Using Fluorescence Spectroscopy to Diagnose Breast Cancer

Optical spectroscopy methods have had considerable impact in the field of biomedical diagnostics, providing novel methods for the early or noninvasive diagnosis of various medical conditions. Among them, fluorescence spectroscopy has been the most widely explored mainly because fluorescence is highly sensitive to the biochemical makeup of tissues. It has been shown that tumors were easily detected on account of altered fluorescence properties with respect to fluorescence of ordinary tissue. Breast cancer is one of the most commonly diagnosed cancers among women in the world and also it is one of the leading causes of deaths from cancer for the female population. However, when detected in early stage, it is one of the most treatable forms of cancer. Therefore, fluorescence technologies could be highly beneficial in early detection and timely treatment of cancer. This chapter presents main results and conclusions that have been reported on the use of fluorescence spectroscopy for the investigation of breast cancer. It also gives an overview on the instruments and methodology of measurements, on the main endogenous fluorophores present in tissues, on the tissue fluorescence, and on the statistical methods that aid interpretations of fluorescence spectra. Finally, examples of using various fluorescence techniques, such as excitation, emission and synchronous spectroscopy, excitation-emission matrices, and lifetimes, for the breast cancer diagnosis are presented

\u3cem\u3eIn vivo\u3c/em\u3e Imaging of Human Retinal Microvasculature Using Adaptive Optics Scanning Light Ophthalmoscope Fluorescein Angiography

The adaptive optics scanning light ophthalmoscope (AOSLO) allows visualization of microscopic structures of the human retina in vivo. In this work, we demonstrate its application in combination with oral and intravenous (IV) fluorescein angiography (FA) to the in vivo visualization of the human retinal microvasculature. Ten healthy subjects ages 20 to 38 years were imaged using oral (7 and/or 20 mg/kg) and/or IV (500 mg) fluorescein. In agreement with current literature, there were no adverse effects among the patients receiving oral fluorescein while one patient receiving IV fluorescein experienced some nausea and heaving. We determined that all retinal capillary beds can be imaged using clinically accepted fluorescein dosages and safe light levels according to the ANSI Z136.1-2000 maximum permissible exposure. As expected, the 20 mg/kg oral dose showed higher image intensity for a longer period of time than did the 7 mg/kg oral and the 500 mg IV doses. The increased resolution of AOSLO FA, compared to conventional FA, offers great opportunity for studying physiological and pathological vascular processes

INVESTIGATING INVASION IN DUCTAL CARCINOMA IN SITU WITH TOPOGRAPHICAL SINGLE CELL GENOME SEQUENCING

Synchronous Ductal Carcinoma in situ (DCIS-IDC) is an early stage breast cancer invasion in which it is possible to delineate genomic evolution during invasion because of the presence of both in situ and invasive regions within the same sample. While laser capture microdissection studies of DCIS-IDC examined the relationship between the paired in situ (DCIS) and invasive (IDC) regions, these studies were either confounded by bulk tissue or limited to a small set of genes or markers. To overcome these challenges, we developed Topographic Single Cell Sequencing (TSCS), which combines laser-catapulting with single cell DNA sequencing to measure genomic copy number profiles from single tumor cells while preserving their spatial context. We applied TSCS to sequence 1,293 single cells from 10 synchronous DCIS patients. We also applied deep-exome sequencing to the in situ, invasive and normal tissues for the DCIS-IDC patients. Previous bulk tissue studies had produced several conflicting models of tumor evolution. Our data support a multiclonal invasion model, in which genome evolution occurs within the ducts and gives rise to multiple subclones that escape the ducts into the adjacent tissues to establish the invasive carcinomas. In summary, we have developed a novel method for single cell DNA sequencing, which preserves spatial context, and applied this method to understand clonal evolution during the transition between carcinoma in situ to invasive ductal carcinoma

Surface Fluorescence Studies of Tissue Mitochondrial Redox State in Isolated Perfused Rat Lungs

