SignificanceLabel-free, two-photon excited fluorescence (TPEF) imaging captures morphological and functional metabolic tissue changes and enables enhanced understanding of numerous diseases. However, noise and other artifacts present in these images severely complicate the extraction of biologically useful information.AimWe aim to employ deep neural architectures in the synthesis of a multiscale denoising algorithm optimized for restoring metrics of metabolic activity from low-signal-to-noise ratio (SNR), TPEF images.ApproachTPEF images of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavoproteins (FAD) from freshly excised human cervical tissues are used to assess the impact of various denoising models, preprocessing methods, and data on metrics of image quality and the recovery of six metrics of metabolic function from the images relative to ground truth images.ResultsOptimized recovery of the redox ratio and mitochondrial organization is achieved using a novel algorithm based on deep denoising in the wavelet transform domain. This algorithm also leads to significant improvements in peak-SNR (PSNR) and structural similarity index measure (SSIM) for all images. Interestingly, other models yield even higher PSNR and SSIM improvements, but they are not optimal for recovery of metabolic function metrics.ConclusionsDenoising algorithms can recover diagnostically useful information from low SNR label-free TPEF images and will be useful for the clinical translation of such imaging.
Multi-photon excited intensity and lifetime fluorescence images relying on endogenous contrast can be analyzed to quantify contributions from key metabolic co-enzymes and associated metabolic function and mitochondrial organization metrics. The high spatio-temporal resolution and context of these non-destructive measurements can be used to provide important insights related to a wide range of samples, conditions and disease models. Corresponding images are acquired from mitochondria, engineered tissues, excised and in vivo human tissues. Recent studies highlight the value of multi-parametric, label-free, metabolic assessments to improve our understanding of traumatic brain injury, (pre)cancer development, and vitiligo lesions.
The potential to differentiate between diseased and healthy tissue has been demonstrated through the extraction of morphological and functional metrics from label-free, two-photon images. Acquiring such images as fast as possible without compromising their diagnostic and functional content is critical for clinical translation of two-photon imaging. Computational restoration methods have demonstrated impressive recovery of image quality and important biological information. However, access to large clinical datasets has hampered advancement of denoising algorithms. Here, we seek to demonstrate the application of denoising algorithms on depth-resolved two-photon excited fluorescence (TPEF) images with specific focus on recovery of functional metabolic metrics. Datasets were generated through the collection of images of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavoproteins from freshly excised rat cheek epithelium. Image datasets were patched across depth, generating 1012, 256-by-256 patches. A well-known U-net architecture was trained on 6628 low-signal-to-noise-ratio (SNR) patches from a previously collected large dataset and later retrained on a smaller 620 low-SNR patches dataset before being validated and evaluated on 88 and 304 low-SNR patches, respectively, using a structural similarity index measure (SSIM) loss function. We demonstrate models trained on larger datasets of human cervical tissue could be used to successfully restore metabolic metrics with an improvement in image quality when applied to rat cheek epithelium images. These results motivate further exploration of weight transfer for denoising of small clinical two-photon microscopy datasets.
Two-photon excited fluorescence (TPEF) can provide metabolic information of the cellular microenvironment. Metabolic changes are intimately associated with numerous diseases. It has been suggested that the metabolic interpretations from fresh tissue imaging can be maintained in tissue samples post-fixation. Given the large availability of fixed tissues for various conditions, we sought to evaluate the impact of fixation on several TPEF-extracted metabolic indicators of unstained tissues based on endogenous intensity and lifetime TPEF measurements. Our results indicate that the metabolic interpretations of fixed tissue cannot be used as a representation of the in vivo environment or that of freshly excised specimens.
Fiber endoscopes capable of making two-photon (2P) autofluorescence measurements, time-resolved fluorescence decay measurements, and collagen second harmonic generation (SHG) measurements have been applied to animal models. Clinical translation of such devices to internal human organs has the potential to overhaul conventional methods of disease diagnosis and monitoring. Previous work by our lab has established the potential to diagnose high-grade cervical precancers using 2P autofluorescence measurements. Other groups have demonstrated that 2P-based fluorescence lifetime imaging microscopy (FLIM) measurements of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and collagen SHG measurements have the potential to discriminate between cancerous and benign tissues. In this work, we demonstrate the potential to discern high-grade cervical precancerous lesions (HSILs) from benign tissues using fluorescence intensity measurements of NAD(P)H and oxidized flavoproteins, FLIM NAD(P)H measurements, and collagen SHG measurements. Consistent with previous results, benign tissues demonstrated increased depth-dependent heterogeneity in mitochondrial clustering, and increased overall and intrafield heterogeneity of oxido-reductive state relative to HSILs. FLIM phasor analysis demonstrated a relative decrease in NAD(P)H short and long lifetime, and a relative increase in NAD(P)H bound fraction for benign tissues compared to HSILs. Collagen SHG intensity in benign tissues was greater than that of HSIL tissues, along with overall intrafield variations in collagen fiber orientation. This work motivates the functionalization of a clinical 2P fiber endoscope capable of making SHG, autofluorescence intensity and lifetime measurements of metabolic coenzymes in the human cervix.
