Comparative Evaluation of LLMs in Clinical Oncology.

BACKGROUND: As artificial intelligence (AI) tools become widely accessible, more patients and medical professionals will turn to them for medical information. Large language models (LLMs), a subset of AI, excel in natural language processing tasks and hold considerable promise for clinical use. Fields such as oncology, in which clinical decisions are highly dependent on a continuous influx of new clinical trial data and evolving guidelines, stand to gain immensely from such advancements. It is therefore of critical importance to benchmark these models and describe their performance characteristics to guide their safe application to clinical oncology. Accordingly, the primary objectives of this work were to conduct comprehensive evaluations of LLMs in the field of oncology and to identify and characterize strategies that medical professionals can use to bolster their confidence in a model's response. METHODS: This study tested five publicly available LLMs (LLaMA 1, PaLM 2, Claude-v1, generative pretrained transformer 3.5 [GPT-3.5], and GPT-4) on a comprehensive battery of 2044 oncology questions, including topics from medical oncology, surgical oncology, radiation oncology, medical statistics, medical physics, and cancer biology. Model prompts were presented independently of each other, and each prompt was repeated three times to assess output consistency. For each response, models were instructed to provide a self-appraised confidence score (from 1 to 4). Model performance was also evaluated against a novel validation set comprising 50 oncology questions curated to eliminate any risk of overlap with the data used to train the LLMs. RESULTS: There was significant heterogeneity in performance between models (analysis of variance, P<0.001). Relative to a human benchmark (2013 and 2014 examination results), GPT-4 was the only model to perform above the 50th percentile. Overall, model performance varied as a function of subject area across all models, with worse performance observed in clinical oncology subcategories compared with foundational topics (medical statistics, medical physics, and cancer biology). Within the clinical oncology subdomain, worse performance was observed in female-predominant malignancies. A combination of model selection, prompt repetition, and confidence self-appraisal allowed for the identification of high-performing subgroups of questions with observed accuracies of 81.7 and 81.1% in the Claude-v1 and GPT-4 models, respectively. Evaluation of the novel validation question set produced similar trends in model performance while also highlighting improved performance in newer, centrally hosted models (GPT-4 Turbo and Gemini 1.0 Ultra) and local models (Mixtral 8×7B and LLaMA 2). CONCLUSIONS: Of the models tested on a standardized set of oncology questions, GPT-4 was observed to have the highest performance. Although this performance is impressive, all LLMs continue to have clinically significant error rates, including examples of overconfidence and consistent inaccuracies. Given the enthusiasm to integrate these new implementations of AI into clinical practice, continued standardized evaluations of the strengths and limitations of these products will be critical to guide both patients and medical professionals. (Funded by the National Institutes of Health Clinical Center for Research and the Intramural Research Program of the National Institutes of Health; Z99 CA999999.).

Automated Whole Slide Imaging for Label-Free Histology Using Photon Absorption Remote Sensing Microscopy.

OBJECTIVE: Pathologists rely on histochemical stains to impart contrast in thin translucent tissue samples, revealing tissue features necessary for identifying pathological conditions. However, the chemical labeling process is destructive and often irreversible or challenging to undo, imposing practical limits on the number of stains that can be applied to the same tissue section. Here we present an automated label-free whole slide scanner using a PARS microscope designed for imaging thin, transmissible samples. METHODS: Peak SNR and in-focus acquisitions are achieved across entire tissue sections using the scattering signal from the PARS detection beam to measure the optimal focal plane. Whole slide images (WSI) are seamlessly stitched together using a custom contrast leveling algorithm. Identical tissue sections are subsequently H&E stained and brightfield imaged. The one-to-one WSIs from both modalities are visually and quantitatively compared. RESULTS: PARS WSIs are presented at standard 40x magnification in malignant human breast and skin samples. We show correspondence of subcellular diagnostic details in both PARS and H&E WSIs and demonstrate virtual H&E staining of an entire PARS WSI. The one-to-one WSI from both modalities show quantitative similarity in nuclear features and structural information. CONCLUSION: PARS WSIs are compatible with existing digital pathology tools, and samples remain suitable for histochemical, immunohistochemical, and other staining techniques. SIGNIFICANCE: This work is a critical advance for integrating label-free optical methods into standard histopathology workflows.

