Elasto-optical refractive index modulation due to photoacoustic initial pressure transients produced significant reflection of a probe beam when the absorbing interface had an appreciable refractive index difference. This effect was harnessed in a new form of non-contact optical resolution photoacoustic microscopy called photoacoustic remote sensing microscopy. A non-interferometric system architecture with a low-coherence probe beam precludes detection of surface oscillations and other phase-modulation phenomenon. The probe beam was confocal with a scanned excitation beam to ensure detection of initial pressure-induced intensity reflections at the subsurface origin where pressures are largest. Phantom studies confirmed signal dependence on optical absorption, index contrast and excitation fluence. In vivo imaging of superficial microvasculature and melanoma tumors was demonstrated with ~2.7±0.5 μm lateral resolution.

Histological visualizations are critical to clinical disease management and are fundamental to biological understanding. However, current approaches that rely on bright-field microscopy require extensive tissue preparation prior to imaging. These processes are both labor intensive and contribute to creating significant delays in clinical feedback for treatment decisions that can extend to 2–3 weeks for standard paraffin-embedded tissue preparation and interpretation, especially if ancillary testing is needed. Here, we present the first comprehensive study on the broad application of a novel label-free reflection-mode imaging modality known as photoacoustic remote sensing (PARS) for visualizing salient subcellular structures from various common histopathological tissue preparations and for use in unprocessed freshly resected tissues. The PARS modality permits non-contact visualizations of intrinsic endogenous optical absorption contrast to be extracted from thick and opaque biological targets with optical resolution. The technique was examined both as a rapid assessment tool that is capable of managing large samples (> 1 cm2) in under 10 min, and as a high contrast imaging modality capable of extracting specific biological contrast to simulate conventional histological stains such as hematoxylin and eosin (H&E). The capabilities of the proposed method are demonstrated in a variety of human tissue preparations including formalin-fixed paraffin-embedded tissue blocks and unstained slides sectioned from these blocks, including normal and neoplastic human brain, and breast epithelium involved with breast cancer. Similarly, PARS images of human skin prepared by frozen section clearly demonstrated basal cell carcinoma and normal human skin tissue. Finally, we imaged unprocessed murine kidney and achieved histologically relevant subcellular morphology in fresh tissue. This represents a vital step towards an effective real-time clinical microscope that overcomes the limitations of standard histopathologic tissue preparations and enables real-time pathology assessment.

Photoacoustic imaging (PAI) takes advantage of both optical and ultrasound imaging properties to visualize optical absorption with high resolution and contrast. Photoacoustic microscopy (PAM) is usually categorized with all-optical microscopy techniques such as optical coherence tomography or confocal microscopes. Despite offering high sensitivity, novel imaging contrast, and high resolution, PAM is not generally an all-optical imaging method unlike the other microscopy techniques. One of the significant limitations of photoacoustic microscopes arises from their need to be in physical contact with the sample through a coupling media. This physical contact, coupling, or immersion of the sample is undesirable or impractical for many clinical and pre-clinical applications. This also limits the flexibility of photoacoustic techniques to be integrated with other all-optical imaging microscopes for providing complementary imaging contrast. To overcome these limitations, several non-contact photoacoustic signal detection approaches have been proposed. This paper presents a brief overview of current non-contact photoacoustic detection techniques with an emphasis on all-optical detection methods and their associated physical mechanisms.

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Malignant brain tumors are among the deadliest neoplasms with the lowest survival rates of any cancer type. In considering surgical tumor resection, suboptimal extent of resection is linked to poor clinical outcomes and lower overall survival rates. Currently available tools for intraoperative histopathological assessment require an average of 20 min processing and are of limited diagnostic quality for guiding surgeries. Consequently, there is an unaddressed need for a rapid imaging technique to guide maximal resection of brain tumors. Working towards this goal, presented here is an all optical non-contact label-free reflection mode photoacoustic remote sensing (PARS) microscope. By using a tunable excitation laser, PARS takes advantage of the endogenous optical absorption peaks of DNA and cytoplasm to achieve virtual contrast analogous to standard hematoxylin and eosin (H&E) staining. In conjunction, a fast 266 nm excitation is used to generate large grossing scans and rapidly assess small fields in real-time with hematoxylin-like contrast. Images obtained using this technique show comparable quality and contrast to the current standard for histopathological assessment of brain tissues. Using the proposed method, rapid, high-throughput, histological-like imaging was achieved in unstained brain tissues, indicating PARS’ utility for intraoperative guidance to improve extent of surgical resection.

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Surgical oncologists depend heavily on visual field acuity during cancer resection surgeries for in-situ margin assessment. Clinicians must wait up to two weeks for results from a pathology lab to confirm a post-operative diagnosis, potentially resulting in subsequent treatments. Currently, there are no clinical tools that can visualize diagnostically pertinent tissue information in-situ. Here, we present the first microscopy capable of non-contact label-free visualization of human cellular morphology in a reflection-mode apparatus. This is possible with the recently reported imaging modality called photoacoustic remote sensing microscopy which enables non-contact detection of optical absorption contrast. By taking advantage of the 266-nanometer optical absorption peak of DNA, photoacoustic remote sensing is efficacious in recovering qualitatively similar nuclear information in comparison to that provided by the hematoxylin stain in the gold-standard hematoxylin and eosin (H&E) prepared samples. A photoacoustic remote sensing system was employed utilizing a 266-nanometer pulsed excitation beam to induce photoacoustic pressures within the sample resulting in refractive index modulation of the optical absorber. A 1310-nanometer continuous-wave interrogation beam detects these perturbed regions as back reflected intensity variations due to the changes in the local optical properties. Using this technique, clinically useful histologic images of human tissue samples including breast cancer (invasive ductal carcinoma), tonsil, gastrointestinal, and pancreatic tissue images were formed. These were qualitatively comparable to standard H&E prepared samples.