The World Health Organization (WHO) called for a global fight against cervical cancer. There are an estimated 569,000 new cases and 310,000 deaths annually. Searching for practical approaches to deal with cervical cancer screening and treatment has been an urgent research subject. One solution could be to use label-free two-photon excited fluorescence (TPEF) imaging to address this need. The colposcopy-guided biopsy method is being used for cervical precancer detection relying primarily on morphological and organization cell and tissue feature changes. However, the overall performance of colposcopy and biopsy remains unsatisfactory. Label-free TPEF provides images with high morphological and functional (metabolic) content and could lead to enhanced detection of cervical pre-cancers. This paper uses the cell texture and morphology features to classify stacks of such TPEF images acquired from freshly excised healthy and pre-cancerous human cervical tissues. Herein, an automated denoising algorithm and a parametrized edge enhancement method is used for pre-processing the images in the stack. The computer simulations performed on the privately available dataset of 10 healthy stacks, 53 precancer stacks, and the recall and specificity of 100 %, respectively, were observed for both texture and morphology features. However, the dataset used to acquire these results is small. The presented model can be used as a base model for further research and analysis of a larger data set to identify early cervical cancerous changes and potentially significantly improve diagnosis and treatment.
Vitiligo is an immune skin disorder consisting of depigmented skin patches caused by the destruction of epidermal melanocytes. Vitiligo treatment represents a clinical challenge since the re-pigmentation mechanism is not fully understood. In this pilot study, we employ in-vivo multiphoton microscopy to evaluate epidermal keratinocyte metabolic state before and during treatment, in-vivo reflectance confocal microscopy to track melanocyte migration after treatment initiation, and single cell transcriptomics to identify unique cell populations more abundant in stable vitiligo lesions compared to normal skin. The findings provide insights into the role of certain cell populations in the viability of micro-grafting treatments.
Vitiligo is a disorder characterized by the loss of skin pigment due to the autoimmune destruction of epidermal melanocytes. Treatments that aim to repigment vitiligo regions are long, incomplete, and suffer from a highly variable success rate. This study aims to use non-invasive, depth-resolved, label-free, two-photon detection of reduced nicotinamide adenine dinucleotide (NADH) to quantitatively evaluate mitochondrial dynamics in vitiligo micrografting patients during the repigmentation process. This study indicates differences in the depth-dependent mitochondrial profiles between healthy and vitiligo tissues. This study also indicates that vitiligo regions responsive to treatment exhibit depth-dependent shifts in mitochondrial dynamics prior to repigmentation.
KEYWORDS: Image restoration, Tissues, Signal to noise ratio, In vivo imaging, Biopsy, Two photon imaging, Microscopy, Luminescence, Imaging systems, Image quality
High signal-to-noise ratio (SNR) images are necessary for analyzing sub-cellular features in biomedical images. Acquisition of such images may be limited by temporal or photon-budget-based imaging constraints. This study aims to use deep-learning-based image restoration methods to extract morpho-functional information from low-SNR, depth-resolved, label-free, two-photon images of human cervical tissue. A deep convolutional autoencoder model was trained using single-frame image inputs and multiple-frame averaged ground-truth image pairs. Automated analysis of restored reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) two-photon excitation fluorescence (TPEF) images extracts depth-dependent, morpho-functional information otherwise lost in single-frame images.
Metabolism plays a critical role in the function of cells and tissues. Changes in metabolic function are hallmarks of numerous conditions, including aging, cancer, obesity and neurodegenerative diseases. Such changes are highly dynamic and heterogeneous at the microscopic scale. Label-free, two-photon microscopic imaging offers opportunities to monitor and characterize such aspects of metabolic function non-destructively. Intensity and lifetime fluorescence measurements from endogenous NAD(P)H and FAD can serve as sensitive indicators of metabolic changes associated with redox state and mitochondrial organization. In fact, a combination of optical metabolic readouts including the redox ratio, NADH bound fraction, and mitochondrial clustering can provide important insights on the specific nature of the metabolic pathway perturbation that yielded the optical changes. Assessment of such information has the potential to improve our understanding, detection, and treatment of numerous diseases. For example, our studies highlight that the use of multiple optical metabolic readouts enables sensitive and specific detection of changes that are associated with adipose tissue type (brown vs beige vs white) and responses to stimuli. In addition, we have discovered that our ability to characterize depth-dependent variations within epithelial tissues, such as the skin and cervix, plays a key role in identifying alterations that occur at the onset of cancer and may be used to develop improved, non-invasive methods for cancer diagnosis. Monitoring of immune system cell activation is another important application area. Such findings pave the way for exploiting label-free, metabolic imaging techniques to understand dynamic cell interaction and their role in the development and treatment of a number of human diseases.
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