🧬 biology

Widefield Nanodiamond Quantum Sensing Based on Light-Sheet Microscopy

This paper presents a widefield quantum sensing method using light-sheet microscopy to overcome the trade-off between high throughput and low phototoxicity in nanodiamond-based biological applications, enabling fast, three-dimensional, and sensitive optically detected magnetic resonance imaging with minimal out-of-focus illumination.

Original authors: Ren-Bao Liu, Shuo WANG, Ming-Zhong Ai, Jing-Wei Fan, Junchen Ye, Chao Lin, Quan Li

Published 2026-06-25

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CC BY 4.0

Original authors: Ren-Bao Liu, Shuo WANG, Ming-Zhong Ai, Jing-Wei Fan, Junchen Ye, Chao Lin, Quan Li

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

Imagine you are trying to listen to a tiny, whispering radio station (a nanodiamond) inside a very noisy, crowded room (a living cell). The problem with previous methods is that to hear the whisper, you had to turn on a giant, blinding spotlight that flooded the entire room. While this helped you hear the radio, the blinding light also burned the furniture and scared the people in the room, eventually causing the room to fall apart.

This paper introduces a clever new way to listen to these tiny radio stations without burning down the house.

The Problem: The "Floodlight" vs. The "Flashlight"

The researchers are working with nanodiamonds containing special defects called Nitrogen-Vacancy (NV) centers. Think of these nanodiamonds as tiny, super-sensitive thermometers and magnetic sensors that can fit inside a living cell. To read their data, scientists usually shine a laser on them.

The Old Way (Widefield): Imagine shining a giant floodlight through a window, illuminating the entire room at once. This is fast, but it creates a lot of "noise" (background light from other parts of the cell) and, more importantly, it cooks the cell with too much light (phototoxicity). The cell gets stressed and dies quickly.
The Confocal Way (Point-by-Point): Imagine using a tiny laser pointer and scanning the room dot-by-dot. This is gentle, but it takes forever to map the whole room, so you can't see fast-moving processes.

The Solution: The "Light Sheet"

The authors, led by researchers at The Chinese University of Hong Kong, developed a system called Light-Sheet Microscopy (LSM).

The Analogy:
Imagine the cell is a loaf of bread.

Old methods either drenched the whole loaf in water (floodlight) or tried to poke a hole in every single crumb (point-by-point).
The Light-Sheet method is like sliding a very thin, flat sheet of light through the bread, slicing it like a deli slicer. You only light up the specific slice you are looking at right now.

How It Works

The Slice: They shine a thin, flat sheet of green laser light from the side of the cell. This sheet is only about 1.5 micrometers thick (thinner than a human hair).
The View: They look at the cell from the top (perpendicular to the light sheet). Because the light only hits the slice they are looking at, they don't see the blurry, glowing mess from the rest of the cell.
The Scan: They move this light sheet up and down, slicing through the cell layer by layer, building a 3D picture very quickly.

What They Discovered

Using this "light sheet" approach, the team achieved three major things:

Clearer Hearing (Better Sensitivity): Because the light sheet ignores the rest of the cell, there is no background noise. It's like listening to a whisper in a quiet room instead of a noisy party. They found they could measure temperature changes inside the cell much more accurately than before.
Gentler Touch (Less Damage): This is the biggest win. Because they aren't blasting the whole cell with light, the cells stay alive much longer.
- The Test: When they used the old "floodlight" method, all the cells died in 20 minutes. With the "light sheet," the cells stayed healthy for over an hour, and some even survived for three hours while being monitored.
3D Mapping: They could take a 3D "photo" of the nanodiamonds inside the cell, showing exactly where they were and what the temperature was at that specific spot, all without hurting the cell.

The "Relaxometry" Trick

They also invented a way to measure magnetic noise (specifically, something called $T_1$ relaxation) inside the cell. Think of this as checking how "tired" the tiny radio stations are getting. They used a special pulse of light (like a quick flash) rather than a constant beam. This was even gentler on the cells, allowing them to watch the same living cell for three hours straight without it dying.

The Bottom Line

This paper doesn't claim to cure diseases or diagnose patients yet. Instead, it provides a new, gentler, and sharper tool for scientists to study living cells. It allows them to watch what happens inside a cell in 3D, for hours at a time, without the "flashlight" of the microscope killing the subject they are trying to study. It's like upgrading from a sledgehammer to a scalpel for looking inside living things.

