MICROSCOPY RESEARCH AND TECHNIQUE 51:464 – 468 (2000)
Single-Pinhole Confocal Imaging of Sub-Resolution Sparse
Objects Using Experimental Point Spread Function and
Image Restoration
A. DIASPRO,* S. ANNUNZIATA,
AND
M. ROBELLO
INFM, Biophysical Section, Genoa Research Unit and Department of Physics, University of Genoa, Via Dodecaneso 33,
16146 Genova, Italy
KEY WORDS:
confocal fluorescence microscopy; subresolution imaging; non linear image restoration; three-dimensional microscopy; cellular biophysics
ABSTRACT
Sparse fluorescent pointlike subresolution objects have been imaged using a diffraction limited single-pinhole confocal fluorescence microscope. A Maximum likelihood image
restoration algorithm has been used in conjunction with a measure of the experimental point
spread function for improving the three-dimensional imaging of subresolution sparse objects. The
experimental point-spread-function profiles have been improved by a factor of 1.95 in lateral
direction and 3.75 in axial direction resulting in full-width half maximum (FWHM) values of 91 ⫾
11 nm and 160 ⫾ 26 nm. This amounts to 1.43 and 2.15 in optical units, respectively. The lateral
and axial FWHM of the sparse pointlike subresolution objects is about 5 and 3 times smaller than
the wavelength. This result points to the attractive possibility of utilising a compact confocal
architecture for localising punctuate fluorescent objects having subresolution dimensions. The key
resides in the utilisation of the measured point spread function coupled to an appropriate image
restoration approach, and, of course, in the stability of the confocal system being used. Microsc. Res.
Tech. 51:464 – 468, 2000. © 2000 Wiley-Liss, Inc.
INTRODUCTION
The three-dimensional localisation of various subcellular components is one of the major determinants
for understanding the delicate and complex relationship existing between structure and function in biological systems (Shotton, 1989). The confocal microscope
provides a practical, non-invasive, and well-established
method to obtain microscopic three-dimensional images and to perform both functional and structural
studies of biological systems and related biostructures
(Pawley, 1995; Wilson, 1990). Two of the most important properties of the confocal microscope are given by
the improvement of resolution by a factor of 1.4 over its
conventional counterpart (Brakenhoff et al., 1989) and
by the optical sectioning ability of fluorescent objects
(Wilson, 1990). Because of diffraction, the image of a
fluorescent point, which is in focus, is not the very same
point but a small patch, called diffraction pattern,
whose intensity distribution is more precisely defined
as the point spread function or impulse response function of the instrument (Bianco and Diaspro, 1989;
Sheppard, 1989). This spreading over a certain spatial
volume depends on the geometry of the aperture of the
optical system. The related physical constraint imposed to resolution cannot be surpassed in far-field
acquisition schemes such as those commonly used in
wide field and in confocal optical microscopy (Bertero
and Boccacci, 1998; Wilson, 1990).
Subdiffraction resolution has been achieved by disabling the fluorescence from the outer part of the
focal spot in terms of stimulated-emission depletion
(STED) (Klar and Hell, 1999), by using especially
calculated image-plane masks (Brand et el., 1999) or
©
2000 WILEY-LISS, INC.
structured illumination (Heinzmann and Cremer,
1998) combined with an interferometric technique
(I5M) in which the sample is observed and/or illuminated from both sides simultaneously using two opposing objective lenses (Gustafsson et al., 1999). Also
4Pi-confocal microscopy can provide a focus that is
sharpened up by physical methods (Hänninen et al,
1995; Hell and Stelzer, 1992; Hell et al., 1994). Unfortunately, the above-mentioned methods need to
modify the microscope architecture significantly and
are not of immediate access for the majority of users.
Moreover, any technology, such as I5M or 4Pi-confocal, which accesses focal plane within the sample
from both sides, is inherently limited to a class of
reasonably thin and transparent samples able to
minimize possible perturbations coming from refractive index variations, too (Gustafsson et al., 1999).
