1. Introduction

The laser scanning fluorescence microscope (LSM) [1,2] is one of the promising techniques for observing living cells, and has been an intense research subject in various fields, including biomedical science, spectroscopy, and micro sensing. However, its spatial resolution is determined by the Rayleigh diffraction limit, which is comparable to the wavelength, even with a high NA lens, such as an oil immersion lens.

For breaking the diffraction limit, studies have demonstrated such techniques as apodization [3] and interferometric superposition [4]. The interferometric superposition technique can allow super resolution by computation of the diffraction patterns on the focal plane. However, it requires relatively complex data processing. To date, two-photon-absorption-and second-harmonic-generation- LSM have been well established and have entered commercial use [5, 2]. However, the spatial resolution is limited to 1/√2 times the diffraction limit, determined by the pump laser wavelength.

In recent years, several researchers have presented two-color super-resolution LSM based on stimulated-emission- [6, 7] or up-conversion- fluorescence-depletion [8, 9, 10]. Watanabe et. al have demonstrated a super-resolution fluorescence spot with a diameter of half the diffraction limit by using nano-second pulse lasers [11, 12]. Theoretically this technique has no limitation for the resolution. According to previous reports, the spatial resolution of <60 nm can be expected [13].

Previous reports have discussed mainly the shrinkage of the fluorescence spot beyond the diffraction limit, but have only little mentioned the spatial separation between two individual fluorescence spots, which is more important in a 2-dimensional practical imaging system [14].

In this report, we present the pronounced separation (~750nm) between two individual fluorescence spots due to fluorescence depletion in a novel super-resolution LSM based on an up-conversion-fluorescence-depletion technique.

2. Basic concept of the “super-resolution” microscope

Figure 1(a) describes the basic concept of the two-color up-conversion fluorescence depletion technique. An upper level population of dye molecules is provided by the first laser (pump laser). If a second laser (erase laser) is also introduced on the molecules at the same time, the excited molecules will be up-converted to higher excited levels. Since a part of these molecules decay to the ground level through a non-radiative process, due to internal conversion, the fluorescence signal from the molecules is depleted. The application of the up-conversion-fluorescence-depletion technique to the LSM can allow a spatial resolution beyond the diffraction limit, i.e. “super-resolution”.

Consider the situation shown in Fig. 1(b). When a diffraction-limited pump laser as well as an annular erase laser (“doughnut” beam) is focused onto a sample, the fluorescence signal from the region where two lasers are spatially overlapped is depleted through an up-conversion process. As a result, the fluorescence spot is shrunken to be a “super-resolution” spot with dimensions smaller than the diffraction limit.

 

Fig. 1. (a) Excitation scheme of a two-color up-conversion fluorescence depletion technique. (b) Scheme for super-resolution microscopy based on a two-color up-conversion fluorescence depletion technique. The pump and “doughnut-shape” erase light are coaxially introduced to the sample.

Download Full Size | PDF

3. Experiments

3.1 Experimental setup

A schematic diagram of the experimental setup is shown in Fig. 2. A ϕ175 nm fluorescent micro-sphere (540 (excitation) / 560 (emission) / Molecular Probes, Inc.) containing rhodamine-6G dye molecules was used as an object. The micro-sphere exhibits relatively strong emission in the region from 540nm to 580nm. The pump laser used was a frequency-doubled Q-switched Nd³⁺:YAG laser. Also, a solid state Raman laser (599nm) pumped by another frequency-doubled Q-switched Nd³⁺:YAG laser was used as the erase laser. The pulse-repetition frequency of the pump and erase lasers was 10 Hz. The erase laser wavelength was sufficiently far from the fluorescence band of the micro-sphere to mainly contribute not to the stimulated emission, but to the up-conversion process [15]. The erase laser wavefront was modulated by a spatial light modulator (Hamamatu, PAL-SLM) [16], thereby generating an annular beam [17, 18]. The pump and erase lasers were co-axially combined, and focused on the object by an objective lens (Olympus, UPlan APO×20, NA=0.7). The time delay between the pump and the erase laser pulses was controlled, thereby yielding efficient depletion of the fluorescence signal.

