Multimodal microscopy for biological tissue imaging

Laser-scanning microscopy offers promising solutions for tissue imaging at sub-cellular level and it can provide both morphological and functional information in a label-free modality. The combination of non-linear optical (NLO) microscopy techniques providing morphological information, together with those providing functional information is a crucial issue for tissue classification and pathological assessment. In fact, an important feature in understanding the development of a tissue disease is the relationship between morphological features such as architecture, shape, symmetry, and function. The proper combination of NLO microscopy techniques with Raman-based techniques in a morpho-chemical multimodal imaging approach can image and classify tissues in a fast, reliable, and label-free way. The experiment is aimed at developing two different advanced laser scanning microscopy platforms. A first platform that combines non-linear imaging techniques (Two-photon fluorescence, Second-harmonic generation, Fluorescence Lifetime Imaging) with Raman microspectroscopy will allow having an instrument able to provide a morpho-functional characterization of the examined samples. A second platform that combines Two-photon fluorescence and Second-harmonic generation microscopy with Brillouin light scattering micro-spectroscopy will enable the study of tissue morpho-mechanics, targeting both tissue morphology and biomechanics by means of an all-optical non-invasive contact-less correlative approach. The instruments are used for various research activities, including:

– Non-linear laser microscopy applied to digital pathology
The growing need for more accurate, objective and faster diagnosis has driven researchers to combine digital imaging with routine diagnostics. The term “Digital Pathology” refers to clinical practice focused on the use of computer technology for the development of high-resolution images, from slides containing tissue samples. Although “Digital Pathology” is transforming diagnostic procedures, the largely used H&E staining allows morphological classification of tissues, while functional aspects require immunohistochemical procedures. Non-linear optical (NLO) microscopy offers promising solutions for tissue imaging at a sub-cellular level and can provide both morphological and functional information in a label-free modality. In this experiment, we plan to combine the advantages offered by “Digital Pathology” with the capabilities of NLO microscopy to provide a label-free digital platform for the automatic analysis and diagnostics of human biological tissues in a rapid, objective and reliable manner. The approach will be used for diagnostic purposes for both cancer and non-cancer diseases.

– Morpho-mechanical imaging of the cornea
The experiment is aimed at using a correlative imaging approach that combines SHG microscopy with Brillouin light scattering micro-spectroscopy for targeting tissue morpho-mechanics, with particular attention to corneal imaging. In particular, second-harmonic generation (SHG) microscopy will be used with both forward and backward detection as well as with a polarization scanning excitation scheme. The main goal is to take advantage of the strong SHG signal emitted by collagen to characterize its organization in both healthy and diseased samples of cornea. The morphological characterization of the sample, provided by SHG microscopy, will be correlated with the corresponding viscoelastic properties, highlighted by Brillouin light scattering micro-spectroscopy. This activity is aimed at advancing in the field of ophthalmic diagnostic imaging by developing new cutting-edge techniques, their applications to specific diseases, and finally their perspective of integration in a single platform, useful for clinical ophthalmic diagnostics. Further, The synergic integration of Brillouin and SHG microscopy will yield an unprecedently comprehensive insight into the morpho-mechanical features of collagenous samples, opening a new frontier in bio-imaging with a strong potential for impact in all clinical procedures requiring a non-invasive assessment of collagen morphology and biomechanics.

SHG images (on the left) and maps of relative variations of Brillouin frequency (on the right), acquired at in depth into the corneal stroma below Bowman’s membrane. Scale bars: 10 um.

Personale INO dipendente:
D’Uva Massimo, Cicchi Riccardo,

Personale associato:
Quercioli Franco, Pavone Francesco Saverio,