Label-free Vibrational Imaging Research Articles

High-sensitivity hyperspectral vibrational imaging of heart tissues by mid-infrared photothermal microscopy.

Visualizing the spatial distribution of chemical compositions in biological tissues is of great importance to study fundamental biological processes and origin of diseases. Raman microscopy, one of the label-free vibrational imaging techniques, has been employed for chemical characterization of tissues. However, the low sensitivity of Raman spectroscopy often requires a long acquisition time of Raman measurement or a high laser power, or both, which prevents one from investigating large-area tissues in a nondestructive manner. In this work, we demonstrated chemical imaging of heart tissues using mid-infrared photothermal (MIP) microscopy that simultaneously achieves the high sensitivity benefited from IR absorption of molecules and the high spatial resolution down to a few micrometers. We successfully visualized the distributions of different biomolecules, including proteins, phosphate-including proteins, and lipids/carbohydrates/amino acids. Further, we experimentally compared MIP microscopy with Raman microscopy to evaluate the sensitivity and photodamage to tissues. We proved that MIP microscopy is a highly sensitive technique for obtaining vibrational information of molecules in a broad fingerprint region, thereby it could be employed for biological and diagnostic applications, such as live-tissue imaging.

Wide-field broadband CARS microscopy

Coherent anti-Stokes Raman scattering is an extremely powerful non-linear optical (NLO) microscopy technique for label-free vibrational imaging allowing for a detailed study of biological samples in their native state. To overcome the long acquisition times associated with raster sample scanning required in NLO microscopy, which impair real-time investigation of fast biological dynamics, we employ here wide-field signal generation over a large field of view, covering tens of micrometers. To this aim, we exploit an innovative approach based on the use of an amplified femtosecond ytterbium laser source delivering high energy (≈μJ) pulses in the near infrared. This enables the generation of stable broadband Stokes pulses to measure the entire fingerprint region of the molecular vibrational spectrum, the richest in chemical information. Our results pave the way for future translational applications and clinical diagnostics with video-rate imaging capabilities. ©2022 The Authors

Distinguishing Amyloid β-Protein in a Mouse Model of Alzheimer's Disease by Label-Free Vibrational Imaging.

Due to the increase in the average age of humans, Alzheimer’s disease (AD) has become one of the disorders with the highest incidence worldwide. Abnormal amyloid β protein (Aβ) accumulation is believed to be the most common cause of AD. Therefore, distinguishing the lesion areas can provide clues for AD diagnosis. Here, we present an optical spectroscopy and imaging approach based on coherent anti-Stokes Raman scattering (CARS). Label-free vibrational imaging of Aβ in a mouse model of AD was performed to distinguish the lesion areas by studying the spectra of regions with and without Aβ plaques. Raman spectra in Aβ and non-Aβ regions exhibited a specific difference in the intensity ratio of the wave peaks detected at 2850 and 2930 cm−1. In the non-Aβ region, the ratio of the peak intensity at 2850 cm−1 to that at 2930 cm−1 was approximately 1, whereas that in the Aβ region was 0.8. This label-free vibrational imaging may provide a new method for the clinical diagnosis and basic research of AD.

Microsecond fingerprint stimulated Raman spectroscopic imaging by ultrafast tuning and spatial-spectral learning

Label-free vibrational imaging by stimulated Raman scattering (SRS) provides unprecedented insight into real-time chemical distributions. Specifically, SRS in the fingerprint region (400–1800 cm−1) can resolve multiple chemicals in a complex bio-environment. However, due to the intrinsic weak Raman cross-sections and the lack of ultrafast spectral acquisition schemes with high spectral fidelity, SRS in the fingerprint region is not viable for studying living cells or large-scale tissue samples. Here, we report a fingerprint spectroscopic SRS platform that acquires a distortion-free SRS spectrum at 10 cm−1 spectral resolution within 20 µs using a polygon scanner. Meanwhile, we significantly improve the signal-to-noise ratio by employing a spatial-spectral residual learning network, reaching a level comparable to that with 100 times integration. Collectively, our system enables high-speed vibrational spectroscopic imaging of multiple biomolecules in samples ranging from a single live microbe to a tissue slice.

Label-free imaging of fibroblast membrane interfaces and protein signatures with vibrational infrared photothermal and phase signals.

Label-free vibrational imaging of biological samples has attracted significant interest due to its integration of structural and chemical information. Vibrational infrared photothermal amplitude and phase signal (VIPPS) imaging provide label-free chemical identification by targeting the characteristic resonances of biological compounds that are present in the mid-infrared fingerprint region (3 µm - 12 µm). High contrast imaging of subcellular features and chemical identification of protein secondary structures in unlabeled and labeled fibroblast cells embedded in a collagen-rich extracellular matrix is demonstrated by combining contrast from absorption signatures (amplitude signals) with sensitive detection of different heat properties (lock-in phase signals). We present that the detectability of nano-sized cell membranes is enhanced to well below the optical diffraction limit since the membranes are found to act as thermal barriers. VIPPS offers a novel combination of chemical imaging and thermal diffusion characterization that paves the way towards label-free imaging of cell models and tissues as well as the study of intracellular heat dynamics.

