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Introduction to interstitial cells of Cajal

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MICROSCOPY RESEARCH AND TECHNIQUE 47:210–214 (1999)
Effect of a Confocal Pinhole in Two-Photon Microscopy
R. GAUDERON, P. B. LUKINS, AND C. J. R. SHEPPARD*
Physical Optics Department, School of Physics A28, University of Sydney, NSW 2006, Australia
KEY WORDS
fluorescence; second-harmonic generation; resolution
ABSTRACT
We investigated the effect of a finite-sized confocal pinhole on the performance of
nonlinear optical microscopes based on two-photon excited fluorescence and second-harmonic
generation. These techniques were implemented using a modified inverted commercial confocal
microscope coupled to a femtosecond Ti:sapphire laser. Both the transverse and axial resolutions are
improved when the confocal pinhole is used, albeit at the expense of the signal level. Therefore, the
routine use of a confocal pinhole of optimized size is recommended for two-photon microscopy
wherever the fluorescence or harmonic signals are large. Microsc. Res. Tech. 47:210–214, 1999.
r 1999 Wiley-Liss, Inc.
INTRODUCTION
Confocal fluorescence microscopy is a well-established laser scanning technique for three-dimensional
(3D) fluorescence imaging in both reflection and transmission modes (Pawley, 1989; Paddock, 1999). Normally, a visible CW laser is used together with a pinhole
in front of the detector. This pinhole allows optical
sectioning by preferentially detecting the light from the
focal region, and also leads to an improvement in
resolution by spatial filtering of the image on the
pinhole. That is, the optical sectioning and hence the
3D capability of confocal microscopy arises from the
design of the optical detection system.
More recently, multi-photon optical microscopes have
been developed (Denk et al., 1990; Gannaway and
Sheppard, 1978; Hell et al., 1996; Mueller et al., 1998)
based on interactions between nonlinear optical properties of the specimen and the high peak power of the
(normally) pulsed illuminating laser. The nonlinear
nature of this interaction means that most of the optical
signal of interest is generated at the focus of the laser
beam in the specimen where the intensity is at a
maximum. The spatial properties of this interaction
provide a useful mechanism for optical sectioning in
such a microscope via a ‘‘soft-aperture’’ effect. Therefore, the optical sectioning arises from the illumination
system and so a confocal pinhole is no longer required.
The collection efficiency of the detection system in a
nonlinear microscope is, therefore, considerably improved because the requirement for an aperture is
removed. This, together with the reasonable efficiency
of certain nonlinear processes, particularly two-photon
excited fluorescence, means that these techniques have
lead to new and useful 3D microscopies. By far the most
important of these techniques is two-photon excited
fluorescence imaging (TPFI), which is now a wellestablished technique with many applications in biomedical, biological, and materials sciences. Secondharmonic generation imaging (SHGI) (Gauderon et al.,
1998) is another nonlinear microscopy but has the
ability to image, in three dimensions, the intrinsic local
second-order nonlinearity of a specimen.
r 1999 WILEY-LISS, INC.
The resolutions obtained in confocal and two-photon
microscopies are, broadly speaking, comparable. Although the focal spot size of the longer wavelength laser
light (usually, but not necessarily, in the near infrared
region) used in two-photon microscopies is larger than
in the visible confocal microscopy case, the point-spreadfunction (PSF) for two-photon imaging is reduced in
width by a factor of 冑2 due to the spatial characteristics
of the nonlinear interaction (Gu and Sheppard, 1995).
The combination of good resolution comparable to
confocal microscopy and the greater penetration into
biological specimens by the longer wavelength radiation used in two-photon microscopies are important
reasons for the current popularity of TPFI in particular.
In view of the above, there is the prospect that implementation of a confocal pinhole in two-photon microscopes will provide enhanced transverse and axial
resolution, albeit at the expense of signal strength. In
this paper, we investigate the effects of adding a
confocal pinhole to two-photon microscopes based on
two-photon excited fluorescence and second-harmonic
generation.
MATERIALS AND METHODS
The effect of the confocal pinhole on TPFI and SHGI
was studied using a modified inverted Leica TCS NT
confocal microscope coupled to a Coherent Mira 900
femtosecond Ti:sapphire laser (⬃ 120 fs pulsewidth,
720–860 nm) pumped by a Coherent 5W Verdi solidstate green laser. For this laser pulse width, an adjustable group velocity dispersion (GVD) precompensation
arrangement was not necessary. This microscope operated in the beam-scanning mode, giving an acquisition
rate of up to 4 images per second. An important
capability of this system is that it can operate simultaneously in transmission and reflection modes. The
system provided several detection options in the reflection mode, a range of dichroic beamsplitters and block-
Contract grant sponsor: Australian Research Council.
