The biopharmaceutical industry relies on selecting high-performing cell lines to meet quality and manufacturability criteria. However, this process is time- and labor-intensive. To address this, label-free multimodal multiphoton microscopy techniques were employed to characterize biopharmaceutical cell lines in early passages. Using a machine learning-assisted single-cell analysis pipeline, over 95% accuracy for monoclonal cell line classification was achieved in all passages. Additionally, Open Set Recognition allowed the differentiation of desired cell lines in polyclonal pools. The study offers a promising solution to expedite the cell line selection process, reducing time and resources while ensuring the identification of high-performance biopharmaceutical cell lines.
Efficient cell line development is crucial for optimizing biopharmaceutical production. We demonstrate the potential of SLAM and FLIM microscopy to optimize this process by correlating metabolism-related features with measured productivity in early CHO cell passages. Eight CHO cell lines were imaged using SLAM and FLIM microscopy, and a pipeline was developed to classify the cells. A linear SVM achieved 95% accuracy in predicting productivity. Important features and their channel affiliations were identified, revealing optical metabolic characteristics from NAD(P)H and FAD associated with productivity. SLAM features correlated with growth and viability, while FLIM features correlated with protein production, highlighting the importance of multimodal label-free imaging.
High speed, three-dimensional optical coherence tomography (3D OCT) at 800nm, 1060nm and 1300nm with approximately 4μm, 7μm and 6μm axial and less than 15μm transverse resolution is demonstrated to investigate the optimum wavelength region for in vivo human skin imaging in terms of contrast, dynamic range and penetration depth. 3D OCT at 1300nm provides deeper penetration, while images obtained at 800nm were better in terms of contrast and speckle noise. 1060nm region was a compromise between 800nm and 1300nm in terms of penetration depth and image contrast. Optimizing sensitivity, penetration and contrast enabled unprecedented visualization of micro-structural morphology underneath the glabrous skin, hairy skin and in scar tissue. Higher contrast obtained at 800 nm appears to be critical in the in vitro tumor study. A multimodal approach combining OCT and PA helped to obtain morphological as well as vascular information from deeper regions of skin.
A noninvasive, multimodal photoacoustic and optical coherence tomography (PAT/OCT) scanner for three-dimensional in vivo (3D) skin imaging is described. The system employs an integrated, all optical detection scheme for both modalities in backward mode utilizing a shared 2D optical scanner with a field-of-view of ~13 × 13 mm(2). The photoacoustic waves were detected using a Fabry Perot polymer film ultrasound sensor placed on the surface of the skin. The sensor is transparent in the spectral range 590-1200 nm. This permits the photoacoustic excitation beam (670-680 nm) and the OCT probe beam (1050 nm) to be transmitted through the sensor head and into the underlying tissue thus providing a backward mode imaging configuration. The respective OCT and PAT axial resolutions were 8 and 20 µm and the lateral resolutions were 18 and 50-100 µm. The system provides greater penetration depth than previous combined PA/OCT devices due to the longer wavelength of the OCT beam (1050 nm rather than 829-870 nm) and by operating in the tomographic rather than the optical resolution mode of photoacoustic imaging. Three-dimensional in vivo images of the vasculature and the surrounding tissue micro-morphology in murine and human skin were acquired. These studies demonstrated the complementary contrast and tissue information provided by each modality for high-resolution 3D imaging of vascular structures to depths of up to 5 mm. Potential applications include characterizing skin conditions such as tumors, vascular lesions, soft tissue damage such as burns and wounds, inflammatory conditions such as dermatitis and other superficial tissue abnormalities.
GSK2894512 is a topically delivered investigational drug being developed for treatment of atopic dermatitis and psoriasis.To investigate, in a phase I clinical trial, the spatial biodistribution and residency of GSK2894512 within the epidermis and dermis of healthy human participants noninvasively using fluorescence lifetime imaging microscopy (FLIM).Two topical drug formulations containing GSK2894512 1% were applied to the right and left forearms of six participants for seven consecutive days, followed by seven days of observation for residency. FLIM images were obtained daily throughout the study, approximately every 24 h. During the treatment phase of the study, images were collected from each participant pretreatment, reflecting the residual dose from the previous day. Three punch biopsies from each participant of one formulation was obtained from the treated region during the post-treatment follow-up period between days 8 and 14 for comparison with FLIM results.Cellular and subcellular features associated with different epidermal and dermal layers were visualized noninvasively, down to a depth of 200 μm. Results yielded three-dimensional maps of GSK2894512 spatial distribution and residency over time. This fluorescence data provided a marker that was used as a monitor for day-to-day variance of drug presence and residency postapplication.The results suggest FLIM could be a viable alternative to skin biopsies without the usual patient discomfort and limitations, thereby enabling the direct measurement of skin distribution through longitudinal monitoring. These results are the first step in establishing the unique capabilities that multiphoton imaging could provide to patients through noninvasive drug detection.
A novel non-invasive in vivo multimodal optical coherence tomography (OCT)/photoacoustic tomography (PAT) imaging system capable of obtaining structural and functional information simultaneously has been demonstrated in skin. A 1060 nm OCT system acquiring 47k depth-scans/s with ~ 7 μm axial and ~ 20 μm transverse resolutions has been incorporated into a backward-mode PA system based on a planar, optically-transparent Fabry-Perot interferometer (FPI) sensor. In this study, the excitation wavelength was set to 670 nm and a focused laser beam at 1550 nm was used as the sensor interrogation beam. OCT and PAT images were obtained sequentially and the coregistered images were combined to form the final 3D image. OCT/PAT images obtained in vivo from the skin of a hairless mouse and human palmar skin demonstrated the ability of this multimodal imaging system to provide complementary structural and functional information from deeper depths with increased contrast.
Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.
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