Biomedical Engineering Reference
In-Depth Information
he maximum intensity in the focal spot is proportional to the Strehl ratio. In a CFM, the coher-
ence between excitation and emission light is destroyed in the luorescence process. If one makes the
approximation that the luorescence excitation and emission have the same wavelength, the double-
pass system introduces the same amount of aberration on the way to and from the focal spot (Booth
et al. 2002). Hence, we deine the signal improvement factor,
F
sig
, for a confocal luorescence micro-
scope system to be
2
=
S
S
corr
ini
(4.13)
F
sig
his is equivalent to the improvement in signal when adaptive aberration correction is applied to the
excitation and the emission paths for the case that the CFM uses an ininitely small pinhole. his simple
model of the imaging process allows us to estimate the potential beneit of adaptive optics in a straight-
forward manner. For small but inite-sized pinholes,
F
sig
is expected to be similar.
We note that the Zernike coeicients are a function of wavelength. If no dispersion is present, the
measured Zernike coeicients scale inversely with the wavelength. In this case, it would be a simple
matter to recalculate the factor of improvement for other wavelengths. Furthermore, the expression is
also valid for pinhole-less two-photon microscopes where the
M
i
should be scaled using the excitation
wavelength (Booth et al. 2002).
4.14 Which Level of Correction Is Sensible?
he technical efort in an active correction system increases with the quality of the correction. hus,
we have to ind the best compromise between correction efort and the anticipated efect. here are also
other things to consider that may limit the performance: an adaptive optics system requires measure-
ment of the wavefront aberrations. In a CFM system, this measurement is based on luorescence inten-
sity and can therefore harm (e.g., photobleach) the specimen under investigation. herefore, the quality
of the measurement is also limited by the luorescence light budget.
For the wavefront measurement procedure described in this chapter, the number of photons available
for the measurement does not limit the wavefront detection accuracy because a transmission geometry
is used. his gives access to the full wavefront data. We can predict the wavefront quality for difer-
ent correction levels by simulating the wavefront correction for a certain mode order
O
according to
Equation 4.10 and evaluating the quality of the obtained wavefront ψ
corr
(
r
,θ,
O
) using the Strehl ratio and
signal factor calculation described in
Section 4.13
. Details of this procedure and the results are given in
the following paragraphs.
We quantiied the Zernike mode-based correction by calculating the Strehl ratios of the wavefronts
before (
S
ini
) and ater the correction (
S
corr
) for the correction up to the mode orders
O
= 12, 18, 22, and 37.
he Zernike modes 2 through 4 do not afect the signal in an epi-confocal system as they correspond to
a shit of the focal spot only but do not afect its shape. herefore, the Strehl ratio
S
ini
was calculated for
a wavefront corrected up to mode order
O
= 4.
he results for the mean initial Strehl ratio, the mean-corrected Strehl ratio, and the mean and
median of the confocal
F
sig
are summarized in
Table 4.3
whereas the specimen numbers used are deined
in
Table 4.2
. To investigate the interplay between the correction quality and the number of corrected
Zernike modes, we calculated simulations up to modes 12, 18, 22, and 37 for all the specimens. In
general, the correction of up to 37 modes was found to give only moderate improvement in the Strehl
ratio compared to that of 22 modes. It appears that for these specimens, the correction of 22 or even 18
Zernike modes is a good compromise between the efort required for the correction and the improve-
ment in Strehl ratio. Maps of the Strehl parameters are shown in
Figure 4.18
;
the specimens are again
