1. Introduction

Multi-Photon Microscopy (MPM) is emerging as an important three-dimensional imaging technique with applications in various scientific disciplines. Bio–imaging is amongst the fastest growing fields of study that utilizes the advantages of MPM [1,2]. In-vivo two-photon deep tissue imaging showed the capability of this technique to image to a 1.6 mm depth in a mouse cortex [3]. Three-photon imaging also has been used to image different stages of dysplasia in Barret’s esophagus [4] and detect ovarian cancer in early stages [5]. In addition to biomedical imaging, MPM can be used in material characterization. Two- and three-photon imaging of thin layers of graphene [6], black phosphorous [7], and molybdenum disulphide [8] illustrates properties of these thin layers that have not been seen before. Electro-optical polymer based devices are another area of study which has been taking advantage of the MPM as a powerful characterization method [9]. Moreover, recent studies showed the ability of utilizing MPM in three dimensional micro-fabricated devices including sensors, waveguides, photonic structures, and biomedical devices, using various types of light sensitive polymers [10–15].

Since the first demonstration of the two-photon microscope in 1990 [16], different optical designs have been utilized to improve the imaging depth by using longer wavelengths (less scattering), and overcome the challenges regarding the optical aberrations of their design [17]. However, most of these microscopes use refractive optics and Titanium sapphire femtosecond (fs) lasers, which are expensive, bulky and hard to use as imaging sources. The use of fiber lasers have allowed compact and low cost designs of multiphoton microscopes. Erbium and Ytterbium doped femtosecond mode-locked fiber lasers showed promising results in label-free biomedical multi-photon imaging [18]. As multiphoton microscopes must use ultrafast pulsed lasers to generate appreciable nonlinear signals, traditional microscope designs using refractive elements have several disadvantages. Shorter pulses are advantageous for providing higher peak power. However, a shorter pulse by its very nature must have a larger spectral bandwidth, leading to adverse chromatic and dispersive effects. Lens based systems must consider dispersion compensation in order to remove the dispersive effects of their refractive beam shaping elements. Various methods have been investigated to quantify and compensate the effects of dispersive elements on femtosecond pulses [19, 20]. Additionally, it is desirable to be able to use more than one excitation wavelength, since that allows for investigating different structures in a biological sample targeted by different chromophores. Imaging modalities such as Stimulated Raman (SRS) and Coherent anti-Stokes Raman spectroscopy (CARS) require utilization of multiple wavelengths at the same time and at the same spatial location. It is possible to minimize the effects of chromatic aberration and dispersion in a multiphoton microscope system by various methods [21, 22]. However, it is desirable to be able to remove this concern entirely. In addition, existing mirror based scanning microscopes [23, 24] lack a large field of view and compact system footprint, and they do not have the same performance as the refractive multiphoton microscopes.

In order to overcome these problems, in this paper we demonstrate a compact All-Reflective Multi-Photon Microscope (ARMPM) design with diffraction limited performance over a scan angle of more than 10 degrees. Metal-coated mirrors used in this design have a high reflectance in a wide range of frequencies, which are suitable for sources with wavelengths from 300 nm to 20 µm. The use of reflective optics eliminates the inherent chromatic aberration of the lenses, and multiple broadband sources with a large separation between wavelengths can be deployed simultaneously. Finally, some applications are illustrated that can become feasible with the employment of this design.

2. Microscope design

Reflective elements were deployed for beam shaping and expansion, simultaneously enabling a design with a more compact footprint in comparison with traditional microscopes. The schematic diagram of our MPM is depicted in Fig. 1. The output from the all-fiber femtosecond laser is collimated using a reflective collimator. The laser beam is then raster scanned by a 2D galvo mirror system. All-reflective optical components (mirrors) are utilized to form a telescope to expand the beam. This step is required in order to fill the back aperture of the focusing objective lens, to make use of the full NA of the lens. The telescope also serves to relay the image of the scan mirror onto the back aperture of the objective, ensuring that all the scan angles are supported by the imaging system without vignetting. A shortpass dichroic filter reflects the beam onto the sample through the objective lens and also passes the reflected signal towards the photo multiplier tubes (PMT). Additional dichroics can be used to separate different portions of the signal into multiple channels for detection. While for single source applications, normal refractive infinity corrected objective lenses can be used, utilization of reflective objective lenses [25] allows simultaneous deployment of multiple laser sources with different wavelengths. The reflected signal can be separated into multiple channels using different dichroic mirrors and filters to detect the desired wavelength in each channel. PMTs were used to detect the generated nonlinear signals which include second harmonic generated (SHG) and third harmonic generated (THG), two photon excited fluorescence (2PEF), and three photon excited fluorescence (3PEF) signals.

