Fluorescent Specimens Research Articles

Fluorescence guided surgery using indocyanine green for pulmonary osteosarcoma metastasectomy in pediatric patients: A feasibility study

BackgroundIn osteosarcoma patients, resection of pulmonary metastases is considered necessary for long term survival. Radiologically detected lesions are sometimes difficult to identify intraoperatively, especially when no palpation is possible during thoracoscopy. In adults, fluorescence guided surgery (FGS) using indocyanine green (ICG) has been shown to be a safe method for intra-operative identification of pulmonary metastases of different primary tumors. Our aim is to determine the feasibility of using ICG for identification of pulmonary metastases in pediatric osteosarcoma patients. MethodsNine consecutive patients with pulmonary metastases received an intravenous dose of ICG 24 h preoperatively. We started with the adult dosage of 0.5 mg/kg and also used 1.0 mg/kg to confirm that maximum fluorescent signal was achieved. Intra-operatively and post-operatively, lesions were visualized with a near-infrared camera system. Fluorescence was quantified by calculating a tumor-to-background ratio (TBR). ResultsTwo (22%) patients underwent a thoracoscopy and seven (78%) underwent a thoracotomy. Five (56%) patients had a fluorescent metastasis during surgery. In four (44%) patients there were no fluorescent metastases. In two the metastases were necrotic. In the other two, intraoperative fluorescence was most likely hampered by the depth of the metastases. Ex vivo, all vital metastases were fluorescent and necrotic specimens were not. There was no difference between 1.0 mg/kg and 0.5 mg/kg concerning TBR. No adverse events occurred. ConclusionsICG for fluorescence guided metastasectomy of pulmonary osteosarcoma is a feasible procedure in the pediatric population. However, its additional value in intra-operative guidance still has to be investigated. Levels of evidenceLevel III evidence based on a Diagnostic test study: study without a universally applied “gold” standard.

New method of whiteness calibration of spectrophotometers

AbstractA method is described for determining and calibrating the CIE Whiteness Index (WI) of an OBA‐treated specimen without incorporating into the measurement system a mechanically adjustable UV filter. The method requires spectrophotometric measurement of the specimen under two lights: UV‐excluded to infer the reflectance r(λ) and UV to infer the fluorescence profile f(λ). Also required is a stored illuminant spectrum Iin(λ) that covers at least the whole visible wavelengths. Only one scalar value k is then needed to mathematically characterize the total spectral radiance factor (TSRF) of the same specimen under Iin(λ). Calibration uses a reference specimen with known WI, and adjusts k until the computed WI matches the reference WI value. If Iin(λ) is the illuminant chosen from ASTM E313 to define the WI, the k adjustment reduces to solving a single quadratic equation for k that involves the reference WI. In general, relative to directly measuring a specimen's TSRF, it is more instrument‐independent to use the current method to characterize TSRF. Therefore, if a fluorescent specimen is measured using UV and UV‐excluded illuminants, a complete, instrument‐stable characterization of the specimen's TSRF can be derived by adjusting k, which can be done through a closed‐form relation of k to the WI.

Detection of peritoneal metastases during cytoreductive surgery using pegsitacianine, a pH sensitive imaging agent: Final results from a phase 2 study.

