We report on a nonlinear optical endoscope that adopts a hollow core photonic crystal fiber for single-mode illumination delivery and a multimode one for signal collection. Femtosecond laser pulses up to 100 mW can be delivered at a centered wavelength of 800 nm. The two-photon fluorescence response of our system is shown to have axial and lateral resolutions of 5.8um and 0.6um respectively. Fluorescence detection was obtained at different wavelengths between 790 and 840 nm which could allow SHG detection for example. The maximal field-of-view of the acquired images is 420 µm×420 µm. Detection efficiency is greater by using an avalanche photodiode in comparison to a photo multiplier tube. Results presented here demonstrate the ability of the system to resolve cellular details and the potential of the device for future in vivo imaging diagnosis
©2008 Optical Society of America
Two-photon fluorescence (TPF) imaging is a powerful imaging modality with unique characteristics that can provide information complementary to that from other biological imaging technologies [1–3]. Advantages of TPF microscopy include intrinsic optical sectioning ability (due to the nonlinear two-photon excitation process), deeper penetration depth into tissue (owing to the use of near-infrared excitation light), and reduced photobleaching and phototoxicity in the out-of-focus regions (due to the confinement of fluorescence excitation to the focal region). Recently, extensive research efforts have focused on developing miniature probes for TPF imaging [3–27]. The use of two-photon fluorescence endoscopy could prove to be an important diagnostic tool, alleviating the need for surgical biopsy in both basic research and clinical pathology and producing spectra and images of tissue at the cellular level. To achieve a compact and miniature microscope such as an endoscope, flexible fiber-optic components such as optical fibers, optical fiber couplers, and gradient-index (GRIN) rod lenses are usually integrated into the imaging system to replace complicated bulk optics as well as compact and small scanner systems. Several scanner configurations have been developed as: fiber bundle [6, 28], distal scanning mechanism as piezoelectric elements [4,8,14,21,25,26] or MEMS [10,11,18,19,20,27] to reduce size for potential application in miniaturized endoscopy. Major challenges for such devices are beam scanning, efficient excitation light delivery, TPF signal collection, and probe miniaturization. Fu et al give a relative complete overview of the state of the art and references in nonlinear micro- and endoscopy . We present in this paper a fiber-optic two-photon endoscope based on a compact two axes piezo scanner system and a miniature high NA GRIN lens objective. A PCF fiber is used for excitation and ultrashort laser pulses can be delivered with typical power up to 100 mW in the wavelength range from 790 to 830 nm. Two-photon fluorescence signal is collected by the use of a multimode fiber. The novelty of this concept is that the high NA GRIN lens objective coupled to the fibers with a beam splitter cube represent a rigid miniaturized optical system block which is scanned as a whole by a piezo scanner allowing always an on-axes beam irradiation of the optical system. This helps to minimize off-axes aberrations and to increase the field of view, but limits the possible frame rate because of the larger inertia of the scanned mass.
2. Experimental setup
The design of the compact piezo-scanner two-photon endoscope is given in Fig. 1. Near-infrared laser pulses (λ=760–970 nm, τ≈100 fs) generated by an ultrafast Ti:Sapphire tuneable laser system (MaiTai, Spectra-Physics, CA) are coupled into a hollow-core photonic crystal fiber (PCF, HC-800-02, Crystal Fibre A/S, Denmark) for efficient pulse delivery with minimal distortions and high damage threshold (Fig. 1(b)) . The laser beam is expanded and collimated with a 5x telescope. It is coupled to the PCF with a microscope objective 5X, NA: 0.15 (Zeiss, Germany) matching the numerical aperture (NA) of the PCF for maximum coupling efficiency. The fiber is held in a fiber chuck and placed on a 3 axes nano-positioning stage to optimize the coupling of the light in the fiber. Laser pulses are guided through the PCF to the ultra-compact fiberscope headpiece (Fig. 1(c)). As shown in Fig. 1(d), the laser beam coming out of the PCF fiber is coupled in a custom designed assembly of GRIN-lenses with 1-mm high NIR reflexion 90 ° prism and 1.3 mm dichroic beam-splitting cube (GRINTECH GmbH, Germany) [28,30–31]. The dichroic coating was designed to reflect ~93% of the near-IR excitation light while transmitting ~95% of the fluorescence emission light in the visible regime (~530 nm). The designed GRIN objective has a maximum NA of 0.65 in water with a working distance of 170 µm. In air the working distance is shorten to 95 µm. Fluorescence