 

Fig. 1 Schematic diagram of the All-Reflective Multiphoton Microscope. The collimated laser beam is raster scanned using a 2D galvo scanner. The beam is later expanded by reflective optics to fill the back aperture of the objective lens. Dichroic mirrors are utilized to separate the excitation laser light from the back-scattered nonlinear signals from the sample, and also to separate the different generated wavelengths from the sample into appropriate PMT channels.

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The birds eye rendered view of the microscope depicted in Fig. 2(a) shows the compact footprint of the all-reflective design. The overall size of the system including laser sources is 18”x18”x18”, which is considerably smaller in comparison with the conventional Ti-Sapphire multiphoton imaging systems. An image of the assembled ARMPM system along with two femtosecond fiber laser sources (1040nm (II) and 1560nm (I)) is shown in Fig. 2(b). Each laser can be used separately for imaging and writing applications, or they can be employed at the same time for simultaneous imaging and writing applications.

 

Fig. 2 Fabricated ARMPM image. (a) birds eye view of the rendered design of the ARMPM. The red color represents the laser light path. Total volume of the system including laser source and galvo mirror drivers is 18in*18in*18in. (b) The whole system is placed inside a black enclosure to minimize the room light and other sources of noise going into the photomultiplier tubes (PMTs). Numbered parts are as follows: (I) 1550 nm femtosecond fiber laser, (II) 1040 nm femtosecond fiber laser, (III) galvo mirrors, (IV) dichroic mirrors, (V) PMTs, and (VI) translation stage and sample.

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Our design is based on the design family of three mirror tilted component telescopes [26]. The large scanning angle required in scanning microscopes and the use of spherical mirrors instead of parabolic surfaces differentiates the design from known types of this class of telescopes such as the Tri-Schiefspiegler [27]. The telescope system is designed to achieve the appropriate beam size to fill the back aperture of the objective lens by expanding the 2.2mm collimated input beam to an 8mm diameter (~4 times beam expansion). After optimizing the design to achieve the minimum aberration, off-the-shelf components close to the optimum design were chosen. A secondary optimization on the tilt angles and separations using these components was performed to achieve a diffraction-limited system. Off-the-shelf mirror components satisfied both of the above-mentioned conditions, depicted in Fig. 3(a). Mirror 1 is a two-inch diameter 150mm focal length concave mirror, which acts as a telescope in conjunction with a concave mirror 4 (f = 300mm). Mirrors 2 and 3 are flat silver coated mirrors that fold the beam to reduce the overall footprint of the system. Finally, the low power convex mirror 5 corrects the coma and astigmatism, which are the two main sources of aberration in the system due to the off-axis nature of the reflective telescope design. Since the diameter of the input beam is small, the aberrations are corrected by using spherical mirrors, which reduces the overall system cost by avoiding the use of custom made parabolic or aspheric mirrors. Table 1 details the specifications of the design, as well as describing the mirror components used. Distances and radii of curvature follow the standard optics convention of changing sign after reflection.

 

Fig. 3 ARMPM design using off-the-shelf optics. (a) The compact arrangement is achieved by employing two flat mirrors to fold the beam (colors represent different scanning angles). The mirror numbering follows the microscope schematic depicted in Fig. 1. (b) OPD of less than 0.35 waves is achieved throughout the scanning field before the objective lens for 800nm, 1040nm, 1550nm, 1700nm laser sources. (c) Diffraction limited spot size is shown for the simulated all reflective afocal system. The Airy disk is drawn for 1040nm wavelength. (d) Ray fan diagram at four different wavelengths. The rays form different wavelengths overlap each other due to the reflective design of the system.

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Table 1. Design specifications and component details.

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The Zemax modeling results are illustrated in Figs. 3(b)-3(d) for 800nm, 1040nm, 1550nm, and 1700nm source wavelengths, which are the main fiber laser sources that are used for our multiphoton imaging and writing applications. Different colors in the image illustrates different angles of incidence coming from scanning galvo mirrors while in Figs. 3(b)-3(d), different wavelengths are shown by different colors. The optical path difference (OPD) of the wavefront (less than 0.35 waves) and the spot size radius shows diffraction limited performance of the system over 10 degrees of scan angle from the galvo mirrors. The ray fan aberrations (less than ± 0.2mr) of different wavelengths overlap each other due to the reflective design of the system. A triangular function is used to drive the galvo scanners to reduce the artifacts caused by high-speed movement of the mirrors at large scanning angles [28].

3. Results and applications

To test the ARMPM, two fs laser sources with ~150fs pulse width, 8MHz repetition rate, and the wavelength 1040nm and 1550nm with average power of ~60mW were used. A reflective microscope objective (36X 0.5 NA) was used to make the system entirely reflective in nature. Figure 4 depicts euphorbia cactus leaf structures, which were imaged with a 1550nm laser (Fig. 4(a)), and a 1040nm laser (Fig. 4(b)) without any change in the position or focus of the sample. Red and green colors represent the 2PEF/SHG, and 3PEF/THG signals respectively. As it can be seen, both of the samples are in focus despite the change in source wavelength. The same test with conventional refractive microscope would result in a 100µm shift in focus between the two laser sources, which would cause one of images to be completely out of focus (no signal).