3003 Background: For patients with peritoneal metastatic disease, extent of cytoreductive surgery (CRS) is a major prognostic factor for long-term survival, regardless of primary tumor type. Pegsitacianine is a systemically delivered pH-sensitive polymeric micellar fluorescent nanoprobe developed to intraoperatively identify residual disease and augment the completeness of CRS following standard of care radiographic imaging, visualization, palpation, and intended resection. Methods: NCT04950166 was a Phase 2, non-randomized, open-label, multi-center U.S. study. Patients eligible for CRS were administered a single intravenous dose of pegsitacianine at 1 mg/kg within 24-72 hours prior to surgery. After completion of intended CRS, the peritoneal cavity was systematically re-examined under near-infrared (NIR) illumination to evaluate for fluorescent tissue. Post-standard of care fluorescent tissue detected by pegsitacianine guidance was excised and sent for evaluation for presence of residual tumor by histopathology. Clinically significant events (CSE) were defined as detection of histologically confirmed residual disease excised under pegsitacianine guidance following standard CRS, resulting in additional excision or a change in the assessment of completeness of CRS. Treatment-related and treatment-emergent adverse events were coded by CTCAE (v5.01). Results: A total of 40 patients were enrolled and evaluable for CSE. Evaluable patients were defined as patients who completed pegsitacianine dosing, underwent CRS with post-SOC evaluation of the peritoneal cavity, and had evidence of disease upon histopathological evaluation of surgical specimens. These 40 patients represented 6 different primary tumor types (appendiceal, colorectal, endometrial, mesothelioma, ovarian, pancreatic). At the patient level, CSEs were detected in 20 of 40 (50%) patients. Patient-level false positive rate – wherein all pegsitacianine-guided fluorescent tissue specimens resected post-CRS were determined to be negative for cancer by histopathology – occurred in 12.5% of patients. Pegsitacianine was well tolerated with no SAEs. Treatment-related, non-anaphylactic infusion reactions occurred in 26% of patients and were transient, with a median duration of 9 minutes. Conclusions: Pegsitacianine received Breakthrough Therapy Designation from the U.S. Food and Drug Administration as a result of data collected in this trial. Pegsitacianine is well-tolerated and facilitates the recognition of occult residual disease following CRS. The high rate of residual disease detection suggests pegsitacianine has the potential to augment surgeon performance during CRS, leading to improved completeness of cytoreduction and patient outcomes. Clinical trial information: NCT04950166 .

Optical projection tomography of fluorescent microscopic specimens using lateral translation of tube lens.

Optical projection tomography (OPT) is a three-dimensional (3D) fluorescence imaging technique, in which projection images are acquired for varying orientations of a sample using a large depth of field. OPT is typically applied to a millimeter-sized specimen, because the rotation of a microscopic specimen is challenging and not compatible with live cell imaging. In this Letter, we demonstrate fluorescence optical tomography of a microscopic specimen by laterally translating the tube lens of a wide-field optical microscope, which allows for high-resolution OPT without rotating the sample. The cost is the reduction of the field of view to about halfway along the direction of the tube lens translation. Using bovine pulmonary artery endothelial cells and 0.1 µm beads, we compare the 3D imaging performance of the proposed method with that of the conventional objective-focus scan method.

Phasor S-FLIM for the physiological profiling of living cells

Fluorescence Lifetime Imaging Microscopy (FLIM) and spectral imaging are two broadly applied methods for increasing dimensionality in microscopy. However, their combination is typically inefficient and slow in terms of both acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased Spectral FLIM (S-FLIM) system capable of extracting detailed and precise information about the photophysics of fluorescent specimens at optical resolution. The data are processed with the phasor approach to provide unbiased and fast analysis of S-FLIM data.

Fluorescein sodium fluorescence: role in stereotactic brain biopsy

Background Stereotactic brain biopsy for eloquent area brain tumours is associated with complications like hemorrhage due to multiple sampling and increased operative time due to dependence on neuropathologist for frozen section assessment. In order to curb these limitations, we investigated the use of Fluorescein sodium fluorescence in confirming pathological tissue samples in stereotactic brain biopsy of gadolinium-enhancing tumours. Methods This prospective observational study included 23 consecutive patients who underwent stereotactic biopsy of gadolinium-contrast-enhancing brain lesions with intravenous fluorescein sodium administration. 93 specimens were obtained and examined for the presence of fluorescence using a microscope with fluorescence visualisation capability. Later the nature of the samples was confirmed on histopathology. The sensitivity and specificity of flourescein sodium fluorescence were calculated based on histopathological confirmation. Results 58 (62.3%) fluorescent and 35 (37.6%) nonfluorescent samples were obtained. All fluorescent specimens contained diagnostic tissue appropriate for tumor grading. Of 35 nonfluorescent specimens, 12 (34.3%) did not contain tumor, 11 (31.4%) contained minor hypercellularity or gliosis, and 12 (34.3%) contained tumor with a high proportion of necrosis. The sensitivity and specificity for fluorescein fluorescence were 83% and 100%, respectively. Conclusions Fluorescein fluorescence is a handy tool to detect the pathological tissue in stereotactic brain biopsy and may improve its diagnostic accuracy and expedite the procedure.

