Infrared spectroscopy offers molecular vibration fingerprint information essential to understanding how molecules work inside a living cell and how molecular changes occur in diseases. In 2016, a research group at the Photonics Center at Boston University, led by professor Ji-Xin Cheng, realized a milestone in IR microscopy technology with its invention of mid-infrared photothermal (MIP) microscopy. Described in a Science Advances paper that year, and featured in the September 2018 issue of Biophotonics magazine, MIP microscopy opened new possibilities for chemical, biological, and materials research by enabling infrared (IR) spectroscopic imaging at submicron spatial resolution. Building on its advancement in photothermal imaging technology, a Cheng-led team has introduced mid-IR photothermal dynamic imaging (PDI). Compared to MIP microscopy, PDI provides better signal-to-noise ratio (SNR) and imaging speed. Moreover, it enables users to collect quantitative photothermal dynamics information about a sample. This information in unavailable from current photothermal microscopy. MIP microscopy uses a visible laser beam to probe the IR absorption-induced thermal lensing effect. The absorption of the modulated IR beam induces a local temperature change, and the temperature gradient around the focusing area becomes the refractive index gradient. The propagation direction of the visible beam (i.e., the probe) changes because of the lensing effect. The signal is acquired by detecting the intensity change of the probe beam with a photodiode and a lock-in amplifier. PDI detects the photothermal signal at the single-pulse level, which is not possible with the lock-in approach. “In photothermal microscopy, it is common sense to use lock-in to extract the signal,” Cheng said. “Our current work, however, breaks with this conventional wisdom. Instead, we use a fast photodiode and fast electronics to digitize the low duty cycle photothermal signal at the single-pulse level. This not only improves SNR and speeds up the imaging process, but also allows us to extract the photothermal dynamics information, which is not available using the lock-in method.” In the PDI system, a pulsed mid-IR beam from a QCL is focused on the sample with a reflective objective lens. The counter-propagated probe beam from a continuous-wave laser is focused by a water immersion objective lens. Backscattered probe photons are collected with a 50-50 beamsplitter. Forward-scattered probe photons are collected by the reflective objective and separated by a dichroic mirror. Both forward and backward probe photons are collected and sent to silicon photodiodes, which are connected to a wideband voltage amplifier. The researchers developed the new lock-in-free, mid-IR PDI system using megahertz (MHz) digitization and match filtering at the harmonics of frequency modulation. Using the amplifier and MHz digitizer, the PDI system can record the photothermal dynamic response at the level of a single IR pulse excitation. The PDI system acquires photothermal dynamic information at nanosecond-scale temporal resolution and covers a bandwidth larger than 25 MHz. Single-pulse excitation, combined with digital signal processing to filter out the noise, enables the PDI system to achieve a greater than fourfold enhancement in SNR over its lock-in predecessor. To characterize the system’s performance and evaluate its capability to analyze transient thermal dynamics with chemical specificity, the researchers performed photothermal dynamic imaging on PMMA particles. In addition to demonstrating improvements in SNR, the PDI system revealed dynamics that enabled accurate thermal property characterization. Using E. coli as a test bed, the researchers also investigated the photothermal dynamic imaging of carbohydrate conversion into biomass at a single bacterium level. Results showed that photothermal dynamic imaging provides high throughput and a high level of sensitivity for single-cell metabolism imaging, at an imaging speed that is nearly two orders of magnitude faster than the original MIP system. The researchers also used PDI to map the photothermal dynamics of organelles inside brain cancer cells. With the lock-in method, the signals from small organelles are buried in the water background. With PDI, they are detectable. Using PDI, the researchers detected a heterogeneous, chemically-dependent thermal environment inside the cells that was distinct from the thermal response of the cell as a whole. Cheng’s group further found that by exploiting the difference in thermal decay between water and biomolecules, PDI was able to extract extremely weak signals from a water background, which allowed the researchers to separate the signal from the water. PDI separated live cell samples from their water background, identifying the cellular components that were buried in a water background based on their time-resolved signatures. These small lipids remained completely buried in the water background when lock-in detection was used, the researchers said. The capability to nondestructively probe a sample’s chemically specific photothermal dynamics, which is not available in the lock-in version of the system, will support the characterization of biological and material specimens. To better understand this capability in PDI, the researchers performed a Fourier analysis of the photothermal dynamic signals. -1;. (c): Photothermal dynamics of the D=300 nm PMMA particle. The red curve is the transient PDI signal. The green dash line is the fitted exponential decay function, with a decay constant of 300 ns. The blue curve is the derivative of photothermal intensity over time. The dash-dot line indicates a thermal equilibrium state. The black curve is the synchronously acquired IR pulse. Courtesy of Jiaze Yin, Photonics Center, Boston University." style="float: left; margin-top: 7px; margin-right: 10px; margin-bottom: 7px; border-width: 1px; border-style: solid; width: 470.545px; height: 301.091px;" /> (a) Principle and schematic of photothermal dynamic imaging. A pulsed mid-IR beam is provided by a QCL and focused on the sample with a reflective objective lens. The counter-propagated probe beam provided by a continuous-wave visible laser is focused by a water immersion objective lens. Backscattered probe photons are collected with a 50-50 beamsplitter; forward-scattered probe photons are collected by the reflective objective and separated by a dichroic mirror. Both forward and backward probes are collected and sent to silicon photodiodes connected with a wideband voltage amplifier. The low duty cycle photothermal signal contains strong components at harmonics of modulation frequency, which can be recovered by the wideband PDI detection scheme. (b) Mid-infrared photothermal dynamic imaging of D=300 nm PMMA nanoparticles at the absorption peak at 1729 cm-1. (c) Photothermal dynamics of the D=300 nm PMMA particle. The red curve is the transient PDI signal. The green dashed line is the fitted exponential decay function, with a decay constant of 300 ns. The blue curve is the derivative of photothermal intensity over time. The dash-dot line indicates a thermal equilibrium state. The black curve is the synchronously acquired IR pulse. Courtesy of Jiaze Yin, Photonics Center, Boston University. The mid-IR PDI approach allows for the investigation of a sample’s intrinsic chemical and physical properties simultaneously, the researchers said. This quality applies broadly both to biology and materials science, they said. “We have demonstrated a photothermal dynamic imaging microscope that can sense transient photothermal modulation with nanosecond temporal resolution, and enable concurrent detection of IR absorption and photothermal dynamics at submicron spatial resolution,” Cheng said. “Compared with conventional photothermal heterodyne imaging via a lock-in amplifier, PDI increases the sensitivity to low duty cycle photothermal signals by more than fourfold. Our PDI method is able to capture all the harmonics and produce an accurate thermal decay constant, which is beyond the reach of the multichannel lock-in or the boxcar averaging approach.” Beyond the mid-IR photothermal process, the PDI system could be used in research related to the imaging of short-lifetime events or complex photothermal decay processes. It could be used to examine intracellular organelles’ thermal response together with their chemical composition, or to monitor transient cell responses to fast perturbation, providing an entirely new perspective with which to understand the cell’s machinery. A U.S. patent for PDI has been filed through Boston University. The technology has attracted attention from a number of companies and could emerge as the next generation of MIP, although it will take time for PDI to be adopted by labs and commercialized. Meanwhile, MIP microscopy, which has been converted into a product, MIRage, by Photothermal Spectroscopy Corp., continues to play a significant role in photothermal microscopy. The research was published in Nature Communications (www.doi.org/10.1038/s41467-021-27362-w).