Abstract
We characterize long (up to 285 mm) gradient index (GRIN) lens endoscope systems for multiphoton imaging. We fabricate a portable, rigid endoscope system suitable for imaging unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The portable device is capable of imaging a ~200 µm diameter field of view at 4 frames/s. The lateral and axial resolution in water is 0.85 µm and 7.4 µm respectively. In vivo images of unstained tissues in live, anesthetized rats using the portable device are presented. These results show great promise for GRIN endoscopy to be used clinically.
🔬 Techniques
✨ Fluorophores
🧪 Sample Preparation
🔬 Cell Lines
🏭 Microscope Brands
🔴 Lasers
📷 Detectors
🎨 Filters
🏛️ Research Organizations (ROR)
Affiliated research institutions:
📋 Methods
3. Experimental setup Figure 1 Fig. 1 Experimental setup used for the optical characterization of the long gradient index endoscope systems and close-up of the doublet GRIN system design (shown here using a 0.75 relay lens pitch). shows the custom built horizontal multiphoton microscope used to characterize the GRIN lens systems. A mode-locked Ti:sapphire laser (Tsunami, Spectra Physics, Inc.) was used as the excitation source at 800 nm with 10 nm bandwidth. Dual axis (5 mm diameter) galvo based scan mirrors (GVSM002, Thorlabs Inc.), and two scan lenses of 10 cm and 30 cm focal length (respectively, AC508-100-B-ML and AC508-300-B-ML, Thorlabs Inc.) were used to scan the beam angle at the overfilled back aperture of a 0.1 NA microscope objective. The focus of the objective was raster scanned by the galvo mirrors across the proximal face of the GRIN lens systems. The GRIN lens systems were mounted on a three axis manual linear translational stage close to the focal plane of the objective to aid alignment. All optical characterizations were conducted by moving the sample mounted on a 3D stage (MP-285, Sutter Instruments) allowing axial scanning of the sample while maintaining the GRIN lens system in a fixed position. The fluorescent signal from the sample was epi-collected through the GRIN lenses and the microscope objective. Collected light is reflected by a dichroic beam splitter (FF-665-Di01, Semrock Inc.). After passing through two 575/250 bandpass filters (HQ575_250 2p, Chroma Technology Corp.) separated by a colored glass (FGS900, Thorlabs Inc.), the fluorescence is detected by a photo-multiplier tube (PMT) (HC125-02, Hamamatsu Photonics). Data acquisition and motion control were implemented using a DAQ card (PCI-6115, National Instruments Corp.) and MPScan software [ 25 ]. The axial resolution of the GRIN lens systems was characterized in air using the full width at half maximum (FWHM) two-photon excited fluorescence signal from a 500 nm thin film of Rhodamine B (RhB) dye, while the lateral resolution was characterized using FWHM two-photon excited fluorescence from subresolution (0.2 µm) fluorescent beads. The field of view (FOV) was characterized by raster-scanning the proximal face of the GRIN lens system and measuring the one-photon transmission using a photodiode (SM05PD1A, Thorlabs Inc.), and was defined as the FWHM of the resulting intensity profile.
Show full methods section
3. Experimental setup Figure 1 Fig. 1 Experimental setup used for the optical characterization of the long gradient index endoscope systems and close-up of the doublet GRIN system design (shown here using a 0.75 relay lens pitch). shows the custom built horizontal multiphoton microscope used to characterize the GRIN lens systems. A mode-locked Ti:sapphire laser (Tsunami, Spectra Physics, Inc.) was used as the excitation source at 800 nm with 10 nm bandwidth. Dual axis (5 mm diameter) galvo based scan mirrors (GVSM002, Thorlabs Inc.), and two scan lenses of 10 cm and 30 cm focal length (respectively, AC508-100-B-ML and AC508-300-B-ML, Thorlabs Inc.) were used to scan the beam angle at the overfilled back aperture of a 0.1 NA microscope objective. The focus of the objective was raster scanned by the galvo mirrors across the proximal face of the GRIN lens systems. The GRIN lens systems were mounted on a three axis manual linear translational stage close to the focal plane of the objective to aid alignment. All optical characterizations were conducted by moving the sample mounted on a 3D stage (MP-285, Sutter Instruments) allowing axial scanning of the sample while maintaining the GRIN lens system in a fixed position. The fluorescent signal from the sample was epi-collected through the GRIN lenses and the microscope objective. Collected light is reflected by a dichroic beam splitter (FF-665-Di01, Semrock Inc.). After passing through two 575/250 bandpass filters (HQ575_250 2p, Chroma Technology Corp.) separated by a colored glass (FGS900, Thorlabs Inc.), the fluorescence is detected by a photo-multiplier tube (PMT) (HC125-02, Hamamatsu Photonics). Data acquisition and motion control were implemented using a DAQ card (PCI-6115, National Instruments Corp.) and MPScan software [ 25 ]. The axial resolution of the GRIN lens systems was characterized in air using the full width at half maximum (FWHM) two-photon excited fluorescence signal from a 500 nm thin film of Rhodamine B (RhB) dye, while the lateral resolution was characterized using FWHM two-photon excited fluorescence from subresolution (0.2 µm) fluorescent beads. The field of view (FOV) was characterized by raster-scanning the proximal face of the GRIN lens system and measuring the one-photon transmission using a photodiode (SM05PD1A, Thorlabs Inc.), and was defined as the FWHM of the resulting intensity profile.
📊 Figures
Fig. 1
Experimental setup used for the optical characterization of the long gradient index endoscope systems and close-up of the doublet GRIN system design (shown here using a 0.75 relay lens pitch).
Fig. 2
Two-photon lateral and axial resolution of GRIN system 2C (285 mm length). (a) Lateral intensity line profile across a subresolution fluorescent bead with a Gaussian fit in black. (b) Axial intensity ...
Fig. 3
Normalized one photon transmission intensity across the field of view for (a) GRIN system 1B and (b) GRIN system 2C.
Fig. 4
Off-axis performance. Axial FWHM of GRIN system 2C in u00b5m plotted (a) Across the FOV and (b) Across a line (dashed blue in (a)) through the center of the FOV. Scale bar is 50u00b5m.
Fig. 5
Portable GRIN endoscope. (a) Optical drawing and (b) Solidworks drawing of the GRIN based endoscope system. Total system length of the portable device is 10.6u201d (including GRIN system).
Fig. 6
Unaveraged in vivo images of unstained rat tissue. The pseudo-color images show red SHG signal (<405 nm) and green intrinsic fluorescent emission (405-700 nm). (a) Image of the superficial kidney r...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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