In terms of linear and space invariant systems
(Castleman, 1996), the image produced by the microscope is the convolution of the object with its own point
spread function. This allows one to consider that a
further image enhancement can be achieved by solving
the deconvolution problem, which consists of the restoration of the characteristics of an object from a given
image and a given point spread function (Bertero and
Boccacci, 1998; Schrader et al., 1996; van der Voort and
Contract grant sponsor: INFM.
*Correspondence to: A. Diaspro, INFM, Biophysical Section, Genoa Research
Unit and Department of Physics, University of Genoa, Via Dodecaneso 33,
16146 Genova, Italy. E-mail: [email protected]
Received 2 February 1999; accepted in revised form 9 May 2000
CONFOCAL IMAGING OF SUB-RESOLUTION SPARSE OBJECTS
Strasters, 1995). This problem is called image restoration. The question then becomes whether it is possible
to improve imaging properties substantially, even in
such a very particular case as the imaging of sparse
beads having a subresolution diameter. It has to be
clearly stated and understood that this is different
from discriminating densely spaced objects below the
resolution. However, image restoration offers a strategy for image enhancement without the need to modify
the optical apparatus significantly or to utilise near
field detection schemes that suffer from the drawback
of being surface analysis techniques (Betzig and Trautman, 1992).
In the present work, a robust maximum likelihood
image restoration algorithm (Bertero and Boccacci,
1998; van Kempen et al., 1996) has been used in conjunction with a measure of the experimental point
spread function (Diaspro et al., 1999) to improve the
discriminating capabilities in three-dimensional imaging of sparse subresolution objects acquired with a
single pinhole confocal laser scanning microscope. We
carried out our experiments with three intentions.
First, we applied mathematical techniques to explore
the extent to which the three-dimensional point spread
function can be reduced and to demonstrate the
achievement of a significant gain in the localisation of
sparse subresolution fluorescent particles. Second, we
aimed to utilise a procedure widely applicable to the
majority of operating confocal microscopes. In order to
fulfill this second intention, we used a measured point
spread function acting as the fingerprint of the microscope and a robust and widely disseminated scheme for
image restoration. The measure of the point spread
function does not need to be repeated for any acquisition session if the operating conditions are quite similar. Third, we wanted to show that a significant enhancement is possible in fluorescence imaging using a
single-pinhole scanning-head. Because of further limitations imposed by the photochemistry of the fluorescent molecules, the results are less spectacular than in
the case of high reflective gold pointlike scatterers
(Schrader et al., 1996).
MATERIALS AND METHODS
We used an advanced and ultracompact laser scanning confocal microscope system (PCM2000, Nikon
SpA, Florence, Italy) based on a galvanometer pointscanning mechanism, a single pinhole optical path, and
a very efficient all-fiber optical system for light delivery, both in excitation and in collection. The all-fiber
solution avoids a dangerous vibration decoupling between the laser source and the scanning head even if
they are placed on different tables. Moreover, by using
three mechanically fixed possible pinhole diameters
(20 ␮m, 50 ␮m, open), it is possible to repeat the same
effect with excellent reproducibility and system stability. Photomultiplier electronic noise is greatly reduced
by their positioning within the control unit, saving and
avoiding background reflection signal collection within
the scanning head. The scanning head is mounted on
an inverted optical microscope Nikon Eclipse TE
300. We used a Plan Fluor oil immersion objective
100X/NA ⫽ 1.3, the 488-nm line of an Argon-ion laser
and standard side-window Hamamatsu R928 photomultiplier tubes. The 20-␮m diameter pinhole resulted
465
in a back-projected radius two times smaller than the
back-projected Airy disk.
Our subresolution pointlike scatterers were 64 ⫾
9-nm diameter fluorescent (excitation at 488 nm, emission at 515 nm) latex beads (Cat no. 17149, Polyscience, Warrington, PA). A drop of dilute sample of
beads suspension was put on a coverslip of nominal
thickness 0.17 mm and air-dried in a dust clean chamber. The beads were then covered with a drop of glycerol. Three-dimensional images of the stack were recorded as a set of 21 images (1,024⫻1,024 pixels) taken
at 100-nm intervals along the z axis, with an integration period for each pixel of 38 ␮s. The pixel size was
26 nm in the optical plane.
