Channprit Kaur, Aria Hajiahmadi, Benjamin R. Ecclestone, James E. D. Tweel, James A. Tummon Simmons, Parsin Haji Reza
The mechanical properties of micro-scale bio-entities are fundamental for understanding their functions and pathological states. However, current methods for assessing elastic properties at single-particle level such as Brillouin and atomic force microscopies exhibit intrinsic limitations, including being often slow, having poor resolution, or involving complicated and invasive setups. In this study, we explore Photon Absorption Remote Sensing (PARS) microscopy as a unique solution for mechanical sensing of single micro-objects. PARS uses probe beam scattering/reflectivity measurements to capture non-radiative relaxation process following the absorption of a pulse of light by a micro-object. In particular, we demonstrate that, when operating at GHz-range bandwidth, PARS can trace the sub-nanosecond dynamics of non-radiative relaxation in individual micro-objects, capturing both photoacoustic (PA) pressure propagation and thermal diffusion. This GHz-range measurement, in conjunction with a developed descriptive model, enables the experimental extraction of a minimally distorted PA temporal profile. The PA temporal profile contain information on the ratio between the absorbing object's sound speed and its characteristic diameter, offering a new dimension in PARS microscopy. This enables the assessment of the object's elastic properties, deduced from its speed of sound. Additionally, it offers the potential for sizing objects with known sound speeds. The proof of principle experiments was conducted using spherical polystyrene absorbers, ranging in size from 1 to 10 micrometers with known properties, embedded in a Polydimethylsiloxane (PDMS) matrix. This technique expands the scope of PARS imaging, opening new perspectives for clinical applications in mechanobiology by demonstrating its potential for mechanical imaging.
James E. D. Tweel, Benjamin R. Ecclestone, James A. Tummon Simmons, Parsin Haji Reza
Histochemical staining is essential for visualizing tissue architecture and cellular morphology but is destructive and limited by the availability of tissue for multiple stains. Virtual staining with label-free microscopy offers a non-destructive alternative, enabling multiple stains to be generated from the same section while reducing stain variability and preserving tissue for downstream assays. Here, a new dual-excitation Photon Absorption Remote Sensing (PARS) system is presented, representing the first application of long-wave ultraviolet A (UVA) 355 nm excitation alongside the established UVC 266 nm source. The addition of 355 nm extends PARS contrast beyond 266 nm, enhancing stromal visualization (e.g., collagen, elastin) and capturing red blood cells, melanin, and other features through complementary radiative and non-radiative absorption. The 266 nm and 355 nm pulses interrogate the sample in an interlaced fashion, enabling concurrent acquisition without compromising imaging speed. Using the RegGAN image-translation framework, this work presents the first demonstration of PARS virtual staining across multiple specialized stains, including Masson's trichrome, periodic acid-Schiff (PAS), and Jones' silver, in addition to hematoxylin and eosin (H&E), across diverse human and murine tissues. A masked evaluation by expert pathologists showed that virtual stains achieved the same diagnostic quality as their chemical counterparts, and pathologists could not reliably distinguish real from virtual stains. By providing label-free multi-stain outputs from a single scan, dual-excitation PARS virtual staining could integrate into digital pathology workflows, expanding diagnostic utility. Real and virtual whole-slide image (WSI) pairs are publicly available at the BioImage Archive (https://doi.org/10.6019/S-BIAD2232).
James A. Tummon Simmons, Sarah J. Werezak, Benjamin R. Ecclestone, James E. D. Tweel, Hager Gaouda, Parsin Haji Reza
Vascular imaging is critical for understanding human health and disease. Most established non-contact and label-free optical techniques capture predominantly structural information about vasculature. However, in many pathologies, functional changes often precede visible morphological changes. This limits the ability of established modalities to prevent negative patient outcomes. In this study, a new in vivo Photon Absorption Remote Sensing (PARS) microscope is proposed. PARS enables the label-free non-contact structural and functional imaging of vascular structures and their microenvironment. PARS aims to capture the dominant light matter interactions surrounding an absorption event including both non-radiative and radiative relaxation. System performance is demonstrated through wide field of view in vivo imaging of vascular contrast in both mouse ear and chicken embryo. Additionally, video rate imaging of chicken embryo capillaries shows first feasibility for blood flow measurement using PARS. This work represents a promising step for vascular imaging techniques where there is significant demand for a method of non-contact label-free functional imaging.