Historical development of FINCH from the beginning to single-shot 3D confocal imaging beyond optical resolution [Invited

We chronicle a 15-year development effort of Fresnel incoherent correlation holography (FINCH) since its first description to its current 3D current microscopic wide-field or confocal imaging that doubles optical resolution beyond the Rayleigh limit to about 100 nm in a single snapshot. The path from the original demonstration of FINCH [Opt. Lett.32, 912 (2007) OPLEDP0146-959210.1364/OL.32.000912] to its current picture-perfect imaging of multicolor fluorescent biological specimens and reference test patterns by fluorescence or reflected light imaging is described.

Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging.

Fluorescence lifetime imaging microscopy (FLIM) and spectral imaging are two broadly applied methods for increasing dimensionality in microscopy. However, their combination is typically inefficient and slow in terms of acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased spectral FLIM (S-FLIM) system. Our method, Phasor S-FLIM, combines true parallel multichannel digital frequency domain electronics with a multidimensional phasor approach to extract detailed and precise information about the photophysics of fluorescent specimens at optical resolution. To show the flexibility of the Phasor S-FLIM technology and its applications to the biological and biomedical field, we address four common, yet challenging, problems: the blind unmixing of spectral and lifetime signatures from multiple unknown species, the unbiased bleedthrough- and background-free Förster resonance energy transfer analysis of biosensors, the photophysical characterization of environment-sensitive probes in living cells and parallel four-color FLIM imaging in tumor spheroids.

Image scanning microscopy with a long depth of focus generated by an annular radially polarized beam.

Image scanning microscopy (ISM) is a promising tool for bioimaging owing to its integration of signal to noise ratio (SNR) and super resolution superior to that obtained in confocal scanning microscopy. In this paper, we introduce the annular radially polarized beam to the ISM, which yields an axially extended excitation focus and enhanced resolution, providing a new possibility to obtain the whole information of thick specimen with a single scan. We present the basic principle and a rigorous theoretical model for ISM with annular radially polarized beam (ISM-aRP). Results show that the resolution of ISM-aRP can be enhanced by 4% compared with that in conventional ISM, and the axial extent of the focus is longer than 6λ. The projected view of the simulated fluorescent beads suspension specimen demonstrates the validity of ISM-aRP to obtain the whole information of volume sample. Moreover, this simple method can be easily integrated into the commercial laser scanning microscopy systems.

3D resolution improvement in confocal microscopy by mirror refection interference and fluorescence emission difference

A method based on mirror refection interference and fluorescence emission difference is provided to improve the three-dimensional resolution and SNR in confocal microscopy. Theoretical analysis and experimental results have been performed to verify the effectiveness of the method. Our numerical simulations predict that lateral resolution close to 0.285λ and axial resolution close to 0.205λ are possible for practical confocal microscopy with visible light. Furthermore, the SNR has also been improved. The experimental results of both fluorescent beads and cell microtubules are presented to verify the applicability and effectiveness of our method. The ability to increase the lateral resolution and axial resolution using mirror refection interference and fluorescence emission difference without increasing the laser power is of great importance for imaging fluorescent biological specimens, which cannot tolerate high laser power.

Intraoperative Molecular Imaging for ex vivo Assessment of Peripheral Margins in Oral Squamous Cell Carcinoma.

Objective: Complete surgical resection is the standard of care for treatment of oral cancer although the positive margin rate remains 15–30%. Tissue sampling from the resected specimen and from the wound bed for frozen section analysis (FSA) remains the mainstay for intraoperative margin assessment but is subject to sampling error and can require the processing of multiple samples. We sought to understand if an ex vivo imaging strategy using a tumor-targeted fluorescently labeled antibody could accurately identify the closest peripheral margin on the mucosal surface of resected tumor specimen, so that this “sentinel margin” could be used to guide pathological sampling.Materials and Methods: Twenty-nine patients with oral squamous cell carcinoma scheduled for surgical resection were consented for the study and received systemic administration of a tumor-targeted fluorescently labeled antibody (Panitumumab IRDye800CW). After surgical resection, the tumor specimen was imaged using a closed-field fluorescent imaging device. Relevant pathological data was available for five patients on retrospective review. For each of these five patients, two regions of highest fluorescence intensity at the peripheral margin and one region of lowest fluorescence intensity were identified, and results were correlated with histology to determine if the region of highest fluorescence intensity along the mucosal margin (i.e., the sentinel margin) was truly the closest margin.Results: Imaging acquisition of the mucosal surface of the specimen immediately after surgery took 30 s. In all of the specimens, the region of highest fluorescence at the specimen edge had a significantly smaller margin distance than other sampled regions. The average margin distance at the closest, “sentinel,” margin was 3.2 mm compared to a margin distance of 8.0 mm at other regions (p < 0.0001).Conclusions: This proof-of-concept study suggests that, when combined with routine FSA, ex vivo fluorescent specimen imaging can be used to identify the closest surgical margin on the specimen. This approach may reduce sampling error of intraoperative evaluation, which should ultimately improve the ability of the surgeon to identify the sentinel margin. This rapid sentinel margin identification improves the surgeon's orientation to areas most likely to be positive in the surgical wound bed and may expedite pathology workflow.

