Editorial Feature

Evaluating Novel Optical Temperature Mapping Modality to Overcome Limitations of Existing Optical Instruments

Photoluminescence lifetime imaging of upconverting nanoparticles is greatly featured in recent progress in optical thermometry. However, the current optical instruments are void of wide-field photoluminescence lifetime imaging. Looking at Liu, X et al's paper published in Nature Communications, this article describes video-rate upconversion temperature sensing in wide-field utilizing single-shot photoluminescence lifetime imaging thermometry (SPLIT).

Image Credit: GiroScience/Shutterstock.com

The crucial parameter linked to numerous chemical, physical, and biological processes is temperature. For scientific research and industrial applications, accurate and real-time temperature sensing at microscopic scales is significant.

Photoluminescence lifetime imaging (PLI) became a promising approach to temperature sensing in the last few decades as it can be excited and detected optically. The resultant non-contact PLI has a high spatial resolution and advantage that overcomes the intrinsic limitation in spatial resolution of imaging thermography.

PLI also brings greater flexibility in sample selection along with numerous other advantages and thus PLI is a prevalent choice for optical thermometry. PLI’s success in temperature is based on two vital constituents—temperature indicators and optical imaging instruments.

Numerous labeling agents were discovered recently for PLI-based temperature sensing of which lanthanide-doped upconverting nanoparticles (UCNPs) are ideal candidates.

The merits of UCNPs make them frontrunners in temperature indicators for PLI.

PLI-based temperature mapping uses another indispensable constituent called advanced optical imaging. The majority of the PLI techniques employ point-scanning time-correlated single-photon counting (TCSPC) to pinpoint photoluminescence on the time scale of microseconds to milliseconds, similar to the ones produced by UCNPs.

However, the existing PLI techniques are inadequate in 2D temperature sensing of mobile samples having a micrometer-level spatial resolution.

This article intends to address the limitations by reporting an optical temperature mapping modality, called single-shot photoluminescence lifetime imaging thermometry (SPLIT).

SPLIT registers wide-field luminescence decay of Yb3+, Er3+, co-doped NaGdF4 UCNPs in real-time, and allows longitudinal 2D temperature monitoring underneath a thin scattering medium and dynamic temperature analysis of a mobile biological sample at single-cell resolution.

Results

Figure 1 illustrates the SPLIT system. A continuous-wave laser is the light source and the laser beam flows through a 4f system having two 50 mm focal length lenses. The optical chopper is located behind the back focal plane of the lens. This illumination scheme creates wide-field illumination to UCNPs at the object plane.

Schematic of the SPLIT system. The illustration shows data acquisition and image reconstruction of luminescence intensity decay in a letter “C”. L1–L5, lens.

Figure 1. Schematic of the SPLIT system. The illustration shows data acquisition and image reconstruction of luminescence intensity decay in a letter “C”. L1–L5, lens. Image Credit: Liu, et al., 2021.

The UCNPs excited in the near-infrared emits light in the visible spectral range. The emitted light is gathered by the same objective lens, transmitted to a dichroic mirror, and filtered by a band-pass filter. The beam splitter splits the light into two components. The spatially encoded scene is pictured by a mechanical streak camera. The SPLIT system’s hardware is synchronized for apprehending both views that are calibrated before data acquisition.

The retrieved datacube has a sequence depth of 12-100 frames and the reconstructed datacube is later converted to a photoluminescence lifetime map. The SPLIT system, depending on the intrinsic frame rate of the EMCCD camera, produces lifetime-determined temperature maps at a video rate of 20 Hz. To demonstrate SPLIT’s abilities, a series of core/shell UCNP samples were prepared (see Figure 2).

Quantification of the performance of the SPLIT system. (a) Images of core/shell UCNPs acquired with a transmission electron microscope. Scale bar: 25 nm. (b) Normalized upconversion spectra of UCNPs are shown in (a). (c) Simplified energy level diagram of Yb3+-Er3+ energy transfer upconversion excitation and emission. (d) Temporally projected image of photoluminescence intensity decay of the 5.6 nm-thick-shell UCNPs covered by a negative resolution target. (e) Comparison of averaged light fluence distribution along the horizontal bars (blue) and vertical bars (orange) of Element 5 in Group 4 on the resolution target. Error bar: standard deviation. (f) Lifetime images of UCNPs with shell thicknesses of 1.9, 3.5, and 5.6 nm covered by transparencies of letters “C”, “A”, and “N” in green emission. (g) Time-lapse averaged emission intensities of the samples. (h) Histograms of photoluminescence lifetimes in the letters shown in (f).

