We report a hybrid miniaturized achromatic objective for nonlinear endomicroscopy that combines two-photon polymerization (2PP)-fabricated polymer optics with a commercial glass lens. The objective achieves 0.6 numerical aperture with dual-wavelength correction at 775 and 860 nm and a 200 µm field of view, all within a 3 mm housing compatible with a hypodermic tube. Fabrication leverages sequential multi-step two-photon polymerization (2PP) to produce a monolithic multi-material triplet with submicron precision. The triplet incorporates two photoresists with different dispersion properties printed in successive steps with post-processing and marker-based realignment between steps, yielding a single monolithic optic that eliminates adhesives while correcting chromatic aberrations. Imaging tests with USAF resolution targets, biological samples, and two-photon fluorescence microscopy of pollen grains demonstrate near-diffraction-limited performance across the design wavelength range. The approach offers a practical method for compact optical systems in biomedical applications.

Significance:Cholesteatomas, benign tumors that grow in the middle ear, can lead to conductive hearing loss. If not completely removed during surgery, these tumors may regrow. Current imaging technologies struggle to detect residual tumors noninvasively due to limitations in contrast and resolution, often necessitating additional surgery for inspection. To address this, we developed a catheter endoscope capable of being inserted through the Eustachian tube, allowing detailed examination of the middle ear without surgery. Using two-photon polymerization (2PP) technology, we fabricated miniature, side-viewing reflective endoscope objectives. This approach enabled the rapid production of single-element objectives with highly repeatable optical properties, easily adaptable to specific imaging needs. Aim:We aim to design, fabricate, and demonstrate a catheter endoscope for optical coherence tomography (OCT) endoscopy of the middle ear via the Eustachian tube. Approach:Side-viewing, reflective lenses were designed in OpticStudio and 3D printed using 2PP followed by sputter coating with gold. Standard metrology techniques were used to verify and optimize the objective's shape. The optical performance of the catheter endoscopes was measured with a beam profiler. Finally, OCT imaging of the middle ear of a pig via the Eustachian tube was completed using the fully assembled catheter endoscope. Results:Metrology showed the printed lenses conformed closely to the design. The catheter endoscope's FWHM spot size had a mean ± standard deviation of 25.3 ± 1.8    μ m with a measured working distance of 1.960 ± 0.057    mm . Volumetric OCT images of the middle ear, inner ear, and Eustachian tube were captured in a postmortem pig head using the catheter endoscope. Conclusions:The 2PP approach is fast and highly repeatable for miniature reflective objective fabrication. OCT catheter endoscopy via the Eustachian tube enabled imaging of the middle ear, Eustachian tube, and surprisingly part of the inner ear.

A microendoscopic objective integrating a commercial glass lens with two-photon polymerization (2PP)-fabricated polymer optics is presented. This ultra-compact, high-NA (0.6) design enables achromatic correction at 775 nm and 860 nm for multi- wavelength nonlinear biomedical imaging.

The work presents examples of two imaging spectrometers enabled with 2-Photon Polymerization 3D printing. The presented spectrometers are field integral devices and image reorganization is achieved with high density waveguide array (waveguide spectrometer) or multifaceted, multi-angle mirror array respectively (Image Mapping Spectrometer - IMS). Fabricated components allow void spaces between image points dedicated for spectral information. High precision and repeatability of the fabricated components simplifies system calibration, improved image quality allows and more compact systems implementations.