Maintenance of Fluorescence During Paraffin Embedding of Fluorescent Protein-Labeled Specimens.

Paraffin embedding is widely used in microscopic imaging for preparing biological specimens. However, owing to significant fluorescence quenching during the embedding process, it is not compatible with fluorescent-labeling techniques, such as transgenic and viral labeling using green fluorescent protein (GFP). Here, we investigate the quenching mechanism and optimize the embedding process to improve the preservation of fluorescence intensity. The results show that dehydration is the main reason for fluorescence quenching during paraffin embedding, caused by the full denaturation of GFP molecules in ethyl alcohol. To evaluate fluorescent and morphological preservation, we modified the embedding process using tertiary butanol (TBA) instead of ethyl alcohol. Fluorescence intensity following TBA dehydration increased 12.08-fold of that observed in the traditional method. We obtained uniform fluorescence maintenance throughout the whole mouse brain, while the continuous apical dendrites, spines, and axon terminals were shown evenly within the cortex, hippocampus, and the amygdala. Moreover, we embedded a whole rat brain labeled with AAV in the prelimbic cortex (Prl). With the axon terminals in different areas, such as the caudate putamen, thalamus, and pyramidal tract, the results showed a continuous tract of Prl neurons throughout the whole brain. This method was also suitable for tdTomota labeled samples. These findings indicate that this modified embedding method could be compatible with GFP and provides a potential turning point for applications in the fluorescent labeling of samples.

Characterizing the heterogeneity in 5-aminolevulinic acid-induced fluorescence in glioblastoma.

5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is an effective surgical adjunct for the intraoperative identification of tumor tissue during resection of high-grade gliomas. The use of 5-ALA-induced PpIX fluorescence in glioblastoma (GBM) has been shown to double the extent of gross-total resection and 6-month progression-free survival. The heterogeneity of 5-ALA-induced PpIX fluorescence observed during surgery presents a technical and diagnostic challenge when utilizing this tool intraoperatively. While some regions show bright fluorescence after 5-ALA administration, other regions do not, despite that both regions of the tumor may be histopathologically indistinguishable. The authors examined the biological basis of this heterogeneity using computational methods. The authors collected both fluorescent and nonfluorescent GBM specimens from a total of 14 patients undergoing surgery and examined their gene expression profiles. In this study, the authors found that the gene expression patterns characterizing fluorescent and nonfluorescent GBM surgical specimens were profoundly different and were associated with distinct cellular functions and different biological pathways. Nonfluorescent tumor tissue tended to resemble the neural subtype of GBM; meanwhile, fluorescent tumor tissue did not exhibit a prominent pattern corresponding to known subtypes of GBM. Consistent with this observation, neural GBM samples from The Cancer Genome Atlas database exhibited a significantly lower fluorescence score than nonneural GBM samples as determined by a fluorescence gene signature developed by the authors. These results provide a greater understanding regarding the biological basis of differential fluorescence observed intraoperatively and can provide a basis to identify novel strategies to maximize the effectiveness of fluorescence agents.

