Ebook: Microscopy Applied to Biophotonics

Biophotonics and microscopy are highly inter-related fields in terms of both technology development and biomedical applications. The tremendous recent advances in microscopy are paralleled by new opportunities for biophotonics—the investigation and manipulation of biological phenomena using light and its application to the biomedicine. This “Enrico Fermi” School aimed to give the students an overview of these rapidly developing fields and enhance their understanding of the technological developments in optical microscopy that are synergistic with the application of photonics to molecular, cell and tissue biology. The School was highly interdisciplinary, involving physicists, biologists, chemists and clinicians and stimulated discussion from both the technological and biological points of view. We hope that other students in the fields of biophotonics and microscopy will also find this volume useful.

The School was designed as a series of short courses, clustering three or four lectures of related topics, with biomedical applications being discussed after the basic photonics concepts had been addressed. The programme began with a review of the fundamentals of microscopy and its related technologies, presenting approaches based on linear and nonlinear interactions of laser light with biological specimens. Lecturers introduced the concepts of fluorescence, phase contrast and nonlinear microscopies, and laser-based light sources. This was followed by lectures focusing on specific fluorophores and other probes and advanced fluorescence microscopy techniques such as structured illumination, FRAP, FLIM, FRET, anisotropy, FCS and spatial correlation microscopy, as well as non-fluorescence techniques utilising phase contrast, autofluorescence, Raman, and second harmonic signals. The students learned how these concepts could be applied to a broad spectrum of applications including multidimensional fluorescence imaging, in vivo cytometry, tissue imaging and clinical diagnosis.

The organizers gave priority to applications from PhD students although post-doctoral researchers were also welcome, and the School attracted international attendees from many continents. Student poster sessions were very impressive and a number of these papers were selected for oral presentation by the students within the lecture programme. The relaxed atmosphere of the School, which took place at the magnificent location of Villa Monastero in Varenna, provided a stimulating environment for scientific exchange between students and lecturers. The local organizers, Barbara Alzani, together with Ramona Brigatti and Marta Pigazzini, did a wonderful job and special thanks are due to Dr. Daniela Selisca who played a major role in the organisation of the School. We also gratefully recognize the contributions of Roberta Comastri, Monica Bonetti, and Marcella Missiroli. Finally, we thank the Italian Physical Society for making this event possible.

F. S. Pavone, P. T. C. So and P. M. W. French

This article is intended to provide a brief overview of fluorescence microscopy and its application to biological samples, with a particular emphasis on fluorescence lifetime imaging (FLIM). It includes an introduction to wide-field and laser scanning confocal and multiphoton microscopy and the analysis of the fluorescence signal with respect to wavelength, lifetime and polarisation. It also reviews recent progress extending fluorescence microscopy and FLIM to super-resolved imaging, to high throughput automated microscopy and to imaging live disease models.

During the last two decades, nonlinear imaging techniques experienced an impressive growth in biological and biomedical imaging applications. New techniques have been developed and applied to several topics in modern biology and biomedical optics. The nonlinear nature of the excitation provides an absorption volume spatially confined to the focal point. The localization of the excitation is maintained even in strongly scattering tissues, allowing deep-tissue high-resolution microscopy. This paper gives a general overview of two-photon fluorescence, second-harmonic generation, and fluorescence lifetime imaging techniques, including a theoretical approach to the physical background, a brief description of the methodologies used, and a list of biological and biomedical applications on tissue imaging.

3D ultramicroscopy (light sheet microscopy) is a versatile imaging technique with a unique combination of capabilities. It provides high imaging speed, high signal-to-noise ratio and low levels of photobleaching and phototoxic effects. These properties are critical in a wide range of applications in the life sciences. When combined with tissue clearing methods, light sheet microscopy allows rapid imaging of large specimens with excellent coverage and high spatial resolution. Even samples up to the size of entire mammalian brains can be efficiently recorded and quantitatively analyzed. Numerous examples of application are given.