 

Fig. 2. Schematic diagram of the experimental setup for two-color super-resolution microscopy using a doughnut beam. PAL-SLM corresponds to a spatial light modulator.

Download Full Size | PDF

3.2 Results and discussion

Figure 3 shows an experimental scanning image of the ϕ175nm micro-sphere. When the erase laser as well as the pump laser was introduced on the object, the diameter of the micro-sphere image was ~300 nm (Fig. 3(a)). This value is very consistent with that of a micro-sphere. For a comparison of our system with a conventional LSM, the erase laser was blocked. Then, the micro-sphere image expanded, and its diameter became ~600 nm (Fig. 3(b)). This value is comparable to the diffraction limit (~500nm) estimated from the numerical aperture of the objective lens. These experiments indicate that our system has a potential to reconstruct a fluorescence image with a spatial resolution of as much as 300 nm; the corresponding spatial resolution has been improved by at least 2 times. The cross sections of the fluorescence images are shown in Fig. 3(c) and (d).

 

Fig. 3. Experimental scanning image of the ϕ175 nm micro-sphere obtained by (a) pump and “doughnut” erase beams, and (b) pump light only. The cross section is also shown below the images (c and d).

Download Full Size | PDF

 

Fig. 4. Experimental scanning image of micro-spheres obtained by (a) pump and “doughnut” erase beams, and (b) pump light only. The cross section profile along the red line is shown below the image (c and d).

Download Full Size | PDF

The spatial separation of two individual micro-spheres was also investigated. Experimental scanning images of two individual micro-spheres are shown in Fig.4. The introduction of an erase laser as well as a pump laser made the two micro-spheres to be separate (Fig. 4(a, c)). The distance between the two micro-spheres was estimated to be 750 nm. When the erase laser was blocked, the separation of the two micro-spheres was vague (Fig. 4(b, d)). This means that a spatial resolution of ~600 nm, determined by the diffraction limit, is not enough to recognize a 750 nm separation of two individual micro-spheres. As can be seen from the cross sectional profile in Fig. 4(c), a small difference in the signal intensity between the neighboring two spheres can be seen. This might be due to the difference of the dye concentration in each sphere. These scanning images can be well reproduced.

These experiments show the possibility of 2-dimensional imaging with a spatial resolution beyond the diffraction limit by our system. It should be emphasized that our results were obtained without applying any numerical data operation, and that the improvement is entirely due to the instrument.

In this work, we investigated the lateral resolution in two-color super-resolution LSM based on up-conversion-fluorescence-depletion. The axial resolution is also important for 3-dimensional imaging. Though, at this stage, we have not yet measured the axial resolution in our system, we expect that there is no difference in the axial resolution between our microscope and a conventional con-focal one.

4. Conclusion

We investigated the pronounced separation between two individual fluorescence spots in a super-resolution scanning laser microscope based on a two-color up-conversion-fluorescence depletion technique. This microscope has the potential to achieve a spatial resolution with 1/2 of the diffraction limit. As a result, 750nm two-point separation, corresponding to the diffraction limit, has been well pronounced.

The spatial separation between two individual fluorescence spots is very important in a 2-dimensional practical imaging system. This work is the first presentation, to the best of our knowledge, of the two-point separation in a scanning laser microscope based on two-color fluorescence depletion technique.

Our system can obtain super-resolution by the use of commercial nano-second lasers, and it requires no ultra-short pulse lasers for achieving super-resolution. We believe that our system is simpler and more practical than that proposed by Hell et.al∸ The benefits of such an approach include minimal cost, easy maintenance, and high reliability. Further improvements in resolution are expected through refinements to the laser system, including the highly repetitive operation.