*Correspondence to: C.J.R. Sheppard, Physical Optics Department, School of
Physics A28, University of Sydney, NSW 2006, Australia. E-mail: c.sheppart@
physics.usyd.edu.au
Received 28 August 1999; accepted in revised form2 September 1999
TWO-PHOTON MICROSCOPY WITH A PINHOLE
211
ing filters, and a confocal pinhole whose size was
adjustable using a motorized actuator. For transmission mode SHGI, the fundamental beam was excluded
from the detector using a 400-nm interference filter.
RESULTS AND DISCUSSION
The effect of the confocal pinhole on the transverse
resolution in TPFI is demonstrated in Figures 1 and 2.
For a test specimen consisting of a small fluorescent
bead with a diameter of 210 nm, images were acquired
for four different sizes of the confocal pinhole at constant detector sensitivity (Fig. 1). The bead was imaged
using excitation at 800 nm and an oil-immersion 63⫻/
1.3 NA microscope objective that has a resolving power
of ⬃260 nm. Since the beads had dimensions smaller
than the diffraction limit, the resulting images represent effectively the intensity PSF of the system. For
each image, a line scan through the middle of the bead
is also shown. The resolution, estimated as the fullwidth half-maximum (FWHM) of the Gaussian fit to the
intensity profile, was calculated for each pinhole size.
Clearly, the transverse resolution is improved when the
confocal pinhole is used. Theoretically, the improvement in transverse resolution using a small pinhole is
approximately 冑2. In practice, we measured an apparent improvement somewhat greater than this. We
attribute this to the detection of scattered radiation for
larger pinhole sizes.
Figure 2 demonstrates this effect for a specimen
comprising an intact spinach chloroplast of width ⬃ 4
µm. Since chlorophyll has a major absorption peak near
415 nm, the laser wavelength was set at 830 nm for
two-photon excitation. The fluorescence near 680 nm
was imaged both without a pinhole (Fig. 2a) and with a
pinhole of optimized size (Fig. 2b). Without a pinhole,
there is little or no structure evident and the crosssectional intensity profile is almost Gaussian. However,
with an optimum confocal pinhole size, the chloroplast
periphery is better resolved, the almost step-like crosssectional intensity profile is more representative of the
chloroplast morphology, and the internal structure is
slightly better resolved. Fine structure of the grana
stacks was not highly resolved but this is due mainly to
the relatively low NA objective used (Leica dry 40⫻/
0.75 NA). In Figure 2b, we also see that the improved
resolution occurs at the expense of a decrease in the
signal level.
The effect of the confocal pinhole on the axial resolution in TPFI is demonstrated in Figure 3. The specimen
consisted of DAPI-stained cultured onion root cells
during the later stages of mitosis. Once completely
separated, the separation between the two groups of
chromosomes will increase and two cells will be formed
(cytokinesis). Because the chromosomes are dispersed
in 3D, the specimen is particularly suited for threedimensional imaging or axial imaging. It can be seen
that the resolution, particularly in the axial direction,
is improved by using a confocal pinhole. This is most
clearly seen for the chromosomes (CH) that are in the
focal plane and give stronger contrast in Figure 3b
when compared with other chromosomes in the images.
Fig. 1. Series of two-photon excited fluorescence images and line
scans of a single fluorescent bead of diameter 210 nm for four different
sizes of the confocal pinhole. a: Confocal pinhole almost fully open rd ⫽
4 Airy units [A.U.]. b: rd ⫽ 2 A.U. c: rd ⫽ 1 A.U. d: rd ⫽ 0.5 A.U. The
FWHMs are quoted relative to the case (a) without a pinhole.
212
R. GAUDERON ET AL.
Fig. 2. Two-photon excited fluorescence images of an intact spinach chloroplast of width ⬃ 4 µm for (a)
a confocal pinhole almost fully open and (b) an optimised pinhole size.
Fig. 3. 2D optical sections of two groups of DAPI-stained onion root chromosomes in a 3D volume
imaged by two-photon excited fluorescence. a: Confocal pinhole almost fully open. b: optimised confocal
pinhole size. The scan size was 50 ⫻ 50 µm and the excitation was at 730 nm.
Because the optical layouts of TPFI and SHGI are
similar, demonstration of the SHGI technique in the
modified Leica microscope and investigations of the
effect of the confocal pinhole were also carried out. For
these experiments, lithium triborate (LBO) specimens
were used since this material has substantial local
second-order nonlinearity, high optical transparency,
and a high laser damage threshold. Since secondharmonic radiation is produced in the forward direction, the microscope was used in the transmission mode
in order to demonstrate the technique. Figure 4 shows a
3D transmission second-harmonic image and its corresponding two-dimensional image obtained with a Zeiss
brightfield microscope for a LBO crystal fragment.