Figure 2. Quantification of the performance of the SPLIT system. (a) Images of core/shell UCNPs acquired with a transmission electron microscope. Scale bar: 25 nm. (b) Normalized upconversion spectra of UCNPs are shown in (a). (c) Simplified energy level diagram of Yb3+-Er3+ energy transfer upconversion excitation and emission. (d) Temporally projected image of photoluminescence intensity decay of the 5.6 nm-thick-shell UCNPs covered by a negative resolution target. (e) Comparison of averaged light fluence distribution along the horizontal bars (blue) and vertical bars (orange) of Element 5 in Group 4 on the resolution target. Error bar: standard deviation. (f) Lifetime images of UCNPs with shell thicknesses of 1.9, 3.5, and 5.6 nm covered by transparencies of letters “C”, “A”, and “N” in green emission. (g) Time-lapse averaged emission intensities of the samples. (h) Histograms of photoluminescence lifetimes in the letters shown in (f). Image Credit: Liu, et al., 2021

SPLIT’s spatial resolution was characterized and found to be 20 µm, and the minimum power density was observed as 0.06 W mm−2. The ability to differentiate various lifetimes was quantified and the results were verified employing a standard TCSPC method. It was noted that the dual-view PnP-ADMM algorithm utilized by SPLIT is more robust in preserving spatial features.

A 5.6-nm-thick-shell UCNPs was used as the temperature indicator for SPLIT and the temperature of the whole sample was evaluated. The reconstructed lifetime images in the temperature range of 20 °C–46 °C are illustrated in Figure 3a–b.

Single-shot temperature mapping using SPLIT. (a, b) Lifetime images of green (a) and red (b) upconversion emission bands under different temperatures. (c, d) Normalized photoluminescence decay curves of green (c) and red (d) emission bands at different temperatures, averaged over the entire field of view. e Relationship between temperature and mean lifetimes of green and red emissions with linear fitting. Error bar: standard deviation from three independent measurements. f Normalized contrast versus chicken tissue thickness for green and red emission bands with single-component exponential fitting. g Longitudinal temperature monitoring through 0.5 mm-thick fresh chicken tissue.

Figure 3. Single-shot temperature mapping using SPLIT. (a, b) Lifetime images of green (a) and red (b) upconversion emission bands under different temperatures. (c, d) Normalized photoluminescence decay curves of green (c) and red (d) emission bands at different temperatures, averaged over the entire field of view. e Relationship between temperature and mean lifetimes of green and red emissions with linear fitting. Error bar: standard deviation from three independent measurements. f Normalized contrast versus chicken tissue thickness for green and red emission bands with single-component exponential fitting. g Longitudinal temperature monitoring through 0.5 mm-thick fresh chicken tissue. Image Credit: Liu, et al., 2021.

SPLIT’s feasibility in a biological environment was analyzed and the results obtained were in agreement. This indicates that SPLIT can map 2D temperatures with great precision beneath biological tissue. When a fresh beef phantom was used, the results affirmed the independence of the measured photoluminescence lifetime of UCNPs.

Dynamic single-cell temperature mapping was examined using a single-layer onion epidermis sample marked by the 5.6 nm-thick-shell UCNPs. It was observed that the temperatures were stable, indicating SPLIT’s resilience to spatial intensity variation. Figure 4 shows SPLITs dynamic single-cell temperature mapping.

Dynamic single-cell temperature mapping using SPLIT. (a) Representative time-integrated images of a moving onion epidermis cell sample labeled by UCNPs. (b) Lifetime images corresponding to (a). (c) Photoluminescence decay profiles at four selected areas [marked by the solid boxes in the first panel of (a)] with varied intensities. (d) Time histories of averaged fluence and corresponding temperature in the four selected regions during the sample’s translational motion.

Figure 4. Dynamic single-cell temperature mapping using SPLIT. (a) Representative time-integrated images of a moving onion epidermis cell sample labeled by UCNPs. (b) Lifetime images corresponding to (a). (c) Photoluminescence decay profiles at four selected areas [marked by the solid boxes in the first panel of (a)] with varied intensities. (d) Time histories of averaged fluence and corresponding temperature in the four selected regions during the sample’s translational motion. Image Credit: Liu, et al., 2021.

Discussion

SPLIT was developed for wide-field dynamic temperature sensing in real-time and it registers the photoluminescence emission over a 2D FOV in two views. The enhanced parallelism in SPLIT’s data acquisition enhances the entire light throughput and it addresses the lasting issue in scanning-based techniques.

SPLIT also enhances the measurement accuracy and, when compared with thermal imaging cameras, SPLIT provides better temperature mapping results with greater image contrast and better resilience to background interference.

The PnP-ADMM algorithm facilitates high imaging quality in SPLIT. It also provides a multifaceted PLI temperature-sensing platform. SPLIT can be used in materials characterization, biomedicine, and studies of temperature-regulated cellular signaling.

Methodology

The optical chopper outputs a transistor-transistor logic (TTL) signal synchronal with the created optical pulses. The GS deflects temporal information to various spatial positions and by rotating during the data acquisition. The angular difference is modified to the lateral shift in space on the EMCCD camera resulting in temporal shearing. SPLITs imaging speed ranged from 4-33 kfps.

Journal Reference:

Liu, X., Skripka, A., Lai, Y., Jiang, C., Liu, J., Vetrone, F., Liang, J. (2021) Fast wide-field upconversion luminescence lifetime thermometry enabled by single-shot compressed ultrahigh-speed imaging. Nature Communications, 12(6401). doi.org/10.1038/s41467-021-26701-1.

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