ABATaRs, sensitive molecular probes for multiplexed bio-imaging in live cells

Seeing chemistry unfold inside living cells is one of the biggest challenges of modern bioimaging. Raman microscopy offers a powerful way to meet this challenge by reading the unique vibrational signatures of molecules. However, cells are extraordinarily complex environments filled with thousands of biomolecules.

To make specific molecules stand out, researchers often attach small chemical probes, such as alkyne tags, that produce signals in a so-called cell-silent spectral window where native cellular components do not scatter light. This allows Raman microscopes to selectively detect the tagged molecules against an otherwise crowded molecular background. Despite this advantage, the widespread adoption of Raman microscopy in biology has been limited by one fundamental problem: Raman signals are extremely weak.

In a new study published in Angewandte Chemie International Edition, researchers report a molecular design strategy that dramatically amplifies Raman signals, enabling the development of Raman sensors that can detect biomolecules using standard spontaneous Raman microscopes. These new Raman sensors enable sensitive, ratiometric imaging of biological molecules and ions, such as hydrogen peroxide molecules, copper ions, and pH, inside living cells. The work is supported by the Department of Atomic Energy, Government of India, and the Japan Science and Technology Agency, with the research team comprising Prof. Ankona Datta and Sujit Das from the Tata Institute of Fundamental Research, Mumbai, and Heqi Xi, Itsuki Yamamoto, and Prof. Katsumasa Fujita from the University of Osaka, Japan.

Raman microscopy works by detecting how light scatters off chemical bonds, producing highly specific spectral signatures. These signatures are narrow and non-overlapping, making Raman imaging well-suited for multiplexed detection of multiple molecules. However, Raman scattering signals are weak—a constraint that has forced researchers to use either very high sensor concentrations or specialized stimulated Raman systems that require complex and costly laser setups.

Instead of modifying instrumentation, the researchers focused on molecular design. They applied a classic concept from physical organic chemistry—the push-pull effect—to Raman sensor design. As the authors note, “The central idea was to develop sensitive Raman probes that can be used for live cell imaging. In ABATaRs, strategic employment of the push-pull effect was the central piece in the design.”

In push-pull systems, the electron-donating and electron-withdrawing groups are placed on opposite sides of a molecule. This internal electronic asymmetry allows increased change in molecular polarizability as a bond vibrates, a key factor that determines how strong the Raman signal is.

By incorporating this design across a single alkyne bond—a commonly used Raman tag—the team created a new class of sensors called Activity-Based Alkyne-Tagged Raman sensors (ABATaRs). “Here, the strategic employment of the donor-acceptor moieties across the triple bond in ABATaRs created a strong push-pull dyad system, which increased the electron cloud density across the triple bond, thus increasing the change in polarisability associated with the bond vibration. This made the signal very strong—stronger than what could be achieved from traditional approaches.”

ABATaRs exhibit up to 10–30 times higher Raman scattering activities compared to widely used standard alkyne tags. As a result, ABATaRs can be detected at low micromolar concentrations using conventional spontaneous Raman microscopy.

Beyond enhanced brightness, ABATaRs function as ratiometric sensors. Each probe is designed to chemically react with a specific biological analyte. When this reaction occurs, the structure of the molecule changes, leading to a measurable shift in Raman frequency.
This frequency shift provides a built-in reference that allows researchers to distinguish genuine chemical transformations from variations in probe concentration or imaging conditions. This provides an essential advantage when working in complex cellular environments. Using this strategy, the researchers developed ABATaRs that selectively respond to hydrogen peroxide, copper ions, and pH.

The team demonstrated that these probes are cell-permeable, non-toxic, and stable in biological media. In live human cells, ABATaRs could detect biologically relevant levels of hydrogen peroxide and copper ions and report on intracellular pH heterogeneity.

In a key advance, the study reports simultaneous, multiplexed Raman imaging of hydrogen peroxide and copper ions, two correlated biological analytes, in living cells using spontaneous Raman microscopy. “In our work, we have highlighted the multiplex imaging of two bio-correlated analytes, namely Cu ions and hydrogen peroxide, which are very essential yet present in low concentration, for the first time using a Raman imaging setup.” Enhanced Raman scattering also reduced the typical duration required for spontaneous Raman imaging and improved compatibility with live-cell studies.

Rather than presenting a single optimized sensor, the authors emphasize that ABATaRs represent a general molecular design framework. “We have diversified the central scheme by making probes for three different analytes, and the same lock-and-key model can be applied for the development of probes for other bio-analytes as well.”

By shifting the focus from instrumentation to molecular design, the work opens new possibilities for chemical imaging in cell biology, disease research, and biomedical diagnostics, using tools already available in many laboratories worldwide.