In this paper, we present a 2-photon imaging probe system featuring a novel fluorescence collection method with improved and reliable efficiency. The system aims to miniaturize the potential of 2-photon imaging in the metabolic and morphological characterization of cervical tissue at sub-micron resolution over large imaging depths into a flexible and clinically viable platform towards the early detection of cancers. Clinical implementation of such a probe system is challenging due to inherently low levels of autofluorescence, particularly when imaging deep in highly scattering tissues. For an efficient collection of fluorescence signals, our probe employs 12 0.5 NA collection fibers arranged around a miniaturized excitation objective. By bending and terminating a multitude of collection fibers at a specific angle, we increase collection area and directivity significantly. Positioning of these fibers allows the collection of fluorescence photons scattered away from their ballistic trajectory multiple times, which offers a system collection efficiency of 4%, which is 55% of what our bench-top microscope with 0.75 NA objective achieves. We demonstrate that the collection efficiency is largely maintained even at high scattering conditions and high imaging depths. Radial symmetry of arrangement maintains uniformity of collection efficiency across the whole FOV. Additionally, our probe can image at different tissue depths via axial actuation by a dc servo motor, allowing depth dependent tissue characterization. We designed our probe to perform imaging at 775 nm, targeting 2-photon autofluorescence from NAD(P)H and FAD molecules, which are often used in metabolic tissue characterization. An air core photonic bandgap fiber delivers laser pulses of 100 fs duration to the sample. A miniaturized objective designed with commercially available lenses of 3 mm diameter focuses the laser beam on tissue, attaining lateral and axial imaging resolutions of 0.66 µm and 4.65 µm, respectively. Characterization results verify that our probe achieves collection efficiency comparable to our optimized bench-top 2-photon imaging microscope, minimally affected by imaging depth and radial positioning. We validate autofluorescence imaging capability with excised porcine vocal fold tissue samples. Images with 120 µm FOV and 0.33 µm pixel sizes collected at 2 fps confirm that the 300 µm imaging depth was achieved.

We present a flexible catheter endoscope using a 3D printed miniature side-viewing reflective lens for OCT imaging. A reflective lens with a depth of field (DOF) of 3mm and a maximum spot size of 80 microns was first designed and optimized in OpticStudio (ZEMAX). The structure of the lens and the fiber mating part of the optic was then constructed using 2-photon polymerization technique, and a gold reflective coating was added as the final step for the reflective lens. The reflective lens was then glued to cleaved optical fiber, and the assembly was covered by a torque coil to allow rotation of the catheter endoscope. This endoscope was used with a fiber-optic rotary joint and custom-built Mach-Zehnder interferometer to acquire OCT images.

We extend the resolution of our 2p autofluorescence imaging probe by incorporation of custom designed and fabricated optics. We will add an NA extender piece, inserted at excitation fiber tip to boost the beam divergence at the excitation optics input. The new custom designed miniaturized objective will achieve a resolution of 0.5 μm, without any changes to the diameter of the objective design. Custom design objective also will allow us to perform autofluorescence imaging at two colors. This will enable the optical redox ratio characterization of tissues, which is an important hallmark in diagnosis of cancers at early stages.

This presentation focuses on examples of devices and components to be used in actual biological and biomedical applications and manufactured in larger quantities. Specifically, we discuss additive manufacturing (full 3D and 2.5D grayscale modes) based on 2PP technology, which allows features of 100nm-150nm and surface roughness of 10-20 nm – sufficient for optical quality components. In addition, the printing volume allows parts of up to 50x50x22 mm3 and thus broader range of possible designs. Here we demonstrate two system examples: (1) high performance (NA=0.6, FOV=200microns, OD = 3.0 mm) hybrid endoscopic microscope objective for 2-photon imaging and diagnostics and (2) image mapping spectrometer for cell signaling in SPIM (Selective Plane Illumination Microscopy) configuration. In both cases we discuss performance of manufactured components and design strategy to optimize both printing time and component/system quality. Presented prototypes demonstrate high level of integration, compact dimensions and design flexibility. Results include high resolution imaging performance (miniature endo-microscopic objective) and snapshot spectral imaging capabilities in cell signaling.

Custom fiber arrays can be used to encode 3-dimensional data for snapshot imaging techniques like imaging spectrometry or volumetric spectral domain OCT. This is achieved if array’s input is dense, while its output creates void spaces for spectral information. Here we present fiber arrays with entirely automatic development process based on 2-Photon Polymerization (2PP) additive manufacturing using Nanoscribe GmbH Quantum X system. Specifically, We developed two types of array prototypes: 10x10 to 1x100 and 20x20 - dense fiber spacing (1-2 microns fiber gap) to 20x20 – sparse fiber spacing (30-40 microns fiber gap). Fiber arrays were incorporated into prism-based imaging spectrometer system to demonstrate proof of concept spectral imaging experiments.

RPPA data showing fold changes in transcriptional regulator expressions in saracatinib treated 10A.B2 cells over vehicle control.