emission photons in the visible regime are coupled to a multimode polyimide fiber with a silica core and cladding of 600 µm and 660 µm respectively (Ceramoptec GmbH, Germany). The photons are detected alternatively by a photomultiplier tube (PMT H7732, Hamamatsu, Japan) and an avalanche photodiode, single photon counting module (APD SPCM-AQR, PerkinElmer, USA) after passing 2×2 mm BG39 emission filters (AHF Analysentechnik, Germany) to ensure no transmission or reflexion of the laser radiation. The total width of the assembly system is 3 mm and was pasted on a 2D scanner prototype based on 2 crossed piezoelectric tri-morph actuators for X and Y axes (Argillon, Germany) (see Fig. 1(e)). The actuators consist of two very thin length expander plates bonded to a metal vane. The polarization of the plates is such that when a voltage is applied to the electrodes the length of one plate will expand, the other contract, causing the element to bend. When the element is mechanically flexed, a voltage will be developed between the electrodes. A rectangular voltage signal is then applied to the short actuator plate (on the left) which will bend and be used for the slow axe Y. A sinusoidal voltage signal is applied to the long actuator plate (on the right) which will bend and be used for the fast axe X. Compare to mono- or bi-morph, the tri-morph technology allow to reduce internal mechanical stresses, extend the lifetime and improve the reliability of piezoelectric bending devices. Several actuators and configurations have been studied to build the most compact system with the best characteristics such as deflection, frequencies, vibrations or mechanical resonances. The piezo scanner is 34 mm long and 1.9 mm wide. The diameter of the complete probe prototype with the optical assembly system is 3.5 mm. A control box including dedicated electronics to drive the piezo system as well as corresponding hardware and software for image acquisition has been specially developed (JenLab, Germany). Advanced functions are allowed. It is possible to control the zoom (i.e. the size of the scanned area). By the maximal deflection the instrument achieves a field of view (FOV) of 420×420 µm2. By minimal deflection (i.e. maximal zoom) it is possible to image an area of 6µm * 6µm2 for high image magnification. Additional options as acquisition parameters for frame rate as speed (up to 400 Hz for the fast axe of the piezo actuators) and resolution (64×64 pixels up to 1024×1024 pixels) are controllable. Region of interest (ROI) is also selectable. A micromotor (Physics Instruments, Germany) allows driving the system on µm scales in the z axe. The complete system has been integrated in an empty commercial endoscope tube with a diameter of 10 mm (the tube was purchased from Richard Wolf, Germany).
3. Results and discussion
3.1 Image quality, time acquisition and detection efficiency
Several image acquisitions have been performed by varying the zoom scale of the software, i.e. the voltage applied to the piezos at a fixed resolution (512×512 pixels) and scan speed (oversampling: 512). The detection has been performed successively using the PMT and the APD with the same laser parameters. A grid target has been home made on glass substrate under lithography with line widths of 1.5 µm and spaced every 13 µm. the grid has been filled with Rhodamin 123 for fluorescence detection. Results are depicted in Fig. 2. Minimization of off-axes aberrations and image field failures are expected when the laser beam go through the GRIN lens optic on-axes. Of course the weight of the whole optical system induce an inertia which can induce mechanical vibrations and slowing the scanning time. The fast axe can be drive at frequencies up to 400 Hz. For relative good results, the piezo scanner can be experimentally drive up to 100 Hz allowing frames in 1,3 second at 128×128 pixels and less than 10 s at 512×512 pixels. Compared to recent developments, as fiber cantilever technique for example, this is quite slower but the field of view is 2 or 3 times bigger. Myaing et al have developed an endoscope which consists of a tubular piezoelectric actuator with an optical fiber cantilever glued to the actuator tip  Theoretically frequencies up to 1300 Hz can be reached, experimentally they made images at a frame rate of 2.6 Hz. Engelbrecht et al using the same method typically used scanning cycles near video rate (25 Hz), producing 128×128 pixel for typical field of view of 200 µm . Using MEMS techniques, Hoy et al reach 10 frames per second at 256×256 pixels with maximal fiel of view of 300 µm with their 10×15×40 mm3 miniaturized two-photon microscope . Piyawattanametha et al have developed a 2D single crystalline silicon mirror and demonstrated a line acquisition rate up to 3.5 kHz (total frame rate not known) for high time-resolution which may be used in miniaturized two-photon endoscope .