Photon Absorption Remote Sensing Imaging of Breast Needle Core Biopsies Is Diagnostically Equivalent to Gold Standard H&E Histologic Assessment.

Photon absorption remote sensing (PARS) is a new laser-based microscope technique that permits cellular-level resolution of unstained fresh, frozen, and fixed tissues. Our objective was to determine whether PARS could provide an image quality sufficient for the diagnostic assessment of breast cancer needle core biopsies (NCB). We PARS imaged and virtually H&E stained seven independent unstained formalin-fixed paraffin-embedded breast NCB sections. These identical tissue sections were subsequently stained with standard H&E and digitally scanned. Both the 40× PARS and H&E whole-slide images were assessed by seven breast cancer pathologists, masked to the origin of the images. A concordance analysis was performed to quantify the diagnostic performances of standard H&E and PARS virtual H&E. The PARS images were deemed to be of diagnostic quality, and pathologists were unable to distinguish the image origin, above that expected by chance. The diagnostic concordance on cancer vs. benign was high between PARS and conventional H&E (98% agreement) and there was complete agreement for within-PARS images. Similarly, agreement was substantial (kappa > 0.6) for specific cancer subtypes. PARS virtual H&E inter-rater reliability was broadly consistent with the published literature on diagnostic performance of conventional histology NCBs across all tested histologic features. PARS was able to image unstained tissues slides that were diagnostically equivalent to conventional H&E. Due to its ability to non-destructively image fixed and fresh tissues, and the suitability of the PARS output for artificial intelligence assistance in diagnosis, this technology has the potential to improve the speed and accuracy of breast cancer diagnosis.

The use of nanomaterials in advancing photodynamic therapy (PDT) for deep-seated tumors and synergy with radiotherapy.

Photodynamic therapy (PDT) has been under development for at least 40 years. Multiple studies have demonstrated significant anti-tumor efficacy with limited toxicity concerns. PDT was expected to become a major new therapeutic option in treating localized cancer. However, despite a shifting focus in oncology to aggressive local therapies, PDT has not to date gained widespread acceptance as a standard-of-care option. A major factor is the technical challenge of treating deep-seated and large tumors, due to the limited penetration and variability of the activating light in tissue. Poor tumor selectivity of PDT sensitizers has been problematic for many applications. Attempts to mitigate these limitations with the use of multiple interstitial fiberoptic catheters to deliver the light, new generations of photosensitizer with longer-wavelength activation, oxygen independence and better tumor specificity, as well as improved dosimetry and treatment planning are starting to show encouraging results. Nanomaterials used either as photosensitizers per se or to improve delivery of molecular photosensitizers is an emerging area of research. PDT can also benefit radiotherapy patients due to its complementary and potentially synergistic mechanisms-of-action, ability to treat radioresistant tumors and upregulation of anti-tumoral immune effects. Furthermore, recent advances may allow ionizing radiation energy, including high-energy X-rays, to replace external light sources, opening a novel therapeutic strategy (radioPDT), which is facilitated by novel nanomaterials. This may provide the best of both worlds by combining the precise targeting and treatment depth/volume capabilities of radiation therapy with the high therapeutic index and biological advantages of PDT, without increasing toxicities. Achieving this, however, will require novel agents, primarily developed with nanomaterials. This is under active investigation by many research groups using different approaches.

High quantum efficiency ruthenium coordination complex photosensitizer for improved radiation-activated Photodynamic Therapy.

