Optical microscopy has a diffraction limited resolution of about 250 nm. Fluorescence methods (e.g. PALM, STORM, STED) beat this, but they are still limited to 10 s of nm, and the images are an indirect pointillist representation of only part of the original object. Here we describe a way of combining a sample preparation technique taken from histopathology, with a probe-based nano-imaging technique, (s SNOM) from the world of Solid State Physics. This allows us to image subcellular structures optically, and at a nanoscale resolution that is about 100 x better than normal microscopes. By adding a tuneable laser source, we also demonstrate mid-infrared chemical nano-imaging (MICHNI) in human myeloma cells and we use it to map the binding sites of the anti cancer drug bortezomib to less than 10 zL sized intracellular components. MICHNI is label free and can be used with any biological material and drugs with specific functional chemistry. We believe that its combination of speed, cheapness, simplicity, safety and chemical contrast promises a transformative impact across the life sciences.
Mid-infrared (mid-IR) spectroscopy provides a unique chemical fingerprint of biomaterials, including DNA and proteins, from single molecules to highly organised structures and, ultimately, to live cells and tissues. However, acquiring good signal–to–noise mid-IR spectroscopic images, at the cellular level, typically involves a synchrotron, with imaging times of order of minutes. Here we use a new laser-based table-top IR spectroscopic micro-imaging system, to obtain vibrational fingerprint signatures of living human ovarian cancer cells at a diffraction limited spatial resolution, and at a spectral resolution (< 20 cm−1) sufficient to map out the spatial distributions of chemical moieties inside the cell itself. The bright laser pulses give very high signal–to–noise images, and ∼100 psec image acquisition times that are roughly 1011 times faster than current mid-IR spectroscopic imaging techniques. The imaging method is quantitative, non-phototoxic, marker-free and easily fast enough to "freeze" moving, living specimens. It can be applied to a range of cell-level biochemical processes, and we believe it could impact on the fields of drug action, cell physiology, pathology and disease as a whole.
A comparative study is made of the laser crystals 50 at. % Er:YAG and 50 at. % Er:YSGG. Both lasers are constructed in the bounce geometry with quasi continuous wave (QCW) diode pumping. In Er:YAG, pulse energies of up to ~31mJ, slope efficiency of 12.6% and a red-shift in laser wavelength are observed with a final and dominant wavelength of 2.936μm. In Er:YSGG, higher performance is achieved with pulse energies of ~55mJ, slope efficiency of 20.5% and a single transition wavelength of 2.797μm observed. The study indicates that diode pumped Er:YSGG is a superior laser source at 3μm than Er:YAG and it has greater energy storage potential for Q-switched operation.
Abstract Purpose Digistain Index (DI), measured using an inexpensive mid-infrared spectrometer, reflects the level of aneuploidy in unstained tissue sections and correlates with tumor grade. We investigated whether incorporating DI with other clinicopathological variables could predict outcomes in patients with early breast cancer. Methods DI was calculated in 801 patients with hormone receptor-positive, HER2-negative primary breast cancer and ≤ 3 positive lymph nodes. All patients were treated with systemic endocrine therapy and no chemotherapy. Multivariable proportional hazards modeling was used to incorporate DI with clinicopathological variables to generate the Digistain Prognostic Score (DPS). DPS was assessed for prediction of 5- and 10-year outcomes (recurrence, recurrence-free survival [RFS] and overall survival [OS]) using receiver operating characteristics and Cox proportional hazards regression models. Kaplan–Meier analysis evaluated the ability of DPS to stratify risk. Results DPS was consistently highly accurate and had negative predictive values for all three outcomes, ranging from 0.96 to 0.99 at 5 years and 0.84 to 0.95 at 10 years. DPS demonstrated statistically significant prognostic ability with significant hazard ratios (95% CI) for low- versus high-risk classification for RFS, recurrence and OS (1.80 [CI 1.31–2.48], 1.83 [1.32–2.52] and 1.77 [1.28–2.43], respectively; all P < 0.001). Conclusion DPS showed high accuracy and predictive performance, was able to stratify patients into low or high-risk, and considering its cost and rapidity, has the potential to offer clinical utility.
