single photon
of the proper excitation wavelength. However, since the energy E of a photon is
described as

E h
,