Traditional external light-based Photodynamic Therapy (PDT)'s application is limited to the surface and minimal thickness tumors because of the inefficiency of light in penetrating deep-seated tumors. To address this, the emerging field of radiation-activated PDT (radioPDT) uses X-rays to trigger photosensitizer-containing nanoparticles (NPs). A key consideration in radioPDT is the energy transfer efficiency from X-rays to the photosensitizer for ultimately generating the phototoxic reactive oxygen species (ROS). In this study, we developed a new variant of pegylated poly-lactic-co-glycolic (PEG-PLGA) encapsulated nanoscintillators (NSCs) along with a new, highly efficient ruthenium-based photosensitizer (Ru/radioPDT). Characterization of this NP via transmission electron microscopy, dynamic light scattering, UV-Vis spectroscopy, and inductively coupled plasma mass-spectroscopy showed an NP size of 120 nm, polydispersity index (PDI) of less than 0.25, high NSCs loading efficiency over 90% and in vitro accumulation within the cytosolic structure of endoplasmic reticulum and lysosome. The therapeutic efficacy of Ru/radioPDT was determined using PC3 cell viability and clonogenic assays. Ru/radioPDT exhibited minimal cell toxicity until activated by radiation to induce significant cancer cell kill over radiation alone. Compared to protoporphyrin IX-mediated radioPDT (PPIX/radioPDT), Ru/radioPDT showed higher capacity for singlet oxygen generation, maintaining a comparable cytotoxic effect on PC3 cells.

Can radiation restore immunotherapy response in metastatic melanoma refractory to checkpoint inhibitors: An institutional experience in salvaging immunotherapy resistant disease.

Melanoma is an immunogenically active tumor with abundantly expressed lymphoid infiltration. Immunotherapy(IO) has proven as a promising treatment option for melanoma but treatment resistance remains as an issue in the majority of patients.There is emerging evidence that radiotherapy (RT) could modulate the tumor microenvironment, increase antigen presentation, and augment adaptive antitumor immunity. Our objective is to evaluate overall treatment response and safety in patients with metastatic melanoma who progressed while on IO, and were treated with RT concurrently with IO for progressive sites.

Dosimetric predictors of toxicity in a randomized study of short-course vs conventional radiotherapy for glioblastoma.

PURPOSE: There is no consensus on appropriate organ at risk (OAR) constraints for short-course radiotherapy for patients with glioblastoma. Using dosimetry and prospectively-collected toxicity data from a trial of short-course radiotherapy for glioblastoma, this study aims to empirically examine the OAR constraints, with particular attention to left hippocampus dosimetry and impact on neuro-cognitive decline. METHODS AND MATERIALS: Data was taken from a randomized control trial of 133 adults (age 18-70 years; ECOG performance score 0-2) with newly diagnosed glioblastoma treated with 60 Gy in 30 (conventional arm) versus 20 (short-course arm) fractions of adjuvant chemoradiotherapy (ClinicalTrials.gov Identifier: NCT02206230). The delivered plan's dosimetry to the OARs was correlated to prospective-collected toxicity and Mini-Mental State Examination (MMSE) data. RESULTS: Toxicity events were not significantly increased in the short-course arm versus the conventional arm. Across all OARs, delivered radiation doses within protocol-allowable maximum doses correlated with lack of grade ≥ 2 toxicities in both arms (p < 0.001), while patients with OAR doses at or above protocol limits correlated with increased grade ≥ 2 toxicities across all examined OARs in both arms (p-values 0.063-0.250). Mean left hippocampus dose was significantly associated with post-radiotherapy decline in MMSE scores (p = 0.005), while the right hippocampus mean dose did not reach statistical significance (p = 0.277). Compared to the original clinical plan, RapidPlan left hippocampus sparing model decreased left hippocampus mean dose by 43 % (p < 0.001), without compromising planning target volume coverage. CONCLUSIONS: In this trial, protocol OAR constraints were appropriate for limiting grade ≥ 2 toxicities in conventional and short-course adjuvant chemoradiotherapy for glioblastoma. Higher left hippocampal mean doses were predictive for neuro-cognitive decline post-radiotherapy. Routine contouring and use of dose constraints to limit hippocampal dose is recommended to minimize neuro-cognitive decline in patients with glioblastoma treated with chemoradiotherapy.

Virtual histological staining of label-free total absorption photoacoustic remote sensing (TA-PARS).