We designed a fiber-optic-based optoelectronic fluorometer to measure emitted fluorescence from the auto-fluorescent electron carriers NADH and FAD of the mitochondrial electron transport chain (ETC). The ratio of NADH to FAD is called the redox ratio (RR = NADH/FAD) and is an indicator of the oxidoreductive state of tissue. We evaluated the fluorometer by measuring the fluorescence intensities of NADH and FAD at the surface of isolated, perfused rat lungs. Alterations of lung mitochondrial metabolic state were achieved by the addition of rotenone (complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor) and/or pentachlorophenol (PCP, uncoupler) into the perfusate recirculating through the lung. Rotenone- or KCN-containing perfusate increased RR by 21 and 30%, respectively. In contrast, PCP-containing perfusate decreased RR by 27%. These changes are consistent with the established effects of rotenone, KCN, and PCP on the redox status of the ETC. Addition of blood to perfusate quenched NADH and FAD signal, but had no effect on RR. This study demonstrates the capacity of fluorometry to detect a change in mitochondrial redox state in isolated perfused lungs, and suggests the potential of fluorometry for use in in vivo experiments to extract a sensitive measure of lung tissue health in real-time

Compact solid-state CMOS single-photon detector array for in vivo NIR fluorescence lifetime oncology measurements

In near infrared fluorescence-guided surgical oncology, it is challenging to distinguish healthy from cancerous tissue. One promising research avenue consists in the analysis of the exogenous fluorophores’ lifetime, which are however in the (sub-)nanosecond range. We have integrated a single-photon pixel array, based on standard CMOS SPADs (single-photon avalanche diodes), in a compact, time-gated measurement system, named FluoCam. In vivo measurements were carried out with indocyanine green (ICG)-modified derivatives targeting the avb3 integrin, initially on a genetically engineered mouse model of melanoma injected with ICG conjugated with tetrameric cyclic pentapeptide (ICG􀀀E[c(RGDfK)4]), then on mice carrying tumour xenografts of U87-MG (a human primary glioblastoma cell line) injected with monomeric ICG􀀀c(RGDfK). Measurements on tumor, muscle and tail locations allowed us to demonstrate the feasibility of in vivo lifetime measurements with the FluoCam, to determine the characteristic lifetimes (around 500 ps) and subtle lifetime differences between bound and unbound ICG-modified fluorophores (10% level), as well as to estimate the available photon fluxes under realistic conditions

Study of Scattering and Polarization of Light in Biological Tissue

Tkáňová optika nabývá rychle na významu a přesná znalost optických vlastností biologických tkání je podstatná pro výzkum v biomedicíně i pro kontrolu kvality potravin. Jestliže je vzorek tkáně osvětlen, dochází k mnohonásobnému odrazu světla. V případě postmortem neživých tkání (maso) je rozměr buněk větší než vlnová délka použitého světla. Dochází k Mieovu rozptylu prošlého nebo zpět odraženého světla, v důsledku čehož se objevují různé polarizační stavy světla. Polarizační stavy světla rozptýleného na difúzním prostředí jsou experimentálně zkoumány a modelovány. V práci jsme provedli dva modifikované experimenty: rozptyl polarizovaného světla, které dvakrát prochází vzorkem (vpřed a vzad) a jen světla, které jen prochází vzorkem. Měření rozptýleného světla ukazuje, že dochází k depolarizaci a ke stáčení polarizační roviny, což obojí závisí na orientaci svalových vláken a stárnutí tkání postmortem. Mimo experimentů byl také proveden teoretický popis difúzní biologické tkáně a byla vypočtena radiační přenosová rovnice pomocí modifikované Monte Carlo metody, která zahrnuje polarizační stav světla (PLMC). Je ukázáno, že stupeň polarizace podstatně závisí na optických vlastnostech rozptylového prostředí. Výsledky ukazují, že stav polarizace světla na výstupu závisí na stavu polarizace světla před vzorkem a na optických vlastnostech a tloušťce vrstvy prostředí v průběhu jejího stárnutí. Je také provedena korelace změn polarizace na čerstvosti masa, i popis dynamického chování polarizace při stárnutí masa.Tissue optics becomes a rapidly expanding field of great interest and a precise knowledge of optical properties of biological tissues is essential for biomedical investigation and food quality control. If the sample of tissue is illuminated, the multiple scattering occurs. In the case of the postmortem tissue (meat) the cell dimensions are larger than the wavelength. Mie scattering of transmitted or reflected light arises and produces various polarization states. Polarization properties of light scattered from a scattering medium have been studied with experiments and modeling. Two modified experiments were performed: scattering of polarized light passing twice the sample (forward and backward) and only transmitted light. The measurements of scattered light display depolarization and rotation of polarized light, which depend both on orientation of the muscle fibers and ageing process of meat. Theoretical description of turbid biological tissue and computing of radiative transfer equation by modified Polarized Light Monte Carlo (PLMC) method has also been executed. It is shown that the degree of polarization is sensitive to the optical properties of the turbid medium. The results demonstrate that polarized light scattered from a scattering medium is sensitive to the state of input polarization and the optical properties and thickness of the tissue during the ageing. The correlations of polarization changes and freshness of meat, as well as dynamic behavior of the polarization in ageing meat are shown.

