Biomedical Engineering Reference
In-Depth Information
are lower, making improvements in signal-to-noise ratio from
brighter sources attractive.
Cairn Research Ltd (Faversham, UK) provides xenon power sup-
plies, lamp housings, and arc lamps with noise that is in the range
of 1 part in 10
4
. A 150 watt lamp yielded 2-3 times more light at
520 + 45 nm than a tungsten filament bulb. The extra intensity
is especially useful for fluorescence measurements from processes
of single neurons where the light intensity is low and the dark
noise is a problem. In that situation, the signal-to-noise ratio will
improve linearly with intensity.
4.2.3. Arc Lamps
Laser illumination can provide the highest illumination intensity
but avoiding photodynamic damage requires careful attention to
minimizing illumination duration. The interference from speckle
noise can be eliminated by reducing the beam coherence (Dejan
Zecevic and Thomas Knopfel, personal communication).
4.2.4. Lasers
Two-photon imaging requires the use of a specific type of light
source, pulsed lasers. These lasers use either a Titanium- sap-
phire (Ti:Sapphire) or a Nd:YAG (neodymium-doped yttrium
aluminium garnet) oscillator. The Ti:Sapphire lasers provide wide
tuning range (from 690 to 1080 nm) whereas Nd:YAG lasers typ-
ically operate at a wavelength of 1064 nm (although they can be
also made to lase at their non-principal wavelengths of 946, 1320
or 1123 nm). At present, mostly Ti:Sapphire lasers are used for
two-photon imaging of living tissues
(67, 71, 72)
.
4.2.5. Two-Photon
Imaging
4.3. Optics
The need to maximize the number of measured photons has been
a dominant factor in the choice of optical components. In wide-
field epifluorescence, both the excitation light and the emitted
light pass through the objective, and the intensity reaching the
photodetector is proportional to the
fourth
power of numeri-
cal aperture
(73)
. In two-photon imaging, only the collection of
emitted light is affected by numerical aperture and thus the inten-
sity reaching the photodetector is proportional to the square of
numerical aperture. Clearly, numerical aperture is an important
consideration in the choice of lenses. However, direct comparison
of the intensity reaching the image plane has shown that the light
collecting efficiency of an objective is not completely determined
by the stated magnification and NA. In wide-field imaging, differ-
ences of a factor of five between lenses of the same specification
have been observed.
4.3.1. Numerical Aperture
Because of the strong light scattering within living tissue, in
vivo two-photon imaging critically depends on detection of scat-
tered photons. Under this condition, the size of back aperture of
the objective (but also the size of all other apertures within the
4.3.2. Back-Aperture of
the Objective