Histopathological visualizations are a pillar of modern medicine and biological research. Surgical oncology relies exclusively on post-operative histology to determine definitive surgical success and guide adjuvant treatments. The current histology workflow is based on bright-field microscopic assessment of histochemical stained tissues and has some major limitations. For example, the preparation of stained specimens for brightfield assessment requires lengthy sample processing, delaying interventions for days or even weeks. Therefore, there is a pressing need for improved histopathology methods. In this paper, we present a deep-learning-based approach for virtual label-free histochemical staining of total-absorption photoacoustic remote sensing (TA-PARS) images of unstained tissue. TA-PARS provides an array of directly measured label-free contrasts such as scattering and total absorption (radiative and non-radiative), ideal for developing H&E colorizations without the need to infer arbitrary tissue structures. We use a Pix2Pix generative adversarial network to develop visualizations analogous to H&E staining from label-free TA-PARS images. Thin sections of human skin tissue were first virtually stained with the TA-PARS, then were chemically stained with H&E producing a one-to-one comparison between the virtual and chemical staining. The one-to-one matched virtually- and chemically- stained images exhibit high concordance validating the digital colorization of the TA-PARS images against the gold standard H&E. TA-PARS images were reviewed by four dermatologic pathologists who confirmed they are of diagnostic quality, and that resolution, contrast, and color permitted interpretation as if they were H&E. The presented approach paves the way for the development of TA-PARS slide-free histological imaging, which promises to dramatically reduce the time from specimen resection to histological imaging.

Three-dimensional virtual histology in unprocessed resected tissues with photoacoustic remote sensing (PARS) microscopy and optical coherence tomography (OCT).

Histological images are critical in the diagnosis and treatment of cancers. Unfortunately, current methods for capturing these microscopy images require resource intensive tissue preparation that may delay diagnosis for days or weeks. To streamline this process, clinicians are limited to assessing small macroscopically representative subsets of tissues. Here, a combined photoacoustic remote sensing (PARS) microscope and swept source optical coherence tomography system designed to circumvent these diagnostic limitations is presented. The proposed multimodal microscope provides label-free three-dimensional depth resolved virtual histology visualizations, capturing nuclear and extranuclear tissue morphology directly on thick unprocessed specimens. The capabilities of the proposed method are demonstrated directly in unprocessed formalin fixed resected tissues. The first images of nuclear contrast in resected human tissues, and the first three-dimensional visualization of subsurface nuclear morphology in resected Rattus tissues, captured with a non-contact photoacoustic system are presented here. Moreover, the proposed system captures the first co-registered OCT and PARS images enabling direct histological assessment of unprocessed tissues. This work represents a vital step towards the development of a rapid histological imaging modality to circumvent the limitations of current histopathology techniques.

Single acquisition label-free histology-like imaging with dual-contrast photoacoustic remote sensing microscopy.

SIGNIFICANCE: Histopathological analysis of tissues is an essential tool for grading, staging, diagnosing, and resecting cancers and other malignancies. Current histopathological imaging techniques require substantial sample processing, prior to staining with hematoxylin and eosin (H&E) dyes, to highlight nuclear and cellular morphology. Sample preparation and staining is resource intensive and introduces potential for variability during sample preparation. AIM: We present a method for direct label-free histopathological assessment of formalin-fixed paraffin-embedded tissue blocks and thin tissue sections using a dual-contrast photoacoustic remote sensing (PARS) microscopy system. APPROACH: To emulate the nuclear and cellular contrast of H&E staining, we leverage unique properties of the PARS system. Here, the ultraviolet excitation PARS microscope takes advantage of DNA's unique optical absorption to provide nuclear contrast analogous to hematoxylin staining of cell nuclei. Concurrently, the optical scattering contrast of the PARS detection system is leveraged to provide bulk tissue contrast reminiscent of eosin staining of cell membranes. RESULTS: We demonstrate the efficacy of this technique by imaging human breast tissue and human skin tissues in formalin-fixed paraffin-embedded tissue blocks and frozen sections, respectively. Salient nuclear and extranuclear features such as cancerous cells, glands and ducts, adipocytes, and stromal structures such as collagen are captured. CONCLUSIONS: The presented dual-contrast PARS microscope enables label-free visualization of tissues with contrast and quality comparable to the current gold standard for histopathological analysis. Thus, the proposed system is well positioned to augment existing histopathological workflows, providing histological imaging directly on unstained tissue blocks and sections.

Towards virtual biopsies of gastrointestinal tissues using photoacoustic remote sensing microscopy.