Photobleaching of ultrashort pulses with different repetition rates in two-photon excitation microscopy

Photobleaching of fluorescent specimens with the excitation of different repetition rate ultrafast fiber lasers was studied and demonstrated in two-photon excitation microscopy. A higher repetition rate is proved to induce a lower grade photobleaching for the same imaging contrast. In detail, 750 MHz can reduce the photobleaching rate by ~3 fold compared to 22 MHz, and by ~2 fold compared to 94 MHz. This was underpinned by the testing of fixed zebrafish heart tissue slices. Furthermore, we highlight the advantages of a high repetition rate fiber laser in two-photon, long-time biological in vivo imaging, with a lower photobleaching rate and suitable fluorescence signal intensity.

Single Semiconductor Nanostructure Extinction Spectroscopy

While emission-based, single-particle microscopies and spectroscopies have been highly successful in revealing the properties of matter hidden by ensemble averages, their limits have now become apparent. To address recognized future needs and, in particular, the need to go beyond fluorescent specimens, single-particle extinction techniques have been developed. Motivating this has been the desire to acquire information about the electronic structure of nanoscale materials difficult to obtain otherwise using either ensemble or emission-based, single-particle measurements. These techniques are, however, nontrivial since single nanostructures attenuate only 0.0001–1% of the incident light. This Review Article describes the challenges associated with overcoming the low signal-to-noise ratios inherent to low-dimensional semiconductor nanostructure extinction measurements. It simultaneously describes the fundamental operating principles and achievements of photothermal heterodyne imaging (PHI) and spatial modula...

Fluorescein-assisted stereotactic needle biopsy of brain tumors: a single-center experience and systematic review

Over the last years, fluorescence-based technology has begun an emergent intraoperative method for diagnostic confirmation of brain tumor tissue in stereotactic needle biopsy. However, the actual level of evidence is quite low, especially about fluorescein sodium (FL) application. This method needs to be further validated and better analyzed about its impact in clinical practice. Retrospective analysis of 11 cases with contrast-enhancing brain tumors, underwent awake stereotactic needle biopsy with intraoperative FL assistance (group 1), was verified under the operative microscope filter. This group was matched with a control group of 18 patients (group 2). In addition, a systematic literature review was performed in PubMed/Medline database according to PRISMA statement. All studies concerning FL or 5-ALA application in stereotactic biopsy as intraoperative confirmation of brain tumor tissue were included. The primary endpoint was the evaluation of diagnostic accuracy. In group 1, all fluorescent specimens were diagnostic. The number of samplings was the useful minimum and non-use of intraoperative neuropathological examination allowed to significantly reduce procedure time (42.09 vs 69.72min of group 2). No complications occurred, and the average hospitalization time after procedure was 1.09days (vs 2.33 of group 2). Literature analysis supports the usefulness of photodiagnosis and its high diagnostic yield especially at the core of high-grade/contrast-enhancing tumors. FL assistance during stereotactic biopsy of contrast-enhancing brain tumors may give a real-time confirmation of tumor tissue, maximizing the diagnostic yield, and reducing time of procedure, morbidity, and hospitalization.

Investigation of the usefulness of fluorescein sodium fluorescence in stereotactic brain biopsy.

Intraoperative frozen section assessment, to confirm acquisition of pathological tissues, is used in stereotactic brain biopsy to minimise sampling errors. Limitations include the dependence on dedicated neuro-oncology pathologists and an increase in operative duration. We investigated the use of intraoperative fluorescein sodium, and compared it to frozen section assessment, for confirming pathological tissue samples in the stereotactic biopsy of gadolinium-contrast-enhancing brain lesions. This prospective observational study consisted of 18 consecutive patients (12 men; median age, 63years) who underwent stereotactic biopsy of gadolinium-contrast-enhancing brain lesions with intravenous fluorescein sodium administration. Twenty-three specimens were obtained and examined for the presence of fluorescence using a microscope with fluorescence visualisation capability. Positive and negative predictive values were calculated based on the fluorescence status of the biopsy samples with its corresponding intraoperative frozen section and definitive histopathological diagnosis. Nineteen specimens (83%) were fluorescent and four (17%) were non-fluorescent. All 19 fluorescent specimens were confirmed to be lesional on intraoperative frozen section assessment and were suitable for histopathological diagnosis. Three of the non-fluorescent specimens were confirmed to be lesional on intraoperative frozen section assessment. One non-fluorescent specimen was non-diagnostic on frozen section and histological assessments. The positive predictive value was 100% and the negative predictive value was 25%. Fluorescein sodium fluorescence is as accurate as frozen section assessment in confirming sampling of pathological tissue in the stereotactic biopsy of gadolinium-contrast-enhancing brain lesions. Fluorescein sodium fluorescence-guided stereotactic biopsy is a useful addition to the neurosurgical armamentarium.

