Photoacoustic microscopy with an enhanced axial resolution of 5.8 μm

The axial resolution of photoacoustic microscopy (PAM) can be enhanced by reducing the speed of sound within the imaging region of interest. This principle was demonstrated on a previously-reported PAM system, which utilized a 125 MHz ultrasonic transducer for signal detection and the Wiener deconvolution for signal processing. With sound slowed by silicone oil immersion, we have achieved a finest axial resolution of 5.8 μm for PAM, as validated by phantom experiments. The axial resolution was also enhanced in vivo when mouse ears injected with silicone oil were imaged. After injection of silicone oil, the blood vessels were resolved more clearly. When tissue-compatible low-speed liquids become available, this approach may find applications in PAM as well as in other imaging modalities, such as photoacoustic computed tomography and ultrasound imaging

Intracellular temperature mapping with fluorescence-assisted photoacoustic-thermometry

Measuring intracellular temperature is critical to understanding many cellular functions but still remains challenging. Here, we present a technique–fluorescence-assisted photoacoustic thermometry (FAPT)–for intracellular temperature mapping applications. To demonstrate FAPT, we monitored the intracellular temperature distribution of HeLa cells with sub-degree (0.7 °C) temperature resolution and sub-micron (0.23 μm) spatial resolution at a sampling rate of 1 kHz. Compared to traditional fluorescence-based methods, FAPT features the unique capability of transforming a regular fluorescence probe into a concentration- and excitation-independent temperature sensor, bringing a large collection of commercially available generic fluorescent probes into the realm of intracellular temperature sensing

Slow-sound photoacoustic microscopy

We propose to enhance the axial resolution of photoacoustic microscopy (PAM) by reducing the speed of sound within the imaging region of interest. With silicone oil immersion, we have achieved a finest axial resolution of 5.8 μm for PAM, as validated by phantom experiments. The axial resolution was also enhanced in vivo when mouse ears injected with silicone oil were imaged. When tissue-compatible low-speed liquid becomes available, this approach may find broad applications in PAM as well as in other imaging modalities, such as photoacoustic computed tomography and ultrasound imaging

Photoimprint Photoacoustic Microscopy for Three-Dimensional Label-Free Subdiffraction Imaging

Subdiffraction optical microscopy allows the imaging of cellular and subcellular structures with a resolution finer than the diffraction limit. Here, combining the absorption-based photoacoustic effect and intensity-dependent photobleaching effect, we demonstrate a simple method for subdiffraction photoacoustic imaging of both fluorescent and nonfluorescent samples. Our method is based on a double-excitation process, where the first excitation pulse partially and inhomogeneously bleaches the molecules in the diffraction-limited excitation volume, thus biasing the signal contributions from a second excitation pulse striking the same region. The differential signal between the two excitations preserves the signal contribution mostly from the center of the excitation volume, and dramatically sharpens the lateral resolution. Moreover, due to the nonlinear nature of the signal, our method offers an inherent optical sectioning capability, which is lacking in conventional photoacoustic microscopy. By scanning the excitation beam, we performed three-dimensional subdiffraction imaging of varied fluorescent and nonfluorescent species. As any molecules have absorption, this technique has the potential to enable label-free subdiffraction imaging, and can be transferred to other optical imaging modalities or combined with other subdiffraction methods

Photo-imprint super-resolution photoacoustic microscopy

Combining the absorption-based photoacoustic effect and intensity-dependent photobleaching effect, we demonstrate a simple method for super-resolution photoacoustic imaging of both fluorescent and non-fluorescent samples. Our method is based on a double-excitation process, where the first excitation pulse partially and inhomogeneously bleaches the molecules in the diffraction-limited excitation volume, thus biasing the signal contributions from a second excitation pulse striking the same region. By scanning the excitation beam, we performed three-dimensional sub-diffraction imaging of varied fluorescent and non-fluorescent species. A lateral resolution of 80 nm and an axial resolution of 370 nm have been demonstrated. This technique has the potential to enable label-free super-resolution imaging, and can be transferred to other optical imaging modalities or combined with other super-resolution methods

Photothermal bleaching and recovery analysis in photoacoustic microscopy

A novel method – photoacoustic recovery after photothermal bleaching (PRAP) – is proposed and implemented to study particle dynamics and medium properties at the micron scale via photoacoustic imaging. PRAP is an intuitive way to visualize as well as quantify dynamic processes in many kinds of media. We demonstrate PRAP first in a phantom study, and then in live cells. PRAP provides high signal-to-noise ratio imaging with minimal bleaching-induced artifacts during the recovery stage, ideal for monitoring the diffusive and kinetic phenomena inside a cell

Single-cell photoacoustic thermometry

A novel photoacoustic thermometric method is presented for simultaneously imaging cells and sensing their temperature. With three-seconds-per-frame imaging speed, a temperature resolution of 0.2°C was achieved in a photo-thermal cell heating experiment. Compared to other approaches, the photoacoustic thermometric method has the advantage of not requiring custom-developed temperature-sensitive biosensors. This feature should facilitate the conversion of single-cell thermometry into a routine lab tool and make it accessible to a much broader biological research community