Label-free vibrational imaging of different A\u03b2 plaque types in Alzheimer\u2019s disease reveals sequential events in plaque development

The neuropathology of Alzheimer’s disease (AD) is characterized by hyperphosphorylated tau neurofibrillary tangles (NFTs) and amyloid-beta (Aβ) plaques. Aβ plaques are hypothesized to follow a development sequence starting with diffuse plaques, which evolve into more compact plaques and finally mature into the classic cored plaque type. A better molecular understanding of Aβ pathology is crucial, as the role of Aβ plaques in AD pathogenesis is under debate. Here, we studied the deposition and fibrillation of Aβ in different plaque types with label-free infrared and Raman imaging. Fourier-transform infrared (FTIR) and Raman imaging was performed on native snap-frozen brain tissue sections from AD cases and non-demented control cases. Subsequently, the scanned tissue was stained against Aβ and annotated for the different plaque types by an AD neuropathology expert. In total, 160 plaques (68 diffuse, 32 compact, and 60 classic cored plaques) were imaged with FTIR and the results of selected plaques were verified with Raman imaging. In diffuse plaques, we detect evidence of short antiparallel β-sheets, suggesting the presence of Aβ oligomers. Aβ fibrillation significantly increases alongside the proposed plaque development sequence. In classic cored plaques, we spatially resolve cores containing predominantly large parallel β-sheets, indicating Aβ fibrils. Combining label-free vibrational imaging and immunohistochemistry on brain tissue samples of AD and non-demented cases provides novel insight into the spatial distribution of the Aβ conformations in different plaque types. This way, we reconstruct the development process of Aβ plaques in human brain tissue, provide insight into Aβ fibrillation in the brain, and support the plaque development hypothesis.

In vivo coherent Raman imaging of the melanomagenesis-associated pigment pheomelanin.

Melanoma is the most deadly form of skin cancer with a yearly global incidence over 232,000 patients. Individuals with fair skin and red hair exhibit the highest risk for developing melanoma, with evidence suggesting the red/blond pigment known as pheomelanin may elevate melanoma risk through both UV radiation-dependent and -independent mechanisms. Although the ability to identify, characterize, and monitor pheomelanin within skin is vital for improving our understanding of the underlying biology of these lesions, no tools exist for real-time, in vivo detection of the pigment. Here we show that the distribution of pheomelanin in cells and tissues can be visually characterized non-destructively and noninvasively in vivo with coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free vibrational imaging technique. We validated our CARS imaging strategy in vitro to in vivo with synthetic pheomelanin, isolated melanocytes, and the Mc1re/e, red-haired mouse model. Nests of pheomelanotic melanocytes were observed in the red-haired animals, but not in the genetically matched Mc1re/e; Tyrc/c (“albino-red-haired”) mice. Importantly, samples from human amelanotic melanomas subjected to CARS imaging exhibited strong pheomelanotic signals. This is the first time, to our knowledge, that pheomelanin has been visualized and spatially localized in melanocytes, skin, and human amelanotic melanomas.

An ex vivo co‐culture model system to evaluate stromal–epithelial interactions in breast cancer

Breast cancer is the most commonly diagnosed cancer among women worldwide. High breast cancer incidence and mortality rates, especially in obese patients, emphasize the need for a better biological understanding of this disease. Previous studies provide substantial evidence for a vital role of the local extracellular environment in multiple steps of tumor progression, including proliferation and invasion. Current evidence supports the role of adipocytes as an endocrine organ, which produces steroid hormones, pro-inflammatory cytokines and adipokines, such as leptin. To further define the role of the mammary microenvironment on tumorigenesis, we have developed an adipose-tumor epithelial cell co-culture system designed to reproduce the in vivo mammary environment. We validate this model through use of coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free vibrational imaging technique. CARS analysis demonstrates the sustained viability of the adipocytes, and that mammary cancer cell morphology parallels that of tumors in vivo. Also, characterized was the influence of mammary adipose tissue on tumor cell growth and migration. Adipose tissue co-cultured with mammary tumor epithelial cells, in the absence of any serum or supplemental growth factors, resulted in substantial increases in growth and migration of tumor cells. In conclusion, this novel co-culture system provides an ideal model to study epithelial-stromal interactions in the mammary gland. Understanding the relationship between adipose tissue, the most abundant and least studied component of the breast stroma and tumor epithelial cells is critical to clarifying the influence of obesity on the development, progression and prognosis of breast cancer.