Single-Pinhole Confocal Imaging of Sub-Resolution Sparse
Objects Using Experimental Point Spread Function and
Image Restoration
A. DIASPRO,* S. ANNUNZIATA,ANDM. 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 res-toration; three-dimensional microscopy; cellular biophysics
ABSTRACT Sparse fluorescent pointlike subresolution objects have been imaged using a dif-fraction 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 sub-cellular components is one of the major determinants for understanding the delicate and complex relation-ship existing between structure and function in biolog-ical systems (Shotton, 1989). The confocal microscope provides a practical, non-invasive, and well-established method to obtain microscopic three-dimensional im-ages and to perform both functional and structural studies of biological systems and related biostructures (Pawley, 1995; Wilson, 1990). Two of the most impor-tant 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 func-tion 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 im-posed 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 dis-abling 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
structured illumination (Heinzmann and Cremer, 1998) combined with an interferometric technique (I5M) in which the sample is observed and/or illumi-nated from both sides simultaneously using two op-posing objective lenses (Gustafsson et al., 1999). Also 4Pi-confocal microscopy can provide a focus that is sharpened up by physical methods (Ha¨nninen et al, 1995; Hell and Stelzer, 1992; Hell et al., 1994). Un-fortunately, 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-confo-cal, 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 refrac-tive index variations, too (Gustafsson et al., 1999).
In terms of linear and space invariant systems (Castleman, 1996), the image produced by the micro-scope 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 resto-ration 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
Strasters, 1995). This problem is called image restora-tion. 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 strat-egy 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 Traut-man, 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 con-junction with a measure of the experimental point spread function (Diaspro et al., 1999) to improve the discriminating capabilities in three-dimensional imag-ing 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 micro-scope 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 acquisi-tion session if the operating condiacquisi-tions are quite simi-lar. Third, we wanted to show that a significant en-hancement is possible in fluorescence imaging using a single-pinhole scanning-head. Because of further limi-tations imposed by the photochemistry of the fluores-cent 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 scan-ning confocal microscope system (PCM2000, Nikon SpA, Florence, Italy) based on a galvanometer point-scanning mechanism, a single pinhole optical path, and a very efficient all-fiber optical system for light deliv-ery, both in excitation and in collection. The all-fiber solution avoids a dangerous vibration decoupling be-tween the laser source and the scanning head even if they are placed on different tables. Moreover, by using three mechanically fixed possible pinhole diameters (20m, 50m, open), it is possible to repeat the same effect with excellent reproducibility and system stabil-ity. 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 photo-multiplier tubes. The 20-m diameter pinhole resulted
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, emis-sion at 515 nm) latex beads (Cat no. 17149, Poly-science, 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 cham-ber. The beads were then covered with a drop of glyc-erol. Three-dimensional images of the stack were re-corded as a set of 21 images (1,024⫻1,024 pixels) taken at 100-nm intervals along the z axis, with an integra-tion 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 suffi-ciently sparse, we can also assume that each of the beads being imaged represents the experimental con-focal 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 the-oretical 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 exper-imental 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 decon-volving 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 resto-ration 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. More-over, 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 direc-tion, resulting in 91⫾11 nm and 161⫾27 nm resolu-tion, 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 wave-length.
We have shown that one is able to perform imaging of subresolution sparse objects with a confocal
micro-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.]
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 mi-croscopy. This applies to a matrix of sparse subresolu-tion objects. Alternative optical designs allow a high-resolution level (Pawley, 1995), and once realized at sufficient signal-to-noise ratio their behaviour can be further enhanced by deconvolution, as for the 4Pi-con-focal scheme (Schrader et al., 1998). Unfortunately, the previously mentioned schemes suffer from the follow-ing drawback: within a reasonable waitfollow-ing time of
10 years they cannot be considered as practicable tech-niques for all those users who nowadays make exten-sive use of the three-dimensional high-resolution capa-bilities of the confocal microscope. This high level of spatial discrimination is necessary for visualisation of local intracellular dynamics, especially transient phe-nomena 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.
Fig. 2. Lateral (top) and axial (bottom) intensity profiles in the focal plane of one of the pointlike objects of Figure 1 from experi-mental (dotted) and deconvolved experimen-tal (solid line) data. Theoretical (circles) ex-pectations calculated by Huygens2 using sca-lar diffraction theory have been reported for comparison. [Color figure can be viewed in the online issue, which is available at www. interscience.wiley.com.]
ACKNOWLEDGMENTS
We acknowledge Cesare Fucilli, Massimo Fazio, and Marco Raimondo for their technical support.
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