Wide-field fluorescent nanodiamond spin measurements toward real-time large-area intracellular thermometry
At a Glance
Section titled âAt a Glanceâ| Metadata | Details |
|---|---|
| Publication Date | 2021-02-19 |
| Journal | Scientific Reports |
| Authors | Yushi Nishimura, Keisuke Oshimi, Yumi Umehara, Yuka Kumon, Kazu Miyaji |
| Institutions | Keio University, Osaka City University |
| Citations | 47 |
| Analysis | Full AI Review Included |
Executive Summary
Section titled âExecutive Summaryâ- Core Achievement: Validation of camera-based wide-field Optically Detected Magnetic Resonance (ODMR) using nanodiamonds (NDs) for real-time, large-area intracellular thermometry in living HeLa cells.
- Sensitivity Comparison: Wide-field ODMR achieved temperature sensitivities (1.7-2.2 K/sqrt(Hz)) comparable to those obtained via traditional photon-counter-based confocal detection (2.1 K/sqrt(Hz)) under the same setup.
- Artifact Management: The study identified and characterized critical artifacts specific to wide-field detection, including contrast degradation due to background fluorescence and frequency shift errors caused by camera pixel saturation (Full-Well Capacity, FWC).
- Positional Robustness: Wide-field detection proved robust against ND z-positional variation (defocusing), but lateral drift required careful selection of the Region of Interest (ROI) to prevent noise and spectral distortion.
- Technological Roadmap: The results provide realistic acquisition parameters necessary for integrating rapid, multi-point ODMR protocols into wide-field systems, paving the way for real-time, large-area thermal live-imaging in biological applications.
Technical Specifications
Section titled âTechnical Specificationsâ| Parameter | Value | Unit | Context |
|---|---|---|---|
| ND Particle Size | 100 | nm | NDNV100nmHi10ml (AdĂĄmas Nanotechnologies) |
| NV Centers per ND | ~500 | - | Estimated concentration per particle |
| Excitation Wavelength | 532 | nm | Continuous-wave laser |
| Excitation Power Density | ~10 | W/cm2 | Typical optical excitation intensity |
| ODMR Frequency Range | 2.810 to 2.93 | GHz | Microwave sweep range |
| Microwave Power (Antenna) | 10 to 50 | mW | Estimated power delivered to the linear antenna |
| Temperature Dependence (dD/dT) | -74 | kHz/K | Zero-field splitting temperature coefficient |
| Wide-Field Sensitivity (in cells) | 1.7 to 2.2 | K/sqrt(Hz) | Measured precision in living HeLa cells |
| Confocal Sensitivity (in cells) | 2.1 | K/sqrt(Hz) | Measured precision in living HeLa cells |
| Camera Type | EMCCD (Evolve Delta) | - | Wide-field detection system |
| Camera FWC (Single Pixel) | 185,000 | e- | Full-Well Capacity |
| Camera ADC Resolution | 16 | bit | Analog-to-Digital Converter |
| Wide-Field Exposure Time (Atexp) | 10 | ms | Standard measurement parameter |
| Wide-Field Accumulations (nacc) | 100 | - | Accumulations per frequency step |
| Confocal Integration Time (Atpc) | 100 | ms | Standard measurement parameter |
| Dish Temperature Stability (Ta) | ±0.25 | K | Stability over 250 min using thermistor |
| Spatial Resolution (Confocal) | 0.24 | ”m | FWHM of fluorescence spot |
| Spatial Resolution (Wide-Field) | 0.55 | ”m | FWHM of fluorescence spot |
Key Methodologies
Section titled âKey Methodologiesâ- Hybrid Microscopy Setup: A single home-built microscope was configured to switch between confocal detection (using an APD gated by a bit pattern generator) and wide-field detection (using an EMCCD camera slaved to the generator).
- Sample Preparation and Environment Control: HeLa cells were labeled with 100 nm NDs and cultured in custom antenna-integrated glass dishes. Phenol red was removed from the medium to eliminate laser absorption and heating artifacts.
- Temperature Calibration and Control: Dish temperature (Ta) was precisely controlled using a stage-top incubator with PID feedback on foil heaters, calibrated against an external Pt100 thermistor (Ta = 5.51 + 0.814Ti).
- Wide-Field Data Acquisition: The EMCCD acquired 16-bit images (10 ms exposure) for both microwave ON (signal) and OFF (reference) states, accumulating 100 images per frequency step to form a 32-bit average image.
- Artifact Characterization: The relationship between camera photon counts (mG-1Iccd) and APD photon counts (Râtpc) was established. Experiments were conducted to map the effects of pixel saturation (FWC limit: 185,000 e-) and positional drift (lateral and z-axis) on ODMR contrast and center frequency.
- Intracellular Thermometry: Wide-field ODMR spectra were measured from multiple NDs simultaneously within living HeLa cells across different dish temperatures (e.g., 35.5 °C and 33.7 °C).
- Spectral Analysis: ODMR spectra were generated by dividing signal images by reference images, and the center frequency shift (ÎÏ) was determined by fitting the spectra with a Gaussian function to calculate the temperature change.
Commercial Applications
Section titled âCommercial Applicationsâ- Quantum Sensing and Imaging: Advancing the development of scalable quantum sensors based on NV centers, moving beyond single-point measurements to parallel, large-area thermal and magnetic imaging.
- Cellular and Subcellular Thermometry: Providing a tool for real-time monitoring of temperature gradients and thermogenesis within living cells, crucial for studying molecular mechanisms related to cell death (e.g., photothermal cancer therapy) and cellular signaling (e.g., thermotaxis).
- High-Throughput Biological Screening: Enabling simultaneous temperature measurements across multiple cells or regions in a large field of view, accelerating research in drug discovery and physiological studies.
- Microscope and Detector Technology: Driving the demand for specialized scientific cameras (e.g., sCMOS or EMCCD) with high FWC (greater than 185,000 e-), high bit depth (greater than 16-bit), and low noise to optimize wide-field quantum sensing performance.
- Nanodiamond Material Science: Requiring the production of highly optimized NDs with high NV concentration, low background fluorescence, and robust functionalization for stable intracellular delivery and targeting of specific organelles (e.g., mitochondria, lysosomes).