Technical Summary: Widefield Nanodiamond Quantum Sensing Based on Light-Sheet Microscopy

Problem Statement
Nanodiamonds (NDs) containing nitrogen-vacancy (NV) centers are powerful quantum sensors for biological applications due to their biocompatibility, sub-micron resolution, and ability to detect temperature, magnetic fields, electric fields, and pressure. However, a critical bottleneck exists in live-cell sensing: the trade-off between high-throughput measurement and low phototoxicity. Optically detected magnetic resonance (ODMR), the standard readout method for NV centers, requires continuous laser irradiation. Conventional widefield ODMR illuminates the entire sample volume, causing significant out-of-focus excitation that leads to phototoxicity (oxidative stress, mitochondrial dysfunction, and DNA damage), limiting long-term viability. Conversely, confocal ODMR minimizes phototoxicity through point-scanning but is too slow for volumetric sensing and cannot capture simultaneous spatial correlations across multiple NDs. Total internal reflection fluorescence (TIRF) reduces excitation volume but restricts sensing to superficial layers, losing 3D information.

Methodology
To address these limitations, the authors developed a widefield quantum sensing platform integrating Light-Sheet Microscopy (LSM) with ND-ODMR (LSM-ODMR).

Optical Configuration: The system employs a side-mounted illumination objective to project a thin, two-dimensional laser sheet (green light at 532 nm for NV excitation) into the sample plane. Fluorescence is collected orthogonally by a detection objective and captured by a scientific CMOS (sCMOS) camera.
Scanning Mechanism: The light sheet is vertically movable along the $z$ -axis, enabling optical sectioning and 3D volumetric scanning without scanning the sample itself.
Sample Handling: To minimize optical aberrations from the coverslip, adherent cell samples are positioned at a tilted angle. Microwave radiation is delivered via an antenna to manipulate NV spin states.
Sensing Modalities: The system supports:
1. ODMR Thermometry: Monitoring temperature via shifts in the zero-field splitting parameter ( $D$ ).
2. $T_1$ Relaxometry: A widefield technique using pulsed laser sequences to measure spin-lattice relaxation times, sensitive to magnetic noise and reactive oxygen species (ROS).

Key Contributions

LSM-ODMR Integration: The first demonstration of a widefield ODMR system based on LSM for live-cell quantum sensing, combining high throughput with optical sectioning.
Phototoxicity Reduction: By eliminating out-of-focus excitation, the system significantly reduces the laser dose delivered to the biological sample compared to conventional widefield illumination.
Enhanced Sensitivity: The optical sectioning capability suppresses out-of-focus background fluorescence (from cell autofluorescence or staining dyes), improving the signal-to-noise ratio and measurement sensitivity.
3D Multi-Point Sensing: The platform enables simultaneous, spatially correlated measurements across multiple NDs within a 3D volume, overcoming the limitations of point-scanning methods.

Results

System Characterization: The light sheet thickness was characterized at approximately 1.5 $\mu$ m (FWHM), maintaining an axial resolution better than 2.1 $\mu$ m over a 24 $\mu$ m lateral range, sufficient for imaging entire HeLa cells.
3D Imaging and Sensing in Agarose: Using NDs embedded in agarose gel, the system achieved 3D imaging and simultaneous multi-point temperature sensing. The temperature sensitivity was measured at approximately 2.9 K $\sqrt{\text{Hz}}^{-1}$ for individual NDs, with a median sensitivity of 4.47 K $\sqrt{\text{Hz}}^{-1}$ across 78 NDs.
Live-Cell Sensitivity Comparison: In BCECF-stained HeLa cells, LSM-ODMR demonstrated superior sensitivity (4.07 K $\sqrt{\text{Hz}}^{-1}$ ) compared to conventional widefield illumination (8.87 K $\sqrt{\text{Hz}}^{-1}$ ) at identical power densities (~5 $\mu$ W $\mu$ m $^{-2}$ ). The LSM method effectively mitigated background interference from the dye.
Phototoxicity Assessment: Under identical laser power densities, HeLa cells illuminated with LSM remained viable for up to 60 minutes (with only one death observed), whereas cells under widefield illumination died within 20 minutes.
Long-Term Monitoring:
- ODMR: The system successfully monitored intracellular temperature changes over one hour, with ND-derived temperature trends closely matching external resistance temperature detector (RTD) readings.
- $T_1$ Relaxometry: A widefield $T_1$ relaxometry method was developed and applied to live cells. Continuous monitoring of $T_1$ values for intracellular NDs was sustained for up to three hours with no significant changes detected, confirming cell viability throughout the experiment.

Significance
The paper claims that this LSM-based widefield nanodiamond sensing platform provides a robust approach for biological studies requiring low phototoxicity and high spatial resolution. By eliminating out-of-focus illumination, the system extends the viable monitoring window for live cells to several hours, enabling the investigation of extended physiological processes such as the mitotic phase of cell division. The ability to perform simultaneous, 3D, multi-modal sensing (temperature and $T_1$ ) with spatial correlation offers a new avenue for quantitative, long-term studies of dynamic intracellular processes, such as tracking radical levels or temperature fluctuations at the subcellular level, without the artifacts associated with single-point measurements or high phototoxicity. The authors note that while Gaussian light sheets face diffraction limits, future implementations could utilize non-scanning Airy light sheets to further optimize performance.