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Adaptive harmonic generation microscopy
of mammalian embryos
Alexander Jesacher,1Anisha Thayil,1Kate Grieve,1Delphine Débarre,1Tomoko Watanabe,2Tony Wilson,1
Shankar Srinivas,2and Martin Booth1,*
1Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK
2Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK
*
Corresponding author: martin.booth@eng.ox.ac.uk
Received July 21, 2008; revised September 8, 2009; accepted September 11, 2009;
posted September 18, 2009 (Doc. ID 114531); published October 9, 2009
Adaptive optics is implemented in a harmonic generation microscope using a wavefront sensorless correction
scheme. Both the second- and third-harmonic intensity signals are used as the optimization metric. Aber-
ration correction is performed to compensate both system- and specimen-induced aberrations by using an
efficient optimization routine based upon Zernike polynomial modes. Images of live mouse embryos show an
improved signal level and resolution. © 2009 Optical Society of America
OCIS codes: 180.0180, 180.4315, 110.1080, 170.3880, 180.6900, 190.4160.
Harmonic generation microscopy (HGM) was pro-
posed as a method for label-free imaging of biological
specimens [1]. Using the nonlinear optical properties
of the tissue, third- and second-harmonic generation
(THG, SHG) are produced at the focus of a short-
pulsed laser beam, revealing cellular structure with
three-dimensional resolution. Implementations of
combined SHG and THG imaging have often used ex-
citation wavelengths around 1200 nm [2–4]. This
choice was influenced by the relatively low absorp-
tion of this wavelength by biological tissues and the
convenient placement of the SHG and THG around
600 and 400 nm, respectively, permitting the use of
standard optics and detectors. A disadvantage of this
excitation wavelength is that high-numerical-
aperture objectives required for such imaging are not
typically specified for this wavelength. One should
therefore expect reduced performance both in terms
of transmission and aberrations. Furthermore, aber-
rations are introduced by the specimens themselves,
particularly by thick specimens to which HGM is of-
ten applied [5]. As the harmonic generation is nonlin-
early dependent on the excitation intensity, these fac-
tors could have a significant effect on the imaging
performance.
One application area where HGM has shown prom-
ise is in imaging for developmental biology [3,2,4]. In
early-stage mouse embryos, THG images reveal cel-
lular structure and sub-cellular features, whereas
SHG has shown mitotic spindles and the zona pellu-
cida. It has also been shown that large aberrations
are induced when focussing through embryos [5].
When combined with the system aberrations arising
from the nonoptimal objective lenses, the effects can
cause a significant reduction in signal level and res-
olution. Adaptive optics has been used to compensate
for aberrations in various microscopes [6]. In this
Letter we demonstrate the use of adaptive optics for
the correction of system and specimen-induced aber-
rations in the HGM of live mouse embryos.
A schematic of the adaptive microscope is shown in
Fig. 1. The chromium forsterite laser (Mavericks, Del
Mar Photonics) emits 65 fs pulses at a repetition rate
of 100 MHz, center wavelength =1235 nm and out-
put power around 200 mW. The expanded beam was
steered by two-axis galvanometer mirrors that are
imaged onto a deformable membrane mirror (MIRAO
52-e, Imagine Eyes). The deformable mirror (DM)
was imaged onto the pupil plane of the microscope
objective. The illumination power in the focus was
approximately 30 mW. The harmonic emission was
collected in trans-configuration by an oil immersion
condenser 共NA=1.4兲. The SHG and THG were sepa-
rated by a dichroic filter and were detected simulta-
neously by using two photomultiplier tubes. Speci-
men scanning in the axial 共z兲direction was enabled
by a piezo actuator attached to the sample stage. The
objective lens (Olympus UApo/340 water immersion,
40⫻, NA=1.15) was chosen because of its high trans-
mission (60% at =1235 nm) compared with other
objectives that were tested. For in situ DM character-
ization, three beam-splitter cubes were inserted into
the optical pathway to form a Mach–Zehnder inter-
ferometer (dashed outline in Fig. 1) using a helium–
neon laser 共=633 nm兲.
Fig. 1. Schematic of the microscope. Lx, lens; BSx, beam
splitter; Mx, mirror; DM, deformable mirror; O, objective;
C, condenser; Dx, detector.
3154 OPTICS LETTERS / Vol. 34, No. 20 / October 15, 2009
0146-9592/09/203154-3/$15.00 © 2009 Optical Society of America
The aberration correction procedure involved the
sequential correction of Zernike polynomial modes
through the maximization of a metric defined as the
total image intensity, i.e., 兺x,yI共x,y兲, where Irepre-
sents the image. A similar approach has been em-
ployed in other adaptive microscopes and provides
rapid correction with a small number of metric mea-
surements [7,8]. The metric value I0was measured
for the initial aberrated image. An amplitude bof a
chosen mode was added to the DM, and the corre-
sponding image metric I+was measured. An amount
−bwas then added, and the value I−was measured.
The correction aberration amplitude was then calcu-
lated through a parabolic maximization as a=b共I+
−I−兲/共2I+−4I0+2I−兲. This was repeated for each mode
of interest. For this demonstration, we chose to cor-
rect 18 low-order Zernike modes (excluding piston,
tip, tilt, and defocus). As the I0measurement is com-
mon to all modes, the correction of nmodes requires
2n+1 measurements. Hence, the total number of
scans per correction cycle was 37.
System aberrations were corrected by optimizing
the THG from the rear glass/air interface of a cover-
glass of 170
m thickness. The objective adjustment
collar was set to the corresponding glass thickness.