Zohreh Hosseinaee, Nicholas Pellegrino, Nima Abbasi, Tara Amiri, James A. Tummon Simmons, Paul Fieguth, Parsin Haji Reza
We have developed a multimodal photoacoustic remote sensing (PARS) microscope combined with swept source optical coherence tomography for in vivo, non-contact retinal imaging. Building on the proven strength of multiwavelength PARS imaging, the system is applied for estimating retinal oxygen saturation in the rat retina. The capability of the technology is demonstrated by imaging both microanatomy and the microvasculature of the retina in vivo. To our knowledge this is the first time a non-contact photoacoustic imaging technique is employed for in vivo oxygen saturation measurement in the retina.
Benjamin R. Ecclestone, James A. Tummon Simmons, James E. D. Tweel, Channprit Kaur, Aria Hajiahmadi, Parsin Haji Reza
Label-free optical absorption microscopy techniques have evolved as effective tools for non-invasive chemical specific structural, and functional imaging. Yet most modern label-free microscopy modalities target only a fraction of the contrast afforded by an optical absorption interaction. We introduce a comprehensive optical absorption microscopy technique, Photon Absorption Remote Sensing (PARS), which simultaneously captures the dominant light matter interactions which occur as a pulse of light is absorbed by a molecule. In PARS, the optical scattering, attenuation, and the transient radiative and non-radiative relaxation processes are collected at each optical absorption event. This provides a complete representation of the absorption event, providing unique contrast presented here as the total absorption (TA) and quantum efficiency ratio (QER) measurements. By capturing a complete view of each absorption interaction, PARS bridges many of the specificity challenges associated with label-free imaging, facilitating recovery of a wider range of biomolecules than independent radiative or non-radiative modalities. To show the versatility of PARS, we explore imaging across a wide range of biological specimens, from single cells to in-vivo imaging of living subjects. These examples of label-free histopathological imaging, and vascular imaging illustrate some of the numerous fields where PARS may have profound impacts. Overall PARS may provide comprehensive label-free contrast in a wide variety of biological specimens, providing otherwise inaccessible visualizations, and representing a new a source of rich data to develop new AI and machine learning methods for diagnostics and visualization.
Benjamin R. Ecclestone, James A. Tummon Simmons, James E. D. Tweel, Deepak Dinakaran, Parsin Haji Reza
Label-free optical absorption microscopy techniques continue to evolve as promising tools for label-free histopathological imaging of cells and tissues. However, critical challenges relating to specificity and contrast, as compared to current gold-standard methods continue to hamper adoption. This work introduces Photon Absorption Remote Sensing (PARS), a new absorption microscope modality, which simultaneously captures the dominant de-excitation processes following an absorption event. In PARS, radiative (auto-fluorescence) and non-radiative (photothermal and photoacoustic) relaxation processes are collected simultaneously, providing enhanced specificity to a range of biomolecules. As an example, a multiwavelength PARS system featuring UV (266 nm) and visible (532 nm) excitation is applied to imaging human skin, and murine brain tissue samples. It is shown that PARS can directly characterize, differentiate, and unmix, clinically relevant biomolecules inside complex tissues samples using established statistical processing methods. Gaussian mixture models (GMM) are used to characterize clinically relevant biomolecules (e.g., white, and gray matter) based on their PARS signals, while non-negative least squares (NNLS) is applied to map the biomolecule abundance in murine brain tissues, without stained ground truth images or deep-learning methods. PARS unmixing and abundance estimates are directly validated and compared against chemically stained ground truth images, and deep learning based-image transforms. Overall, it is found that the PARS unique and rich contrast may provide comprehensive, and otherwise inaccessible, label-free characterization of molecular pathology, representing a new source of data to develop AI and machine learning methods for diagnostics and visualization.