Gastrointestinal (GI) tissue biopsies provide critical diagnostic information for a wide variety of conditions such as neoplastic diseases (colorectal, small bowel and stomach cancers) and non-neoplastic diseases (inflammatory disorders, infection, celiac disease). Endoscopic biopsies collect small tissue samples that require resource intensive processing to permit histopathological analysis. Unfortunately, the sparsely collected biopsy samples may fail to capture the pathologic condition because selection of biopsy sites relies on macroscopic superficial tissue features and clinician judgement. Here, we present the first all-optical non-contact label-free non-interferometric photoacoustic microscopy system capable of performing "virtual biopsies". A modular photoacoustic remote sensing (PARS™) architecture is used facilitating imaging of unprocessed tissues providing information similar to conventional histopathological staining techniques. Prospectively this would allow gastroenterologists to assess subcellular tissue morphology in situ when selecting biopsy location. Tested on preserved unstained human and freshly resected murine tissues, the presented PARS microscope rapidly retrieves images of similar area to current biopsies, while maintaining comparable quality to the current standard for histopathological analysis. Additionally, results show the first label free assessment of subsurface cellular morphology in FFPE GI tissue blocks. Clinically relevant features are recovered including cellular details such as lamina propria within colon tissue and cell nuclear structure in resected smooth muscle. Constructed with a modular architecture, this system facilitates the future development of compact imaging heads. The modular PARS system overcomes many of the challenges with imaging unstained thick tissue in situ, representing a significant milestone in the development of a clinical microscope providing virtual biopsy capabilities.

Histopathology for Mohs micrographic surgery with photoacoustic remote sensing microscopy.

Mohs micrographic surgery (MMS) is a precise oncological technique where layers of tissue are resected and examined with intraoperative histopathology to minimize the removal of normal tissue while completely excising the cancer. To achieve intraoperative pathology, the tissue is frozen, sectioned and stained over a 20- to 60-minute period, then analyzed by the MMS surgeon. Surgery is continued one layer at a time until no cancerous cells remain, meaning MMS can take several hours to complete. Ideally, it would be desirable to circumvent or augment frozen sectioning methods and directly visualize subcellular morphology on the unprocessed excised tissues. Employing photoacoustic remote sensing (PARS) microscopy, we present a non-contact label-free reflection-mode method of performing such visualizations in frozen sections of human skin. PARS leverages endogenous optical absorption contrast within cell nuclei to provide visualizations reminiscent of histochemical staining techniques. Presented here, is the first true one to one comparison between PARS microscopy and standard histopathological imaging in human tissues. We demonstrate the ability of PARS microscopy to provide large grossing scans (>1 cm2, sufficient to visualize entire MMS sections) and regional scans with subcellular lateral resolution (300 nm).

Proposal for 'segmented peer review' of multidisciplinary papers.

We propose a new process for peer review of multidisciplinary journal submissions called 'segmented peer review'. The current translational research environment increasingly requires complex and multidisciplinary studies that span multiple distinct specialties within a single paper. Such papers present logistic and practical barriers to effective peer review. To address these barriers, papers undergoing segmented peer review require authors to explicitly i) identify each of the areas of expertise required to review the paper, ii) direct each reviewer to the relevant portions of the manuscript, and iii) suggest in-field reviewers. This segmentation of the paper is then followed by a 'segmented peer review request' tailored to the expertise of each potential reviewer, with a request to confine his / her review to those 'in-scope' aspects of the paper, while de-emphasizing any optional 'out-of-scope' comments. Each reviewer indicates the fitness for publication, or suitability for revision, of their particular segment of the manuscript. The segmented peer review process is completed when the editors integrate the segmented peer reviews. We propose segmented peer review as a fit-for-purpose process with tangible advantages for authors, reviewers, and journal editors. It should reduce the specific barriers to publication inherent in the evaluation of multidisciplinary research efforts.

Reflection-mode virtual histology using photoacoustic remote sensing microscopy.

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.

Improving maximal safe brain tumor resection with photoacoustic remote sensing microscopy.

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.

The FABP12/PPARγ pathway promotes metastatic transformation by inducing epithelial-to-mesenchymal transition and lipid-derived energy production in prostate cancer cells.