Abstract Several multigene-based, risk scoring methods are available and their use is recommended to support treatment decision making in breast cancer. Although the value of these tests is established, critical challenges remain that limit their use, especially regarding turnaround time for fast decision making and the associated costs. We have developed DigiStain - a novel technology that uses mid-infrared imaging to precisely measure a surrogate of tumour aneuploidy, across unstained biopsy sections. Considering that genomic context shapes the pattern and consequences of aneuploidy during cancer development and progression, aneuploidy may have valuable prognostic or predictive value. Using bespoke software, DigiStain allows a quantitative score ‘DigiStain index’ (DI) to be reproducibly extracted from an objective physical measurement of a cancer i.e., aneuploidy. This information is generated within minutes and is available at the same time as routine H&E staining. We previously showed that DI significantly correlates with tumour grade and survival. To further validate the technology, we have investigated the ability of the DI to predict 5-year survival in a retrospective analysis of patients with oestrogen receptor (ER) positive breast cancer. Methods: Clinical samples were obtained from patients (N=944) treated at the University Hospital of Nottingham UK. Using the DigiStain imager, H&E images were registered before acquiring the DI score for the section. Statistical methodology followed the recommended approach of Royston et al. 2009, The BMJ. A multivariate logistic model modelling the ‘event’ of death by 5 years was generated. The predictive performance of the logistic regression model was assessed using Area Under the receiver operating characteristic Curve (AUC) plots. The predicted vs observed event probability was determined at various cut-offs and the accuracy of the classification was measured by its sensitivity and specificity. Results: Of 944 ER+ patients with follow up information, 77 (8.2%) died in the first 5 years. The age distribution was approximately normal, ranging from 26 to 70 (mean = 54.8 years). Tumour size ranged from 0.2 to 7.5 mm (mean = 1.90 mm and median = 1.70 mm) and had normal distribution when the natural log of size was considered. DI contributed to the prognostic model with (p=0.0018). Odds ratios showed increased odds of death by 5 years with higher DI. The AUC obtained for the model was 0.7722. Due to the number of events the sample was not split into training and validation sets. Instead bootstrapping (n=500) was used to assess performance. When adjusted by bootstrapping the AUC was reduced only slightly to 0.76. Classification tables for sensitivity and specificity across different risk cut-offs and distribution of 5-year predicted probabilities showed the best distinction between groups at low levels of predicted probabilities. As expected, there was a trade-off between sensitivity and specificity when choosing an appropriate cut-off point to define high risk of death by 5 years. Conclusions: The risk score obtained using the DI is a significant prognostic factor for 5-year survival in ER+ breast cancer (p=0.0018) with good diagnostic accuracy. Analysis of 10-year survival data is ongoing and plans to expand the sample collection to include patients from other sites are underway. Citation Format: Hemmel Amrania. A Novel, Rapid and Economical Prognostic Tool For Adjuvant therapy Decisions in Hormone Positive Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-24.
We discuss the potential biomedical applications for a novel infrared spectroscopic microimaging system. A tunable, table top solid-state laser has been coupled to a commercial infrared microscope, fitted with a modified high resolution infrared camera, to create a unique tool for midinfrared imaging. The system is capable of performing broadband imaging at a diffraction-limited spatial resolution, as is demonstrated here by spatially resolved spectroscopy of polymer test samples with a spectral resolution of 20 cm(-1). The large pulse energies (tens of microjoules) offer previously unobtainable combinations of high signal-to-noise levels and rapid data collection times which are superior to current stand-alone laboratory instruments by many decades. Coupled with the short (100 ps) short pulse duration, these characteristics promise to make a wide range of time-resolved and reflection mode imaging experiments possible with live biological systems.
The interaction between plasmonic resonances, sharp modes, and light in nanoscale plasmonic systems often leads to Fano interference effects. This occurs because the plasmonic excitations are usually spectrally broad and the characteristic narrow asymmetric Fano line-shape results upon interaction with spectrally sharper modes. By considering the plasmonic resonance in the Fano model, as opposed to previous flat continuum approaches, here we show that a simple and exact expression for the line-shape can be found. This allows the role of the width and energy of the plasmonic resonance to be properly understood. As examples, we show how Fano resonances measured on an array of gold nanoantennas covered with PMMA, as well as the hybridization of dark with bright plasmons in nanocavities, are well reproduced with a simple exact formula and without any fitting parameters.
We present two new modalities for generating chemical maps. Both are mid-IR based and aimed at the biomedical community, but they differ substantially in their technological readiness. The first, so-called “Digistain”, is a technologically mature “locked down” way of acquiring diffraction-limited chemical images of human cancer biopsy tissue. Although it is less flexible than conventional methods of acquiring IR images, this is an intentional, and key, design feature. It allows it to be used, on a routine basis, by clinical personnel themselves. It is in the process of a full clinical evaluation and the philosophy behind the approach is discussed. The second modality is a very new, probe-based “s-SNOM”, which we are developing in conjunction with a new family of tunable “Quantum Cascade Laser” (QCL) diode lasers. Although in its infancy, this instrument can already deliver ultra-detailed chemical images whose spatial resolutions beat the normal diffraction limit by a factor of ∼1000. This is easily enough to generate chemical maps of the insides of single cells for the first time, and a range of new possible scientific applications are explored.
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