TWO-PHOTON MICROSCOPY WITH A PINHOLE
213
Fig. 5. SHG reflection image of LBO for a confocal pinhole almost
fully open (a) and for the optimised pinhole size (b). A comparison of
line scans made in the region denoted by the circle is shown for 6
different confocal pinhole sizes (c).
Fig. 4. 3D SHG transmission image of a LBO crystal fragment
(a) and a corresponding 2D brightfield image (b).
Features such as crevasses, cracks, and microcrystals
are observable in both images and correlate well. This
offers the interesting possibility of relating the local
second-order nonlinear coefficients of the specimen
with the corresponding refractive and absorption properties.
In order to investigate the effect of the confocal
pinhole, SHGI had to be performed in the reflection
mode. In this experiment, the specimen consisted of a
thin polished plate of LBO coated on one side with
aluminum. Hence, in the reflection mode, the detected
second-harmonic signal arose from both back-scattered
and retro-reflected second-harmonic light, the relative
proportion depending on the specimen transparency.
For confocal operation, the laser beam was focused
close to the surface of the coating in order to guarantee
minimum deviation of the second-harmonic light on its
return path. A set of six second-harmonic images of the
aluminum-coated LBO specimen was recorded. Figure
5a shows the image with no confocal pinhole while
Figure 5b shows the image with an optimum pinhole
size. This optimal size was determined by examining
the line scans for features with strong intensity gradients and comparing their intensity distributions (Fig.
5c). The resolution is optimal for approximately rd ⫽ 1.5
(where rd is the pinhole size expressed in Airy units) as
can best be seen for the two peaks near the centre of the
line scan. Apart from changes in signal level and
resolution, some alteration in the shape of the line
image is apparent. We attribute this to the fact that the
intensity in the plane of the pinhole is not uniform,
perhaps as a result of refractive effects. We would not
expect this to be as evident in two-photon fluorescence
as a result of the isotropic emission. The effect of the
confocal pinhole is more apparent when xz-sections are
taken, as illustrated in Figure 6. Ideally, such sections
should be perfectly thin, i.e., dimensionless lines. In
reality, the lines spread in both directions. However, it
can be observed that the image obtained using an
optimised confocal pinhole (Fig. 6b) exhibits better
axial resolution than the corresponding image without
a pinhole (Fig. 6a).
Previous theoretical studies showed that the signalto-noise ratio and the resolution of multi-photon micro-
214
R. GAUDERON ET AL.
detection sensitivity. On the other hand, if the signal is
large, then substantial resolution improvements can be
obtained by using a confocal pinhole. We also demonstrated that both TPFI and SHGI can be easily carried
out in a modified inverted commercial confocal fluorescence microscope coupled to a femtosecond laser. Equivalently, a confocal configuration could be implemented in
two-photon systems.
Fig. 6. Effect of the confocal pinhole size on SHG reflection
xz-sections of an aluminum-coated LBO thin plate with the confocal
pinhole almost fully open rd ⫽ 3.6 A.U. (a) and with an optimal pinhole
size rd ⫽ 0.25 A.U. (b). The scan size was 30 ⫻ 30 µm.
ACKNOWLEDGMENTS
The authors thank Leica Microsystems, Coherent
Scientific Pty Ltd, the Australian Key Centre for Microscopy and Microanalysis, and Dr Guy Cox. Special
thanks are due to Mrs. Eleanor Kable and Mr. Greg
Stevens who kindly prepared the biological specimens.
REFERENCES
scopes were improved by adding a confocal configuration to the detection path (Gauderon and Sheppard,
1999; Gu and Gan, 1996; Wilson and Sheppard, 1979).
Our experimental results have confirmed that adding a
confocal pinhole gives a further improvement in transverse and axial resolution. We found that the use of a
confocal pinhole slightly improves the transverse resolution, but gives an even greater improvement in the
axial resolution. This particular improvement in the
axial direction is especially beneficial since the unimproved axial resolution is generally inferior to transverse resolution in laser scanning microscopes. The use
of a confocal pinhole is, therefore, most effective for
high-resolution 3D imaging. Unfortunately, the signal
collection efficiency is reduced by using a confocal
pinhole. In addition, if a confocal pinhole is not used,
the emitted radiation need not be descanned so that
even scattered fluorescence can be detected. If the
signal levels are low or if the total laser exposure that
can be tolerated by the specimen is limited, addition of
a confocal pinhole may be problematic in terms of the
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