Unprecedented advancements in fluorescence live imaging have been made in the last two decades. These advancements, coupled with the development of molecular techniques that generate genetically transgenic animals expressing fluorescently tagged proteins, have been instrumental in discovering the molecular mechanisms underlying numerous diseases. Among the available optical techniques, fluorescence microscopy (FM) and optical coherence tomography (OCT) has attracted significant research attention. FM offers a better understanding of cellular processes at the molecular level. OCT enables the evaluation of three-dimensional (3D) images of tissue structures with controllable cellular resolution.
One main advantage of OCT is the possibility to independently control its lateral and axial resolutions, which depend on the numerical aperture of the scanning objective and source parameters, respectively. Despite the advancements to achieve a high axial resolution, most OCT systems operate with a low numerical aperture objective, which limits the lateral resolution. Optical coherence microscopy (OCM) is a promising alternative that circumvents the technical limitations of OCT and provides unique access to fundamental aspects of biological processes, without the requirement for sample pre-processing or labeling. It takes full advantage of the 3D capability of OCT, thereby opening new possibilities for evaluating cellular level information in tissues.
Lately, multimodal imaging techniques involving combining OCT with other imaging techniques have been deemed as an effective approach for evaluating both functional and structural information in tissues. Specifically, the integration of OCM and FM imaging modalities offers great opportunities for evaluating the effects of structural information on various cell processes. Despite the significant research efforts devoted to the realization of effective integration of OCM and FM imaging modalities, achieving simultaneous co-registration of functional and structural information remains the biggest challenge.
Herein, Dr. Reddikumar Maddipatla and Professor Patrice Tankam from Indiana University School of Optometry developed a high-speed dual-modality imaging system through the integration of high-resolution optical coherence microscopy (HR-OCM) system with a dual-channel scanning confocal fluorescence microscopy (DC-SCFM) system to achieve the co-registration of fluorescence and reflectance signals simultaneously. The challenge of the focal plane mismatch associated with the two techniques was addressed to allow co-registration of DC-SCFM and HR-OCM. The performance and capabilities of the integrated system were evaluated by imaging red and green fluorescence microspheres embedded in a multi-layer tape and silicone phantom. The work is currently published in the journal, Optics and Lasers in Engineering.
The authors demonstrated the robustness and efficiency of the integrated system in achieving the simultaneous co-registration of the fluorescence and reflectance signals at high speed of 250 kHz and a high lateral resolution of 2 µm over a 1.1 × 1.1 mm field of view. This was attributed to the combined benefits and advantages of the individual systems. While HR-OCM provided the needed cross-sectional and en face images required to accurately identify the 3D location of the cellular features with a high axial resolution of about 2.4 µm through depth localization, DC-SCFM permitted the discrimination between the red and green microspheres with a high lateral resolution of about 2 µm.
In summary, Dr. Reddikumar Maddipatla and Professor Patrice Tankam reported the successful integration of HR-OCM and DC-SCFM to realize simultaneous co-registration of fluorescence and reflectance signals. The results showed that the integrated system is synergistic in generating functional and structural information of samples. In a statement to Advances in Engineering, the authors said their proposed integrated system may represent a new chapter in advancing the in vivo investigations of different biological processes in live specimens using transgenic animal models. “The structural information provided by HROCM is useful to identify the depth location of fluorophores in the sample and can serve as a reference when tracking molecular changes in tissues over time”, says Dr. Maddipatla. “We are working toward enabling longitudinal studies in transgenic mouse models with this system. The possibility to follow biological processes in the same transgenic mouse over time offers tremendous opportunities to advance the understanding of disease mechanism”, says Dr. Tankam.
Maddipatla, R., & Tankam, P. (2022). Development of high-speed, integrated high-resolution optical coherence microscopy and dual-channel fluorescence microscopy for the simultaneous co-registration of reflectance and fluorescence signals. Optics and Lasers in Engineering, 149, 106823.