Abstract
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.
Highlights
Imaging complex biological samples and biomimetic tissue cultures is of great interest to life scientists [1]
We present Vibrational Infrared Photothermal and Phase Signal (VIPPS) imaging to map the protein distribution in fibroblast cells grown in a collagen matrix as a physiologically relevant model
Vibrational infrared photothermal amplitude and phase signal (VIPPS) imaging of fibroblast cells in collagen and cross registration of subcellular features with fluorescence imaging In Fig. 2, VIPPS imaging of fluorescently labeled and unlabeled fibroblast cells grown on a collagen extracellular matrix is demonstrated. (The sample preparation details can be found in the section on Materials and Methods in Supplement 1.) We focus on the Amide I band whose spectral signature arises from the C = O stretching vibrations from the amide groups of the peptide bonds
Summary
Imaging complex biological samples and biomimetic tissue cultures is of great interest to life scientists [1] Fluorescence microscopy techniques such as confocal and multiphoton have demonstrated high specificity and unique super-resolution capabilities [2]. Due to similar absorption properties between intracellular and extracellular matrix proteins, there are currently few IR studies undertaken on tissue engineered models, in which cells are grown in two and three dimensional extracellular matrices. We present Vibrational Infrared Photothermal and Phase Signal (VIPPS) imaging to map the protein distribution in fibroblast cells grown in a collagen matrix as a physiologically relevant model. VIPPS enables simultaneous study of chemical composition combined with thermal diffusion dynamics that can offer diverse and enriching insights into cell growth and structure as well as intracellular heat dynamics
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