Prior to the correction, the DM surface was flat up to
an rms value of less than /12 at =633 nm. The
plots in Fig. 2(a) show the THG axial response before
and after five repeated correction cycles. The peak in-
tensity increased by almost 50%, and the FWHM de-
creased by 14% to 1.22
m, compared with a calcu-
lated value of 1.15
m for an unaberrated system.
The procedure was repeated to check whether further
improvement could be obtained. The signal rapidly
converged on a maximum, which shows that for this
particular specimen Zernike modes have a mostly in-
dependent effect on the THG signal. Figure 2(b)
shows the signal improvement achieved with each
correction cycle (blue dashed curve), together with
the accordingly retrieved phase aberration. The total
correction aberration had an amplitude of 0.37 rad
rms at =1235 nm. This procedure could be repeated
before imaging each specimen, for example, to miti-
gate the effects of any variations in the coverslip
thickness.
In another experiment, aberrations induced by
mouse embryo specimens were corrected. Figure 3
shows frames from a three-dimensional image stack
of an embryonic day 5.5 stage mouse embryo. The
cover glass in the culture dish had a nominal maxi-
mum thickness of 130
m, which corresponded to the
objective correction collar’s minimum setting. First,
the system aberrations were compensated by the pro-
cedure previously described, resulting in the correc-
tion phase of amplitude 0.58 rad rms. This consisted
mainly of spherical aberration, probably due to in-
complete coverglass compensation in the objective
lens. A THG image stack covering 40
m⫻40
m
⫻20
m within the embryo was acquired from a re-
gion approximately 90
m deep in the sample [Fig.
Fig. 2. (Color online) Correction of system aberrations. (a) THG axial response at a glass/air interface before (blue dashed
line) and after correction. (b) THG signal (blue dashed line) and incremental retrieved phase aberration for subsequently
applied correction cycles.
Fig. 3. (Color online) (a) THG images of a 5.5 day old live
mouse embryo with correction of only system aberrations
(left) and after additional correction of specimen-induced
aberrations (right). The dashed lines show where the xy
and xz planes intersect. The corresponding correction
phase functions are also shown. (b) Intensity profiles along
the solid lines A–B and C–D as drawn in (a).
October 15, 2009 / Vol. 34, No. 20 / OPTICS LETTERS 3155
3(a), left]. There was no significant SHG from this
portion of the embryo. One cycle of the correction pro-
cedure was performed at the x–y section shown in the
figure. The correction aberration amplitude was
0.67 rad rms at =1235 nm. The image stack was re-
acquired with the phase correction applied. The re-
sult is shown in Fig. 3(a) right. Cell boundaries and
organelles are clearly visible. The total signal from
the corrected x–y section increased by 54%. A second
correction cycle, performed to verify the initial correc-
tion, showed no significant improvement. Fig. 3(b)
shows intensity profiles, as indicated by the solid
lines in (a), along the xand zdirections both before
and after correction of the specimen aberrations. Ab-
erration correction has improved both signal and res-
olution. It can be seen that the increase in brightness
of small features is more significant than the in-
crease in background signal.
Both SHG and THG can be used separately as the
optimization metric. Figure 4shows images from a
live embryonic day 2.5 stage mouse embryo. A strong
THG signal is observed from cytoplasmic structures
(possibly lipid vesicles); SHG is seen from mitotic
spindles in one of the cells at a 45
m depth. We per-
formed SHG-based correction using an x–yimage
containing the spindle and the THG-based correction
to a plane in close vicinity, where sufficient THG
could be observed. Two cycles were performed for
each correction procedure. As expected from the
smaller imaging depth, the aberrations had smaller
amplitude than in the 5.5 day old embryo. The corre-
sponding Zernike coefficients, shown in the bar chart,
are similar, which is expected as both SHG and THG
channels are affected by the same aberrations. This
shows that THG and SHG signals can both be used
independently for the correction procedure. After cor-
rection, the total signal increased by 21% and 9% for
SHG and THG, respectively. This difference in im-
provement is attributed to the different specimen
structures. Preliminary studies show that the effect
of aberrations on the image intensity varies, such
that the intensity from fine structures is reduced
more than that from larger features.
Aberration correction leads to more efficient imag-
ing and could permit the use of lower illumination
power for the same image signal-to-noise ratio. This
has important consequences for imaging of live speci-
mens in HGM, where the illumination dose can affect
long-term viability. In this Letter, we have shown
that both system and specimen-induced aberrations
have detrimental effects on the imaging properties of
HGM. Adaptive optics have been used to mitigate
these effects and restore the signal level and reso-
lution of these microscopes. In these experiments
THG and SHG signals have proved to be a suitable
metric for aberration correction, where improved res-
olution corresponds to higher intensity. It is possible
that for certain objects this relationship between res-
olution and intensity is different. For example, when
several interfaces are near the focus, a maximized
THG signal does not necessarily correspond to mini-
mum aberrations. In such situations, other metrics
such as image sharpness or the use of spatial averag-
ing could be incorporated.
Support from the following grants is acknowl-
edged: Biotechnology and Biological Science Re-
search Council (BBE0049461), Engineering and
Physical Sciences Research Council (EP/E055818/1),
Austrian Science Fund (J2826-N20).
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Fig. 4. (Color online) Equivalence of correction using SHG
and THG signals. (a) Overview of the 2.5 day old embryo
showing SHG (solid red) and THG. (b) Sections used for the
SHG- and THG-based corrections. The sections are located
in close vicinity. Upper image pair, before correction; lower
image pair, after correction. (c) Zernike coefficients and
phase functions retrieved by both correction procedures.
3156 OPTICS LETTERS / Vol. 34, No. 20 / October 15, 2009
