Researchers at the Tata Institute of Fundamental Research (TIFR) in Mumbai have achieved a remarkable feat in chemical biology by developing a new class of molecular sensors called ABATaRs, enabling the real-time visualization of chemical reactions inside living cells. This breakthrough, detailed in a recent publication in the renowned journal Angewandte Chemie International Edition, allows scientists to track elusive biomolecules such as hydrogen peroxide (H₂O₂) and copper ions (Cu²⁺) with unprecedented precision and sensitivity. Led by Professor Ankona Datta from TIFR's Department of Chemical Sciences, along with graduate student Sujit Kumar Das and collaborators from Osaka University, the technique leverages spontaneous Raman scattering—a label-free imaging method—to peer into the dynamic chemical world of cells without disrupting their natural processes.

The significance of this innovation cannot be overstated. Living cells are bustling factories where thousands of chemical reactions occur simultaneously, governing everything from metabolism to signaling pathways. However, traditional imaging techniques like fluorescence microscopy often require invasive labels that can alter cellular behavior or photobleach over time. ABATaRs address these limitations, offering a non-invasive, multiplexed approach that operates at low sensor concentrations (as little as 1-5 µM), making it accessible even on standard spontaneous Raman setups rather than expensive stimulated Raman systems.

This development not only highlights TIFR's prowess in chemical biology but also positions Indian higher education institutions at the forefront of global bioimaging research. Supported by the Department of Atomic Energy (DAE), Government of India, and the Japan Science and Technology Agency (JST), the work exemplifies international collaboration in advancing scientific frontiers.

Understanding cellular chemistry in real time has long been a holy grail in biology. Cells maintain delicate balances of reactive oxygen species (ROS) like H₂O₂, which act as signaling molecules at physiological levels but trigger oxidative stress and diseases such as cancer, neurodegeneration, and cardiovascular disorders when dysregulated. Similarly, Cu²⁺ is crucial for enzymatic functions but its imbalance is implicated in Alzheimer's disease and Wilson’s disease.

Prior methods struggled with sensitivity and multiplexing. Fluorescence probes, while popular, suffer from background autofluorescence and the need for genetic engineering or toxic dyes. Raman spectroscopy, which detects molecular vibrations via inelastic light scattering, offers a spectral fingerprint unique to each molecule. However, the Raman cross-section for bio-relevant tags like alkynes (C≡C stretch around 2100-2260 cm^-1, in the cell-silent region) is notoriously low, limiting detection in living systems.

The TIFR team overcame this by engineering ABATaRs—Activity-Based Alkyne Tag Ratiometric sensors. These smart molecules incorporate a 'push-pull' electronic architecture: an electron-donating group pushes density toward the alkyne, while an electron-withdrawing group pulls it, dramatically boosting the Raman scattering cross-section by 12-34 times compared to benchmark EdU (5-ethynyl-2'-deoxyuridine). Experimental intensities surpass EdU by 5-22 fold, with relative cross-sections up to 466 versus DMSO C-H stretch.

ABATaRs are built around a central alkyne tag flanked by donor-acceptor moieties. Upon encountering a target analyte, they undergo a specific chemical reaction that shifts the alkyne Raman peak by 9-18 cm^-1, providing a ratiometric readout (before/after shift ratio) independent of sensor concentration or instrument variations.

• pH sensor: Protonation alters the push-pull balance, shifting the peak.
• H₂O₂ sensor: Oxidation reaction modifies the alkyne environment.
• Cu²⁺ sensor: Coordination or redox reaction induces the shift.

This reaction-based activation ensures high specificity. The sensors are cell-permeable, crossing membranes without carriers, and non-toxic at working concentrations. In proof-of-concept experiments, HeLa cells loaded with 1-5 µM sensors showed clear ratiometric images of cytosolic and organelle-localized analytes under physiological (e.g., H₂O₂ ~1-10 µM) and pathophysiological conditions (e.g., elevated during inflammation).

Crucially, multiplexing was demonstrated: dual imaging of H₂O₂ and Cu²⁺ in the same cell, with distinct peaks resolving spatial dynamics—something fluorescence struggles with due to spectral overlap.

1. Sensor Design: Synthesize push-pull alkynes with analyte-reactive groups, optimizing for ~2200 cm^-1 peak in silent region.
2. Cell Loading: Incubate live cells (e.g., HeLa, neurons) with 1-5 µM sensor for 30-60 min; sensors diffuse in.
3. Stimulation: Induce analyte changes (e.g., growth factors for H₂O₂ burst, Cu chelators).
4. Imaging: Use spontaneous Raman microscope (e.g., 532 nm laser); scan alkyne region pre/post-stimulation.
5. Analysis: Ratiometric maps (shifted peak / unshifted) quantify local concentrations spatiotemporal dynamics.

This workflow accelerates imaging speed, enabling minute-scale resolution versus hours for older methods.

The study's success stems from Indo-Japanese synergy. TIFR handled sensor design and cell biology, while Osaka University's Prof. Katsumasa Fujita's group provided advanced Raman microscopy expertise. Heqi Xi and Itsuki Yamamoto contributed imaging optimizations. Such partnerships underscore TIFR's global outreach, fostering knowledge exchange vital for India's research ecosystem.

For more on Prof. Datta's work, visit her group page.

ABATaRs open doors to dissecting redox signaling networks. Real-time H₂O₂ mapping can reveal oxidative bursts in immune responses or mitochondrial dysfunction in neurodegeneration. Cu²⁺ imaging aids metal homeostasis studies, crucial for neurodegenerative diseases where Cu aggregation in amyloid plaques is key.

Applications span drug discovery (screening antioxidants), cancer research (tumor microenvironment ROS), and personalized medicine. By avoiding labels, it preserves native dynamics, potentially accelerating therapies. In India, with rising metabolic diseases, this tool could inform public health strategies.

Access the full study via DOI: 10.1002/anie.202522980.

Established in 1945, TIFR is India's premier multi-disciplinary research institute, functioning as a deemed university under DAE. Home to luminaries like Homi Bhabha, it excels in chemical sciences, pioneering tools for bioimaging. Prof. Datta's lab focuses on in vivo tracking of metals and ROS, building on prior fluorescence sensors to Raman frontiers.

This publication reinforces TIFR's impact, with alumni and faculty driving national innovation. It inspires students pursuing MSc/PhD in chemical biology, aligning with NEP 2020's research emphasis.

• Sensitivity Boost: Push-pull design yields ultra-bright Raman tags.
• Ratiometry: Self-calibrating, robust to variations.
• Multiplexing: Multiple analytes via peak shifts.
• Accessibility: Spontaneous Raman vs. costly SRS.
• Biocompatibility: Low toxicity, no photobleaching.

Compared to fluorescence, Raman offers deeper tissue penetration and chemical specificity without labels.

Next steps include expanding ABATaR palette for glutathione, Zn²⁺, and hypoxia markers; 3D tissue imaging; and AI integration for automated analysis. In clinics, it could monitor therapy responses in real-time, e.g., ROS in chemotherapy. For India, scaling via DBT-Wellcome or ICMR grants could democratize advanced imaging.

TIFR's innovation signals India's rising bioimaging prowess, attracting global talent and funding.

Photo by Aniket Narula on Unsplash

Bioimaging experts hail it as 'game-changing' for spontaneous Raman. Students benefit from TIFR's GS 2026 admissions, fostering next-gen researchers. Policymakers see potential in Atmanirbhar Bharat for medtech.

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