Multimodal nonlinear microscopy: A powerful label-free method for supporting standard diagnostics on biological tissues

The large use of nonlinear laser scanning microscopy in the past decade paved the way for potential clinical application of this imaging technique. Modern nonlinear microscopy techniques offer promising label-free solutions to improve diagnostic performances on tissues. In particular, the combination of multiple nonlinear imaging techniques in the same microscope allows integrating morphological with functional information in a morpho-functional scheme. Such approach provides a high-resolution label-free alternative to both histological and immunohistochemical examination of tissues and is becoming increasingly popular among the clinical community. Nevertheless, several technical improvements, including automatic scanning and image analysis, are required before the technique represents a standard diagnostic method. In this review paper, we highlight the capabilities of multimodal nonlinear microscopy for tissue imaging, by providing various examples on colon, arterial and skin tissues. The comparison between images acquired using multimodal nonlinear microscopy and histology shows a good agreement between the two methods. The results demonstrate that multimodal nonlinear microscopy is a powerful label-free alternative to standard histopathological methods and has the potential to find a stable place in the clinical setting in the near future

Few Shot Learning in Histopathological Images:Reducing the Need of Labeled Data on Biological Datasets

Although deep learning pathology diagnostic algorithms are proving comparable results with human experts in a wide variety of tasks, they still require a huge amount of well annotated data for training. Generating such extensive and well labelled datasets is time consuming and is not feasible for certain tasks and so, most of the medical datasets available are scarce in images and therefore, not enough for training. In this work we validate that the use of few shot learning techniques can transfer knowledge from a well defined source domain from Colon tissue into a more generic domain composed by Colon, Lung and Breast tissue by using very few training images. Our results show that our few-shot approach is able to obtain a balanced accuracy (BAC) of 90% with just 60 training images, even for the Lung and Breast tissues that were not present on the training set. This outperforms the finetune transfer learning approach that obtains 73% BAC with 60 images and requires 600 images to get up to 81% BAC.This study has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 732111 (PICCOLO project)

Observation of an improved healing process in superficial skin wounds after irradiation with a blue-LED haemostatic device

The healing process of superficial skin wounds treated with a blue-LED haemostatic device is studied. Four mechanical abrasions are produced on the back of 10 Sprague Dawley rats: two are treated with the blue-LED device, while the other two are left to naturally recover. Visual observations, non-linear microscopic imaging, as well as histology and immunofluorescence analyses are performed 8 days after the treatment, demonstrating no adverse reactions neither thermal damages in both abraded areas and surrounding tissue. A faster healing process and a better-recovered skin morphology are observed: the treated wounds show a reduced inflammatory response and a higher collagen content. Blue LED induced photothermal effect on superficial abrasions

Multimodal nonlinear imaging of atherosclerotic plaques differentiation of triglyceride and cholesterol deposits

Cardiovascular diseases in general and atherothrombosis as the most common of its individual disease entities is the leading cause of death in the developed countries. Therefore, visualization and characterization of inner arterial plaque composition is of vital diagnostic interest, especially for the early recognition of vulnerable plaques. Established clinical techniques provide valuable morphological information but cannot deliver information about the chemical composition of individual plaques. Therefore, spectroscopic imaging techniques have recently drawn considerable attention. Based on the spectroscopic properties of the individual plaque components, as for instance different types of lipids, the composition of atherosclerotic plaques can be analyzed qualitatively as well as quantitatively. Here, we compare the feasibility of multimodal nonlinear imaging combining two-photon fluorescence (TPF), coherent anti-Stokes Raman scattering (CARS) and second-harmonic generation (SHG) microscopy to contrast composition and morphology of lipid deposits against the surrounding matrix of connective tissue with diffraction limited spatial resolution. In this contribution, the spatial distribution of major constituents of the arterial wall and atherosclerotic plaques like elastin, collagen, triglycerides and cholesterol can be simultaneously visualized by a combination of nonlinear imaging methods, providing a powerful label-free complement to standard histopathological methods with great potential for in vivo application

Three-dimensional mapping of the orientation of collagen corneal lamellae in healthy and keratoconic human corneas using SHG microscopy