RESULTS AND DISCUSSION
As the size of the bead is less then one-seventh the
wavelength in the medium and the beads are sufficiently sparse, we can also assume that each of the
beads being imaged represents the experimental confocal point spread function.
Figure 1 shows the three-dimensional rendering of a
portion of the field of the imaged subresolution objects
before and after image restoration.
The experimental lateral and axial Full Width at
Half Maximum (FWHM) are determined as 178⫾21
nm and 509⫾49 nm, respectively. These data, obtained
by averaging over more than 30 objects within the
acquired frames, are in good agreement with the theoretical values of 180 nm and 480 nm for the lateral
and axial FWHM calculated using Huygens2 (Scientific
Volume Imaging, The Netherlands) by means of scalar
theory (van der Voort and Strasters, 1995) (Fig. 2).
Moreover, these data are in agreement with an earlier
evaluation of the point spread function of the system
used (Diaspro et al., 1999). In optical units, the experimental lateral and axial FWHM is 2.80 and 6.93,
respectively. We used one of the three-dimensional
point spread functions we measured for our system and
corrected it for a slight broadening using Huygens2.
We used this as the point spread function for deconvolving the stack of subresolved pointlike fluorescent
objects. It is worthy of note that this three-dimensional
point spread function was acquired in an independent
session and represents the fingerprint of our scanning
head under the acquisition conditions described above.
We used the iterative maximum likelihood estimation
algorithm implemented within the Huygens2 software
because it is optimally suited to restore low signal
images (Bertero and Boccacci, 1998). It essentially uses
a positivity constraint and is a non-linear image restoration approach. We checked the processed data in the
Fourier domain or in terms of optical transfer function
in order to identify possible computational artefacts. In
this case, we had no computational artefacts. Moreover, we considered single particles. The experimental
lateral and axial FWHM is improved by a factor of
1.95 in the lateral direction and 3.75 in the axial direction, resulting in 91⫾11 nm and 161⫾27 nm resolution, respectively. This amounts to a lateral and axial
extent of the intensity profile of 1.43 and 2.15 in optical
units and about 5 and 3 times smaller than the wavelength.
We have shown that one is able to perform imaging
of subresolution sparse objects with a confocal micro-
466
A. DIASPRO ET AL.
Fig. 1. Three-dimensional rendering of a portion of the field of the imaged sparse subresolution
objects before (top) and after (bottom) image restoration. [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
CONFOCAL IMAGING OF SUB-RESOLUTION SPARSE OBJECTS
467
Fig. 2. Lateral (top) and axial (bottom)
intensity profiles in the focal plane of one of
the pointlike objects of Figure 1 from experimental (dotted) and deconvolved experimental (solid line) data. Theoretical (circles) expectations calculated by Huygens2 using scalar diffraction theory have been reported for
comparison. [Color figure can be viewed in
the online issue, which is available at www.
interscience.wiley.com.]
scope operating in the fluorescence mode. From the
results obtained, one can infer that one is able to
achieve object detection of less than one third of a
wavelength by fluorescence imaging with far-field microscopy. This applies to a matrix of sparse subresolution objects. Alternative optical designs allow a highresolution level (Pawley, 1995), and once realized at
sufficient signal-to-noise ratio their behaviour can be
further enhanced by deconvolution, as for the 4Pi-confocal scheme (Schrader et al., 1998). Unfortunately, the
previously mentioned schemes suffer from the following drawback: within a reasonable waiting time of
10 years they cannot be considered as practicable techniques for all those users who nowadays make extensive use of the three-dimensional high-resolution capabilities of the confocal microscope. This high level of
spatial discrimination is necessary for visualisation of
local intracellular dynamics, especially transient phenomena or chemical dynamics within living cells. We
think that our results, coupled to a physical spatial
confinement of the excitation volume achievable with
two-photon excitation modalities (Denk et al., 1990;
Diaspro and Robello, 1999), can be of interest in the
field of three-dimensional microscopy applications.
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A. DIASPRO ET AL.
ACKNOWLEDGMENTS
We acknowledge Cesare Fucilli, Massimo Fazio, and
Marco Raimondo for their technical support.
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