A 25 micron-thin microscope for imaging upconverting nanoparticles with NIR-I and NIR-II illumination

Abstract

Rationale: Intraoperative visualization in small surgical cavities and hard-to-access areas are essential requirements for modern, minimally invasive surgeries and demand significant miniaturization. However, current optical imagers require multiple hard-to-miniaturize components including lenses, filters and optical fibers. These components restrict both the form-factor and maneuverability of these imagers, and imagers largely remain stand-alone devices with centimeter-scale dimensions. Methods: We have engineered INSITE (Immunotargeted Nanoparticle Single-Chip Imaging Technology), which integrates the unique optical properties of lanthanide-based alloyed upconverting nanoparticles (aUCNPs) with the time-resolved imaging of a 25-micron thin CMOS-based (complementary metal oxide semiconductor) imager. We have synthesized core/shell aUCNPs of different compositions and imaged their visible emission with INSITE under either NIR-I and NIR-II photoexcitation. We characterized aUCNP imaging with INSITE across both varying aUCNP composition and 980 nm and 1550 nm excitation wavelengths. To demonstrate clinical experimental validity, we also conducted an intratumoral injection into LNCaP prostate tumors in a male nude mouse that was subsequently excised and imaged with INSITE. Results: Under the low illumination fluences compatible with live animal imaging, we measure aUCNP radiative lifetimes of 600 μs - 1.3 ms, which provides strong signal for time-resolved INSITE imaging. Core/shell NaEr_0.6Yb_0.4F₄ aUCNPs show the highest INSITE signal when illuminated at either 980 nm or 1550 nm, with signal from NIR-I excitation about an order of magnitude brighter than from NIR-II excitation. The 55 μm spatial resolution achievable with this approach is demonstrated through imaging of aUCNPs in PDMS (polydimethylsiloxane) micro-wells, showing resolution of micrometer-scale targets with single-pixel precision. INSITE imaging of intratumoral NaEr_0.8Yb_0.2F₄ aUCNPs shows a signal-to-background ratio of 9, limited only by photodiode dark current and electronic noise. Conclusion: This work demonstrates INSITE imaging of aUCNPs in tumors, achieving an imaging platform that is thinned to just a 25 μm-thin, planar form-factor, with both NIR-I and NIR-II excitation. Based on a highly paralleled array structure INSITE is scalable, enabling direct coupling with a wide array of surgical and robotic tools for seamless integration with tissue actuation, resection or ablation.

Keywords: NIR excitation; intraoperative imaging; time-resolved imaging; upconverting nanoparticle.

Figure 1

INSITE imaging of upconverting nanoparticles. (A) Multiphoton energy absorption, transfer, and emission in Yb³⁺/Er³⁺-based aUCNPs following either NIR-I (980 nm) or NIR-II (1550 nm) excitation. (B) Cartoon of INSITE directly integrated onto surfaces, such surgical glove. (C) Diagram of the time-resolved image acquisition scheme. T_exc, pulse excitation time; T_int, emission signal integration time; T_delay, time gating delay. (D) Angle-selective gratings used to achieve lensless image acquisition and block obliquely incident background light.

Figure 2

INSITE configuration for imaging of aUCNP dispersions. Laser beam width is 300 μm.

Figure 3

Emission decays of aUCNPs (T_int = 1 ms, T_exc = 5 ms) as measured by pixel output at (A) 8 W/cm² of 980 nm excitation, or (B) 60 W/cm² of 1550 nm excitation. T_int is integation time; T_exc is duration of exciation light pulse.

Figure 4

Integrated visible emission as a function of aUCNP composition at either 980 and 1550 nm excitation, both with 8 W/cm² excitation power density. Hexane blank is without aUCNPs.

Figure 5

(A) Photograph of CMOS contact imager and PDMS micro-well holding aUCNPs, fabricated for this study. (B) Time-resolved image of the aUCNP-coated surface of the micro-well. Normalized emission intensity shown as in grayscale legend. Section (I) is background, (II) is outer PDMS with aUNCPs diffused into PDMS, and (III) is microwell with aUCNP sample. (C) Cross-section emission profile for the three regions in (B).

Figure 6

Live mouse images of intratumorally-injected NaEr_0.8Yb_0.2F₄ aUCNPs with 8 W/cm² 980 nm excitation. (A) Images of aUCNPs-injected into mouse prostate tumor (left) and non-injected side (right). Emission intensity as in colored legend. (B) Measured emission spectrum of the injected and non-injected sides, showing tumor-specific Er³⁺ emission bands at 545 and 655 nm.

Figure 7

Photograph of the tumor injected with aUCNPs (left side of mouse, from Figure 6) is excised and placed directly on INSITE for imaging. The area within the excised tissue where the aUCNPs are located (aUCNP spot) is circled. The remainder of the tissue does not contain aUCNPs. The path of the illumination laser is drawn in red. *This is for illustrative purposes only, and is not an image of the actual laser beam, which is not visible.

Figure 8

(A) Microscope image of excised tumor injected with 25 μL of a 250 nM aqueous dispersion of aUCNPs, excited with 1 W/cm² 980 nm, showing distinct localization of aUCNPs. (B) Images of NIR-II laser scanning of tumor, from top to bottom in increments of 300 μm (numbered 1-8). At each position, an image of the tumor sample is acquired with INSITE using a 5 millisecond-pulsed 60 W/cm² 1550-nm laser for illumination.