Early stage localized prostate cancer (PCa) has an excellent prognosis; however, patient survival drops dramatically when PCa metastasizes. The molecular mechanisms underlying PCa metastasis are complex and remain unclear. Here, we examine the role of a new member of the fatty acid-binding protein (FABP) family, FABP12, in PCa progression. FABP12 is preferentially amplified and/or overexpressed in metastatic compared to primary tumors from both PCa patients and xenograft animal models. We show that FABP12 concurrently triggers metastatic phenotypes (induced epithelial-to-mesenchymal transition (EMT) leading to increased cell motility and invasion) and lipid bioenergetics (increased fatty acid uptake and accumulation, increased ATP production from fatty acid ß-oxidation) in PCa cells, supporting increased reliance on fatty acids for energy production. Mechanistically, we show that FABP12 is a driver of PPARγ activation which, in turn, regulates FABP12's role in lipid metabolism and PCa progression. Our results point to a novel role for a FABP-PPAR pathway in promoting PCa metastasis through induction of EMT and lipid bioenergetics.

PEG-PLGA nanospheres loaded with nanoscintillators and photosensitizers for radiation-activated photodynamic therapy.

Photodynamic Therapy (PDT) is an effective treatment modality for cancers, with Protoporphyrin IX (PPIX)-based PDT being the most widely used to treat cancers in patients. However, PDT is limited to superficial, thin (few mm in depth) lesions that can be accessed by visible wavelength light. Interstitial light-delivery strategies have been developed to treat deep-seated lesions (i.e. prostate cancer). The most promising of these are X-ray-induced scintillation nanoparticles, which have shown potential benefits for PDT of deep-seated tumors. Herein, the design and use of a new nanoscintillator-based radiation-activated PDT (radioPDT) system is investigated in the treatment of deep-seated tumors. Poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG-PLGA) nanospheres were loaded with a scintillator (LaF3:Ce3+) and photosensitizer (PPIX) to effect radioPDT. UV-Vis spectroscopy and electron microscopy studies demonstrated efficient encapsulation of nanoscintillators and PPIX (>90% efficiency) into the PEG-PLGA nanospheres. The nanoparticles were uniform in size and approximately 100 nm in diameter. They were highly stable and functional for up to 24 h under physiological conditions and demonstrated slow release kinetics. In vitro and in vivo toxicity studies showed no appreciable drug toxicity to human skin fibroblast (GM38), prostate cancer cells (PC3), and to C57/BL mice. Cell uptake studies demonstrated accumulation of the nanoparticles in the cytoplasm of PC3 cells. When activated, fluorescent resonant energy transfer (FRET) was evident via fluorescent spectroscopy and singlet oxygen yield. Determination of stability revealed that the nanoparticles were stable for up to 4 weeks. The nanoparticle production was scaled-up with no change in properties. This nanoparticle represents a unique, optimally designed therapeutic and diagnostic agent (theranostic) agent for radioPDT with characteristics capable of potentially augmenting radiotherapy for deep-seated tumors and integrating into current cancer radiotherapy.

Chromophore selective multi-wavelength photoacoustic remote sensing of unstained human tissues.

Identifying positive surgical margins after resection of cancer often triggers re-excision and adjuvant treatments. Incomplete initial resections result in poorer patient outcomes, psychological and financial stress to the patient and increased healthcare costs. Surgical margins are typically assessed post-operatively using time consuming and expensive slide-based histopathology tissue analysis. Currently, a real-time non-contact virtual histology-like intraoperative margin assessment tool is not available. To address this need, we have developed a non-contact multi-wavelength reflection-mode, photoacoustic remote sensing (PARS) microscope demonstrating chromophore selective contrast in human tissues. We show the capabilities of multi-wavelength PARS microscopy utilizing both 266 nm and 532 nm excitation wavelengths and a 1310 nm detection wavelength. Cell nuclei and hemoglobin were visualized at the cellular scale without the addition of exogenous contrast agents. These works provide a critical step towards a virtual histology tool to provide intraoperative histology-like information in living tissue.

All-optical Reflection-mode Microscopic Histology of Unstained Human Tissues.

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.