As shown in the figures above, the squares of the grid are clearly discernible but image distortions are noticeable at the left side of the image, appearing slightly stretched in the horizontal direction. These distortions are inherent to the piezos actuators experimental response compared to the “ideal” trajectory defined by the drive signals, this can be quite easily corrected, but also by the fact that the laser beam describes a small circular arc. Accordingly the image is slightly out of focus on the borders. By varying the focus check of a couple of microns, a clear image at the image plane can be obtained at the borders of the target. An average laser power of 60 mW measured after the GRIN lens objective was used.
As can be observed the detection is more efficient using the high sensitive avalanche photodiode. Better details and higher fluorescence signals can be detected on both sides of the focus region.
3.2 Spectral range detection
Fluorescence detection of mixed preparation of 1 and 2 µm microspheres has been performed at different wavelengths from 780 nm to 860 nm as shown in figure 3. The PCF fiber has the best efficiency at 800 nm but as can be observed detection is also possible at wavelengths up to 840 nm which is quite interesting for second harmonic generation detection (SHG) for example. Of course more power is also needed. Detection under 800 nm decreases rapidly. At 780 nm no signal is detectable.
3.3 Characterization of spatial resolution
A two-photon image of a mixed preparation of 0.2 and 6 µm fluorescent microspheres is shown in Fig. 4. This image was magnified with a zoom factor. The scanned area is of 60×60 µm2. The image was performed at 512×512 pixels at a frequency of 6.25 Hz for the fast piezo actuator. This is relatively slow but the purpose here was to obtain an image with a high resolution. This represents a frame rate of 80 s. Individual 6 and 0.2 µm microspheres are discernible. The fluorescence signal from the 0.2 µm microspheres is quite low.
Functional imaging of cellular and subcellular activity requires micrometer spatial resolution. We therefore characterized the spatial resolution of the two-photon fiber endoscope by imaging sub- µm resolution (200-nm diameter) fluorescent microspheres. Focusing was achieved by varying the sample in Z direction by the use of the x,y,z micromanipulator. We analyzed the widths of the fluorescence intensity profiles along the lateral (horizontally and vertically) and axial directions. The full widths at half-maximum (FWHM) of the Gaussian fits to the bead images (Fig.5 (a) and (b)) were found to be 0.58 µm vertically (Y) and 0.63 horizontally (X) for lateral dimensions putting in evidence a small distortion of the image. The spatial resolution was found to be 4.7 µm axially (Fig. 5 (c)). It has to be noticed that the two- photon imaging of the samples was done in air. The theoretical spot size of a classical lens with a NA of 0.65 is estimated to be 0.78 µm FWHM. Results obtained here are quite in agreement. Moreover, better results are expected with a better power of focusing in liquids like water. Less laser power will be also necessary. A high improvement of the resolution is shown using our high NA GRIN lens system compared to other recent works even for the axial direction, where the extension can be attributed to spherical aberration from the GRIN lens, as well as a larger maximal field of view [10,17,18,20,21,26,27].
3.4 Two-photon imaging of cells and chromosomes
To demonstrate the suitability of the compact two-photon endoscope for functional measurements of cells, we applied the device to measure labeled mitochondria in cells and DAPI labeled metaphase chromosomes as shown in Fig. 6. As can be observed, the fuorescence detection is low and has to be improved. For example, the multimode fiber induce certainly many losses and some improvements can be expected by testing other fibers for signal collection.
In summary, we have reported on the development of a fiber-optic piezo-scanning two-photon fluorescence endoscope The novelty of the concept is that the fibers and the micro-optics are integrated and scanned as a whole by the two axes piezo scanner allowing always an on-axes beam irradiation of the optical system. We have shown that fluorescence detection is more efficient using a high sensitive avalanche photodiode than a photomultiplier tube. Fluorescence detection was obtained at different wavelengths between 790 and 840 nm which could allow SHG detection. Lateral resolution values for the system were experimentally measured to be 0.58 µm vertically and 0.63 µm horizontally. Axial resolution was found to be 4.7 µm. The endoscope is high flexible and controllable in terms of time acquisition, resolution and magnification. Fluorescence images were acquired over a maximal field-of-view of 420 µm×420 µm2. Results presented here demonstrate the ability of the system to resolve cellular details. Further improvements are necessary as the implementation of a thin hermetically sealed quartz slide at the end of the endoscope tube to perform in vivo endoscsopy. The packaging has to be again reduced in a smaller endoscope diameter to lead to a powerful clinical device. Such a system holds a promising future for application in nonlinear optical endoscopy.
This work was in part supported by the German Federal Ministry of Education and Research (BMBF, grant number 0313661).
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