Development of Bright and Biocompatible Nanoruby and Its Application to Background-Free Time-Gated Imaging of G-Protein-Coupled Receptors.

At the forefront of developing fluorescent probes for biological imaging applications are enhancements aimed at increasing their brightness, contrast, and photostability, especially toward demanding applications of single-molecule detection. In comparison with existing probes, nanorubies exhibit unlimited photostability and a long emission lifetime (∼4 ms), which enable continuous imaging at single-particle sensitivity in highly scattering and fluorescent biological specimens. However, their wide application as fluorescence probes has so far been hindered by the absence of facile methods for scaled-up high-volume production and molecularly specific targeting. The present work encompasses the large-scale production of colloidally stable nanoruby particles, the demonstration of their biofunctionality and negligible cytotoxicity, as well as the validation of its use for targeted biomolecular imaging. In addition, optical characteristics of nanorubies are found to be comparable or superior to those of state-of-the-art quantum dots. Protocols of reproducible and robust coupling of functional proteins to the nanoruby surface are also presented. As an example, NeutrAvidin-coupled nanoruby show excellent affinity and specificity to μ-opioid receptors in fixed and live cells, allowing wide-field imaging of G-protein coupled receptors with single-particle sensitivity.

Multi‐wavelength excitation method for measuring FWA‐treated paper

AbstractColorimetric properties of fluorescent materials depend on the SPD of the illumination. That is why most standards for evaluating them specify the illuminations, which are often hard‐to‐realize daylight illuminants. The presented method using commercially available LEDs enables accurate enough colorimetric measurements of FWA‐treated papers or prints on them illuminated by the specified illuminant. The total spectral radiance factor of a fluorescent specimen, from which most colorimetric values are derived, consists of the luminescent spectral radiance factor and the spectral reflectance factor. This method separately estimates those of FWA‐treated paper to add up to the total spectral radiance factor. The luminescent spectral radiance factor is obtained by estimating the SPD of luminescence excited by the specified illuminant as the weighted sum of the multiple SPDs of luminescence excited by the respective narrow band LED emissions at different wavelengths. The LEDs and their weights are determined optimally for generally used papers. The spectral reflectance factor is derived from the estimated SPD of the radiation with fluorescence excluded from the paper illuminated by visible illumination. The method was applied with five different illumination systems each using two or three narrow band LEDs in the excitation range. They were evaluated by measuring the total spectral radiance factors by D50 of seven FWA‐treated papers and CMYK prints on four papers. The derived colorimetric values were compared to the respective references by the ideal D50.

Acceptance of the 2016 Roebling Medal of the Mineralogical Society of America

President Lange, Friends, and Colleagues: What a thrill to be here today, celebrating mineralogy. And Rus. Thank you for those most kind remarks. Your unwavering support, both professional and personal, has played a significant role through most of my career, and I am more grateful than I can easily express. Receiving a medal named for Washington Roebling has special meaning for me, as I feel a kinship with the great engineer in at least two significant ways. First is our mutual love of mineralogy. My passion for minerals was kindled in eighth grade, when I was fortunate to have Wilfred Welsh as my science teacher. Bill Welsh was a soft-spoken, engaged, but initially unassuming teacher. He lined his classroom with glass cases and filled them with beautiful minerals. He observed me lingering after class, examining one specimen after another, and he offered to teach me more. He gave me a starter collection of 100 specimens, mineral field guides, and mimeographed directions to Paterson and Franklin, New Jersey (favored haunts of Washington Roebling, by the way). I visited those great localities again and again, including a memorable Franklin, New Jersey, date with Margee in 1966—50 years ago. We still have fluorescent specimens collected that night. Needless to say, I was hooked—both by mineralogy and by Margee, who has shared in every step of this journey. Fast forward to freshman year at MIT, where my arrival coincided with that of the ebullient, passionate David R. Wones—my mineralogy teacher and subsequent Masters Thesis adviser. What an inspiration Dave was! I had gone to …