 

Fig. 4 Multiphoton image of the euphorbia cactus leaf using. (a) 1550nm laser and (b) 1040nm laser. Red color represents the two-photon excited fluorescence (2PEF) and second harmonic generated signal (SHG). Green color illustrates three-photon excited fluorescence (3PEF) and third harmonic generated signal (THG). The focus does not change by changing the source wavelength due to the all-reflective design of the system.

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The resolution of the ARMPM was examined via fluorescent microbeads with 500nm diameter. The beads were imaged using the 1040nm laser source and an infinity corrected refractive objective (20X 0.5NA), with the resulting image seen in Fig. 5(a). An experimental Point Spread Function (PSF) was calculated from the image of the beads using deconvolution [29,30] (Fig. 5(b)). The microbead images were averaged in order to achieve higher signal to noise ratio to calculate the PSF. Figure 5(c) illustrates the cross section data of an ideal PSF function [31,32] in comparison with the experimental PSF. The experimental (FHWM = 990nm) data that is fitted with a Gaussian curve matched quite well with the ideal case (FHWM = 910nm) and the small difference is due to the aberrations of the objective lens in the infrared range.

 

Fig. 5 ARMPM image of fluorescent beads. (a) Image of the microbeads with 500 nm diameter. (b) The experimental PSF image exported from averaged individual bead images. (c) The comparison between the ideal vs the experimental PSF functions. The red curve shows the Gaussian function fitted to the experimental data.

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As previously mentioned, Multiphoton Micro and Nano 3D writing processes are a growing field due to the ability to go beyond the diffraction limit using two- and three- photon absorption. Different light absorption regimes in polymers are currently in use for lithography applications, which demands various light sources for each material. As it is not sensitive to the source wavelength used, the ARMPM is a good choice for mask-less, three-dimensional writing applications. Figure 6(b) shows the SHG image of the three-photon printed University of Arizona logo written with ARMPM on positive photoresist using the 1040nm laser source. The dark area shows the refractive index change due to polymerization of the resist. One promising field for utilizing this multiphoton printing method is on-chip waveguide writing [33], which can facilitate the fabrication of all optical photonic components. Figure 6(a) shows the multiphoton image of a ring resonator fabricated using the 1560nm laser source on electro optical polymer (SEO250) on Si substrate. The refractive index on the exposed (dark) areas on the polymer would change (Δn = 0.078 (0.068) for TE (TM)) and act as the cladding for the waveguide. Simultaneous multiphoton writing and imaging using different laser sources is also another potential fabrication method that can benefit from the all-reflective design of the microscope (see Visualization 1).

 

Fig. 6 Multiphoton writing using ARMPM. (a) Multiphoton image of a ring resonator fabricated on electro optical polymer (SEO250) using two-photon polymerization using ARMPM with 1560nm laser source. (b) SHG image of the University of Arizona logo written on positive photoresist using three-photon polymerization with 1040nm laser.

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Additionally, biomedical imaging without slicing and staining the tissue is one the promising applications of multiphoton imaging. Using the ARMPM enables deployment of various source wavelengths to investigate different structures in biological tissues or incorporating different fluorescent dyes to detect specific targets [34]. Figure 7(a) shows the image of an unstained ovary tissue captured using ARMPM. The human ovary tissue was obtained under a protocol approved by the University of Arizona Institutional Review Board, and the patient consented to the use of salpingo-oophorectomy surgical discard tissue for this study. The green color shows three photon fluorescent and third harmonic generated signals from lipid structures and red blood cells inside the tissue. The red color depicts second harmonic generated signal from the collagen fibers. The red blood cells signal and the blood vessel boundary is visible in the enlarged image of Fig. 7(b).

 

Fig. 7 ARMPM image of the unstained human ovary biopsy tissue (a). Red color represents the SHG from collagen fibers, and green color illustrates the THG and 3PEF from red blood cells and lipids. (b) Zoomed in image depicts the fluorescent signal from the red blood cells, and SHG from the collagen structure around the blood vessel.

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4. Conclusion

In this paper, we have demonstrated a multiphoton microscope design using all reflective optical components. The mirror based design enables the use of any wavelength of femtosecond laser, and can perform with the same diffraction limited properties of the traditional designs, while eliminating the chromatic aberration and dispersion introduced by using refractive optics. Although the off-axis design of the system can produce coma and astigmatism, it can be corrected using a negative mirror after the beam expander in the system. We have also illustrated the performance of this design and its unique capabilities to use multiple laser sources in one system.