Chemical imaging, especially mid-infrared spectroscopic microscopy, enables label-free biomedical analyses while achieving expansive molecular sensitivity. However, its slow speed and poor image quality impede widespread adoption. We present a microscope that provides high-throughput recording, low noise, and high spatial resolution where the bottom-up design of its optical train facilitates dual-axis galvo laser scanning of a diffraction-limited focal point over large areas using custom infinity-corrected objectives. The data quality enables applications of modern machine learning and capabilities not previously feasible. Distinct from conventional approaches that focus on morphological investigations or immunostaining, this development makes label-free imaging of minimally processed tissue practical.
Quantum cascade lasers (QCLs) have presented a new chapter for the development of infrared spectroscopy for its high throughput and ability to tune to absorption bands of interest. However, compared to conventional thermal sources, the output stability of QCLs is a limiting factor. Specifically, the noise associated with pulse-to-pulse instability is of specific concern as we design faster and more sensitive spectrometers. Here, we demonstrate a dual-detector approach to reference measurements with the intensity illuminating the sample in real-time per individual laser pulse. We integrate this concept into our custom point-scanning confocal IR microscope. Results show double the SNR and a 10-fold shorter response time with the proposed method versus a single detector approach, all while being lock-in amplifier free. We characterize the controllable parameters and discuss the trade-offs required for a unified system capable of collecting high-quality point spectra as well as whole-slide spectral imaging.
Infrared spectroscopic imaging combines the ability to record molecular content with the ability to visualize chemistry in its spatial diversity. Given the need to record a significantly larger quantity of data than a typical microscopy image (MB vs. GB) and the extensive bandwidth of the spectra (~10 m), trade-offs often have to be made between the closely related considerations of signal to noise ratio, spatial-spectral coverage, resolution and optical arrangements. Here, we present a path from rigorous theory to modeling and design to realizing the advantages offered by new ideas on fundamentally changing these trade-offs. We first describe a new microscope design for increased speed and rapid coverage that is useful for biomedical and clinical tissue imaging. Next, we describe a configuration to measure chirality in samples that promises higher spectral information that present methods. Finally, we present a new approach to nanoscale IR imaging that provides greater fidelity and speed at unprecedented levels of signal to noise ratio. Finally, we show how emerging machine learning approaches can further augment these advances. For each instrumentation advance, examples of use cases will be presented.
Infrared polarimetry is a powerful label-free diagnostic tool to study the molecular alignment and organization in biological tissues and cells. Similar to absorbance images which capture intensity information, polarimetric imaging is essential for capturing the polarization states of the light intensity. Recent advancements in the development of Quantum Cascade Lasers (QCL) sources have opened new avenues in IR imaging with high spatial and spectral resolution while enabling drastic increases in imaging speeds than a corresponding FT-IR approach. We demonstrate improved performance in terms of fast and comprehensive polarimetric image acquisition using a custom-built QCL microscope with point mapping design.
Infrared spectroscopic imaging is an analytical approach that can reveal important molecular information without the need for substantial sample processing. These instruments can provide objective and automated evaluations to aid pathologists improve diagnostic accuracy. The quantum cascade laser, allows for a discrete frequency approach, increasing imaging speeds with superior spatial and spectral resolution. We present our recent progress toward developing new instruments capable of diffraction limited performance at all fingerprint-region wavelengths across the entire field of view. We demonstrate high throughput imaging of tissue sections and tissue microarrays and evaluate the advantages in data quality obtained from a well-corrected system.
Infrared spectroscopic imaging has emerged as a powerful label-free diagnostic tool to study the molecular composition and organization in biological tissues and cells. We report infrared spectroscopic imaging using polarized light to study differential absorption of plane-polarized light by an oriented sample to detect valuable information, such as, birefringence and dichroism. For instance, the organization of collagen, specifically fiber orientation and alignment, is crucial in understanding the progression and metastasis of cancer. Recent advancements in the development of Quantum Cascade Lasers (QCL) sources have opened new avenues for high SNR measurements in the field of IR spectroscopy. In addition, QCL sources are intrinsically polarized and orientation information can be obtained at discrete frequencies with different polarization orientations, allowing much faster acquisition than a corresponding FT-IR approach. We demonstrate improved performance in terms of fast and comprehensive polarimetric image acquisition and analysis using custom-built QCL microscope and evaluate its impact on applications by analyzing the important spectral bands of surgical tissue sections.
Infrared (IR) spectroscopic imaging is an emerging modality for biological tissue analysis that has traditionally employed an interferometer for spectral discrimination. Recent technology developments have made discrete frequency sources, both lasers and filters, practical for imaging. The use of quantum cascade lasers in particular, presents new opportunities as well as challenges. Here we describe results from a novel point scanning confocal IR microscope and demonstrate the performance imaging several important spectral bands of lung tissue. Results show the possibility of discrete frequency (DF) absorbance measurements with RMS noise levels down to 0.34 mAU in 0.25 ms.
Conventional mid-infrared (mid-IR) Fourier transform infrared (FT-IR) spectroscopic imaging systems employ an incoherent globar source and achieve spectral contrast through interferometry. While this approach is suitable for many general applications, recent advancements in broadly tunable external cavity Quantum Cascade Lasers (QCL) offer new approaches to and new possibilities for mid-IR micro-spectroscopic imaging. While QCL-based devices have yet to achieve the wide spectral range generally employed by spectroscopists for molecular analyses, they are starting to be used for microscopy at discrete frequencies. Here, we present a discrete frequency IR (DFIR) microscope based on a QCL source and explore its utility for mid-IR imaging. In our prototype instrument, spectral contrast is achieved by tuning the QCL to bands in a narrow spectral region of interest. We demonstrate wide-field imaging employing a 128x128 pixel liquid nitrogen cooled mercury cadmium telluride (MCT) focal plane array (FPA) detector. The resulting images demonstrate successful imaging as well as several unique features due to coherence effects from the laser source. Here we discuss the effects of this coherence and compare our instrument to conventional mid-IR imaging instrumentation.
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