Hardware implementation algorithm and error analysis of high-speed fluorescence lifetime sensing systems using center-of-mass method

A new, simple, high-speed, and hardware-only integrationbased fluorescence-lifetime-sensing algorithm using a center-of-mass method CMM is proposed to implement lifetime calculations, and its signal-to-noise-ratio based on statistics theory is also deduced. Compared to the commonly used iterative least-squares method or the maximum-likelihood-estimation–based, general purpose fluorescence lifetime imaging microscopy FLIM analysis software, the proposed hardware lifetime calculation algorithm with CMM offers direct calculation of fluorescence lifetime based on the collected photon counts and timing information provided by in-pixel circuitry and therefore delivers faster analysis for real-time applications, such as clinical diagnosis. A real-time hardware implementation of this CMM FLIM algorithm suitable for a single-photon avalanche diode array in CMOS imaging technology is now proposed for implementation on field-programmable gate array. The performance of the proposed methods has been tested on Fluorescein, Coumarin 6, and 1,8- anilinonaphthalenesulfonate in water/methanol mixture

Supercontinuum radiation in fluorescence microscopy and biomedical imaging applications

Compact, high brightness supercontinuum sources have already made a big impact in the fields of fluorescence microscopy and other biomedical imaging techniques, such as optical coherence tomography and coherent anti-Stokes Raman scattering. In this review, we provide a brief overview on the generation and properties of supercontinuum radiation for imaging applications. We review specific uses of supercontinuum sources and their potential for imaging, but also their limitations and caveats. We conclude with a review of recent advances in UV supercontinuum generation, near-IR microscopy, exciting new potentials for the use of hollow-core PCFs, on-chip supercontinuum generation, and technologies to improve supercontinuum stability for certain applications.H2020 Marie Skłodowska-Curie Actions (MSCA) (722380); MedImmune; Infinitus (China) Ltd.; Engineering and Physical Sciences Research Council (EPSRC) (EP/H018301/1, EP/L015889/1); Wellcome Trust (089703/Z/09/Z, 3-3249/Z/16/Z); Medical Research Council (MRC) (MR/K015850/1, MR/K02292X/1)

Temporal metabolic partitioning of the yeast and protist cellular networks:the cell is a global scale-invariant (fractal or self-similar) multioscillator

Britton Chance, electronics expert when a teenager, became an enthusiastic student of biological oscillations, passing on this enthusiasm to many students and colleagues, including one of us (DL). This historical essay traces BC’s influence through the accumulated work of DL to DL’s many collaborators. The overall temporal organization of mass-energy, information, and signaling networks in yeast in self-synchronized continuous cultures represents, until now, the most characterized example of in vivo elucidation of time structure. Continuous online monitoring of dissolved gases by direct measurement (membrane-inlet mass spectrometry, together with NAD(P)H and flavin fluorescence) gives strain-specific dynamic information from timescales of minutes to hours as does two-photon imaging. The predominantly oscillatory behavior of network components becomes evident, with spontaneously synchronized cellular respiration cycles between discrete periods of increased oxygen consumption (oxidative phase) and decreased oxygen consumption (reductive phase). This temperature-compensated ultradian clock provides coordination, linking temporally partitioned functions by direct feedback loops between the energetic and redox state of the cell and its growing ultrastructure. Multioscillatory outputs in dissolved gases with 13 h, 40 min, and 4 min periods gave statistical self-similarity in power spectral and relative dispersional analyses: i.e., complex nonlinear (chaotic) behavior and a functional scale-free (fractal) network operating simultaneously over several timescales