Modern trends in Raman spectroscopy for biomedical applications will be presented. It will be shown that Raman spectroscopy and various Raman based technologies like, e.g., Raman microscopy, SERS, TERS, CARS are powerful tools to address a broad spectrum of bioanalytical and biomedical problems like, e.g., rapid pathogen identification, sensitive monitoring of low concentrated molecules (e.g., drugs, metabolites) or objective clinical cell and tissue diagnostics for an early diagnosis of diseases like, e.g., cancer.

The clinically relevant transmittance digital microscopy (TDM) and photothermal (PA)/photoacoustic (PT) in vivo flow cytometry platform for blood and lymph cell analysis in flows is described. PA/PT techniques allowing for identification of non-fluorescent cells with different natural absorptive properties, such as counting of white blood cells (WBCs) in blood flow or rare red blood cells (RBCs) in lymph flow, are presented and their applicability for investigation of pathologic conditions is discussed. The capability of these techniques to detect cancer cells and small bodies in blood and lymph streams, such as platelets, chylomicrons, and bacteria, is demonstrated. Advantages of non-invasive TDM/PT/PA in vivo flow cytometry that include the possibility to measure flow velocity without the help of any probes, labels or scattering effects, to study cell-cell interactions in flows (cell aggregation, deformability, rolling, etc.) and cellular responses to the impact of different environmental agents (e.g., drugs, laser and gamma-radiation) are analyzed.

Resonant nonlinear optical microscopy is a field that uses resonant nonlinear optics for spatial imaging on a microscopic scale. Enabled by the development of suitable laser sources and detectors, this field has brought a host of new contrast techniques to light. In this article we describe vibrationally resonant techniques such as coherent anti-Stokes Raman scattering (CARS) or stimulated Raman scattering (SRS), and electronically resonant techniques such as four-wave mixing imaging, with specific emphasis on generalization of the underlying concepts.

The strength of any microscopy method lies in the ability of its applied contrast mechanism to visualise and quantify properties in biological structures without interfering with normal physiological behaviour. Second-harmonic generation (SHG) is one of the several available label-free nonlinear contrast mechanisms that can be utilised in the microscope. Since SHG requires a noncentrosymmetric media, only certain biological structures can inherently generate the signal. Collagen, starch granules and the thick myosin filaments of striated muscle are examples of biological structures that generate SHG. Investigating the SHG intensity dependence on the incident polarisation allows the relative magnitudes of the second-order nonlinear susceptibility tensor components to be determined. For example, collagen and muscle myosin can be modeled with cylindrical symmetry, which results in three surviving tensor components. Since the samples have different material composition the tensor component magnitudes and the resulting polarisation dependence differ drastically. In this work the outgoing polarisation of the generated second harmonic is also measured for higher accuracy fitting of data obtained from biological samples. The polarisation-in polarisation-out (PIPO) surface plots can be used to obtain values for the nonlinear susceptibility tensor components. In addition, birefringence in the fundamental beam and emitted second harmonic can be accounted for in the PIPO theory, thereby providing more accurate results of the nonlinear susceptibility tensors in birefringent biological structures. The birefringence in a single skeletal muscle fibre was determined to be Δn(1028 nm)=0.0018±0.0004 and Δn(514 nm)=0.0021±0.0003.

We present a chemical sensor design, based on the co-integration of microfluidic and optical functions on a planar glass substrate. Borosilicate glasses, thanks to their high chemical resistance allow the use of the sensor in a nuclear environment, such as the nuclear industry, in which analysis miniaturization is one of the most active research fields. The working principle of this sensor is based on absorptiometry, sensitivity-enhanced by the introduction of segmented waveguides into a resonant cavity.

Second-harmonic generation (SHG) microscopy has a great potential for the clinical investigation of human skin and skin diseases, especially in combination with others nonlinear optical modalities. However, such multimodal approaches generate immense datasets, which requires automated data handling. In this contribution, the potential of an SHG-image processing algorithm for the automated classification of skin into normal or keloid is demonstrated. The classification of the tissue implemented in the algorithm employs the geometrical features of collagen patterns that differ depending on the constitution, i.e., physiological status of the skin.