SHG image acquired with sagittal optical sectioning (A) of a healthy cornea and (B) of a keratoconic cornea. Scale bars: 30 μm. Keratoconus is an eye disorder that causes the cornea to take an abnormal conical shape, thus impairing its refractive functions and causing blindness. The late diagnosis of keratoconus is among the principal reasons for corneal surgical transplantation. This pathology is characterized by a reduced corneal stiffness in the region immediately below Bowman's membrane, probably due to a different lamellar organization, as suggested by previous studies. Here, the lamellar organization in this corneal region is characterized in three dimensions by means of second-harmonic generation (SHG) microscopy. In particular, a method based on a three-dimensional correlation analysis allows to probe the orientation of sutural lamellae close to the Bowman's membrane, finding statistical differences between healthy and keratoconic samples. This method is demonstrated also in combination with an epi-detection scheme, paving the way for a potential clinical ophthalmic application of SHG microscopy for the early diagnosis of keratoconus

Multidimensional non-linear laser imaging of Basal Cell Carcinoma

We have used a multidimensional non-linear laser imaging approach to visualize ex-vivo samples of basal cell carcinoma (BCC). A combination of several non-linear laser imaging techniques involving fluorescence lifetime, multispectral two-photon and second-harmonic generation imaging has been used to image different skin layers. This approach has elucidated some morphological (supported by histopathological images), biochemical, and physiochemical differences of the healthy samples with respect to BCC ones. In particular, in comparison with normal skin, BCC showed a blue-shifted fluorescence emission, a higher fluorescence response at 800 nm excitation wavelength and a slightly longer mean fluorescence lifetime. Finally, the use of aminolevulinic acid as a contrast agent has been demonstrated to increase the constrast in tumor border detection. The results obtained provide further support for in-vivo non-invasive imaging of Basal Cell Carcinoma

Photothermally-induced disordered patterns of corneal collagen revealed by SHG imaging

The loss of organization of the corneal collagen lattice induced by photothermal effects was analyzed by using second-harmonic generation (SHG) imaging. Porcine cornea samples were treated with low-power laser irradiation in order to get localized areas of tissue disorganization. The disorder induced within the irradiated area of corneal stroma was quantified by means of Discrete Fourier Transform, auto-correlation and entropy analyses of the SHG images. Polarization modulated SHG measurements allowed to probe the changes in the structural anisotropy of sub-micron hierarchical levels of the stromal collagen. Our results emphasize the great potential of the SHG imaging to detect subtle modifications in the collagen assembly. The proposed analytical methods may be used to track several genetic, pathologic, accidental or surgical-induced disorder states of biological tissues

Protein conformation and molecular order probed by second-harmonic-generation microscopy

Second-harmonic-generation (SHG) microscopy has emerged as a powerful tool to image unstained living tissues and probe their molecular and supramolecular organization. In this article, we review the physical basis of SHG, highlighting how coherent summation of second-harmonic response leads to the sensitivity of polarized SHG to the three-dimensional distribution of emitters within the focal volume. Based on the physical description of the process, we examine experimental applications for probing the molecular organization within a tissue and its alterations in response to different biomedically relevant conditions. We also describe the approach for obtaining information on molecular conformation based on SHG polarization anisotropy measurements and its application to the study of myosin conformation in different physiological states of muscle. The capability of coupling the advantages of nonlinear microscopy (micrometer-scale resolution in deep tissue) with tools for probing molecular structure in vivo renders SHG microscopy an extremely powerful tool for the advancement of biomedical optics, with particular regard to novel technologies for molecular diagnostic in vivo. (C) 2012 Society of Photo-Optical Instrumentation Engineers (SPIE)

Spectral morphological analysis of skin lesions with a polarization multispectral dermoscope.

Dermoscopy is the conventional technique used for the clinical inspection of human skin lesions. However, the identification of diagnostically relevant morphologies can become a complex task. We report on the development of a polarization multispectral dermoscope for the in vivo imaging of skin lesions. Linearly polarized illumination at three distinct spectral regions (470, 530 and 625 nm), is performed by high luminance LEDs. Processing of the acquired images, by means of spectral and polarization filtering, produces new contrast images, each one specific for melanin absorption, hemoglobin absorption, and single scattering. Analysis of such images could facilitate the identification of pathological morphologies. (C) 2013 Optical Society of Americ