Advances in 2-photon lithography have enabled in-lab production of sub-micron resolution and millimeter scale 3D optical components. The potential complex geometries are well suited to rapid prototyping and production of waveguide structures, interconnects, and waveguide directional couplers, furthering future development and miniaturization of waveguide-based imaging technologies. System alignment is inherent to the 2-photon process, obviating the need for manual assembly and allowing precise micron scale waveguide geometries not possible in traditional fused fiber coupler fabrication. Here we present the use of 2-photon lithography for direct printing of multi-mode waveguide couplers with air cladding and single mode waveguide couplers with uncured liquid photoresin cladding. Experimental results show reproducible coupling which can be modified by selected design parameters.

We report on a proof-of-concept snapshot imaging spectrometer developed using an array of optical fibers fabricated with 2-photon polymerization (2PP). The dense input array maps to an output array with engineered void spaces for spectral information. Previously, the development and fabrication of custom fiber arrays for imaging spectrometers have been a complex, time-consuming, and costly process, requiring a semi-manual assembly of commercial components. This work applies an automatic development process based on 2PP additive manufacturing with the Nanoscribe GmbH Quantum X system. The technique allows printing of arbitrary optical quality structures with submicron resolution with less than 5 nm roughness, enabling small core fibers/integrated arrays. Specifically, we developed an array prototype of 40 × 80 with 6-micron pitch at the input and 80-micron pitch at the output. The air-clad fibers had a core diameter of 5 µm. Fabricated optical fiber arrays were incorporated into a prism-based imaging spectrometer system with 48 spectral channels to demonstrate multi-spectral imaging. Imaging of a USAF target and color printed letter C as well as spectral comparisons to a commercial spectrometer were used to validate the performance of the system. These results clearly demonstrate the functionality and potential applications of the 3D-printed fiber-based snapshot imaging spectrometer.

A design and fabrication technique for making high-precision and large-format multifaceted mapping mirrors is presented. The method is based on two-photon polymerization, which allows more flexibility in the mapping mirror design. The mirror fabricated in this paper consists of 36 2D tilted square pixels, instead of the continuous facet design used in diamond cutting. The paper presents a detailed discussion of the fabrication parameters and optimization process, with particular emphasis on the optimization of stitching defects by compensating for the overall tilt angle and reducing the printing field of view. The fabricated mirrors were coated with a thin layer of aluminum (93 nm) using sputter coating to enhance the reflection rate over the target wave range. The mapping mirror was characterized using a white light interferometer and a scanning electron microscope, which demonstrates its optical quality surface (with a surface roughness of 12 nm) and high-precision tilt angles (with an average of 2.03% deviation). Finally, the incorporation of one of the 3D printed mapping mirrors into an image mapping spectrometer prototype allowed for the acquisition of high-quality images of the USAF resolution target and bovine pulmonary artery endothelial cells stained with three fluorescent dyes, demonstrating the potential of this technology for practical applications.

Imaging modalities capable of detecting functional changes over small areas can increase sensitivity and specificity of early cancer detection. Label-free imaging of metabolic activity at cellular level resolution over full thickness of cervix epithelium is possible with 2p imaging. However, low probability of 2p excitation and scattering nature of tissues limit autofluorescence levels in 2p imaging. We present a 2p autofluorescence imaging endoscope system for detection of metabolic changes in cervix in a clinical setting, with an increased collection efficiency in scattering media. Collection of autofluorescence signals is done with a multitude of high NA fibers arranged around a miniaturized excitation objective. By cleaving the collection fibers at a specific angle, we increase the directivity of the collection and the collection efficiency per fiber. The endoscope performs imaging at 775 nm, which is capable of exciting NAD(P)H and FAD molecules. Laser pulses of 100 fs duration are delivered to the sample with an air core photonic bandgap fiber. Fiber is scanned in spiral pattern via a piezo actuator tube. Scanning at different tissue depths is possible with the axial actuation of the endoscope via a linear stepper motor. Benchtop tests indicate that the endoscope system has lateral and axial resolutions of 0.65 μm and 4.33 μm, respectively. Fluorescence images of pollen cores are presented to demonstrate the imaging quality of the endoscope system.

Saracatinib increases tumor free and overall survival in ER- mammary mouse models.