Native fluorescence spectra of retinoic acid (RA)-treated and untreated human breast cancer cells were measured using selective wavelengths of 300 nm and 340 nm for excitation. The spectral data of the two types of cells were analyzed using machine learning algorithms for linear unmixing and classification which yielded high accuracy. The results show that the concentrations of the native fluorophores such as tryptophan, NADH and flavins in the human malignant breast cells change when they are treated with RA. The study shows the dual-wavelength fluorescence spectroscopy aided by machine learning has potential clinical applications in drug development and chemotherapeutic studies.
We present here a novel full field pump-probe photothermal dynamics microscopy (PTDM) which uses a numerical lockin mechanism for capturing full field photothermal responses and is capable of imaging 2D thermal dissipation dynamics by varying the time delay between the probing and pump nano-second pulses. PTDM may find interesting applications in biology and medicine. As one example, we report PTDM imaging nuclei contained on intact hematoxylin and eosin (H&E) stained prostate cancer specimens may potentially be used to distinguish low grade and high grade prostate cancer.
Cybesin, a smart contrast agent to target cancer cells, was investigated using a near infrared (NIR) spectral polarization imaging technique for prostate cancer detection. The approach relies on applying a contrast agent that can target cancer cells. Cybesin, as a small ICG-derivative dye-peptide, emit fluorescence between 750 nm and 900 nm, which is in the “tissue optical window”. Cybesin was reported targeting the over-expressed bombesin receptors in cancer cells in animal model and the human prostate cancers over-expressing bombesin receptors. The NIR spectral polarization imaging study reported here demonstrated that Cybesin can be used as a smart optical biomarker and as a prostate cancer receptor targeted contrast agent.
Time-resolved fluorescence emission from normal breast tissue, benign and malignant breast tumors were measured
using excitation of 100 fs pulses at 310nm. Fluorescence relaxation decay parameters were extracted from the measured
temporal emission profiles. The decay times and amplitudes of fast and slow profiles of malignant tissues were different
from normal and benign tissues, which allows the malignant tumor to be separated from non-malignant breast tissue. The
differences between the decay times of slow and fast component can be explained by tissue structure changes during
breast cancer evolution.
A portable near infrared scanning polarization imaging unit with an optical fiber-based rectal probe, namely Photonic
Finger, was designed and developed o locate the 3D position of abnormal prostate site inside normal prostate tissue. An
inverse algorithm, Optical Tomography using Independent Component Analysis (OPTICA) was improved particularly
to unmix the signal from targets (cancerous tissue) embedded in a turbid medium (normal tissue) in the backscattering
imaging geometry. Photonic Finger combined with OPTICA was tested to characterize different target(s) inside different
tissue medium, including cancerous prostate tissue embedded by large piece of normal tissue.
Three-dimensional (3D) localization of human cancerous prostate tissue embedded in normal prostate tissue was
demonstrated and characterized using backscattering scanning polarization imaging and independent component analysis
(ICA). Backscattering two-dimensional (2D) images of a tissue sample illuminated with a raster scanning laser beam
were recorded using a CCD camera to obtain multiple angular views of the target embedded inside the tissue medium.
The 3D location of the object was retrieved by matching the leading Independent Component(s) of intensity distribution
of images to the numerical propagation of the target to the surface of the tissue medium.
Time-resolved spectroscopy and near infrared imaging enhanced by receptor-targeted contrast agents for prostate cancer
detection will be presented. Two contrast agents, Cybesin and Cytate, were investigated using time-resolved
spectroscopy in aqueous solution and cancerous and normal prostate tissues. The time evolution of the fluorescent dipole
in solution was studied using a system of first-order linear differential equations containing two main parameters: the
decay rate of emission and the rate of one orthogonal emission component transferring to another. An analytical
polarization model was developed and used to extract rotational times and fluorescence anisotropies of the contrast
agents in prostate tissues. The differences of rotational times and polarization anisotropies were observed for Cybesin
(Cytate) in cancerous and normal prostate tissue, which reflect preferred bond of contrast agents and cancerous tissue
cells. The conjugation of Cybesin (Cytate) to prostate cancerous cells offers high contrast between normal and cancerous
tissues.
The fluorescence spectra of human cancerous and normal prostate tissues obtained by the selective excitation wavelength of 340 nm were measured. The contributions of principle biochemical components to tissue fluorescence spectra were investigated using the method of multivariate curve resolution with alternating least squares. The results show that there is a reduced contribution from the emission of collagen and increased contribution from nicotinamide adenine dinucleotide (NADH) in cancerous tissues as compared with normal tissue. This difference is attributed to the changes of relative contents of NADH and collagen during cancer development. This research may present a potential native biomarker for prostate cancer detection.
The optical birefringence of porcine aortic tissues including heated and non-heated
tissues was studied using polarization technique. The measurements show that a whole
piece of aortic tissue has birefringence properties like a uniaxial crystal. The experiment
results indicate that the birefringence status of tissue have a potential application for
monitoring changes of tissue structure due to burning, plastic surgery, laser tissue welding
and wound healing.
Recently microendoscopes are being developed as a tool to detection cancer or pre-cancerous lesions in the milk ducts of the human breast. The microendoscope can be inserted into the duct through the nipple. Integration of fluorescence spectroscopy into microendoscopy can provide an improved platform for real-time cancer detection followed by immediate intervention. Typically, the optical fibers employed by existing microendoscope systems transmit in the 450
to 900 nm range. A prototype system combining fluorescence spectroscopy with visible imaging by microendoscopy is described and preliminary measurements on ex vivo human breast tissues are presented. Image resolution and distortion are discussed.
An optical fiber needle probe was developed that can be inserted into a hollow metallic needle for tumor diagnosis using fluorescence at key wavelengths for breast, kidney, liver, and brain. The optical fiber needle probe is based on fluorescence ration method which will allow to detect tumor in vivo for a real time evaluation. This method will be couple with other current modalities such as X-ray, ultrasound and MRI.
A real time fluorescence ratio mapping a system for evaluating the surface of tissue can detect changes in tissue state including precancerous conditions. This system, using a personal computer and video camera, functions in near real-time, displaying pseudo color fluorescence intensity ratios maps at 15 frames per second. A fluorescence ratio mapping system functioning at close to video rates can be integrated with an endoscope to give the physician additional information during a patient exam about the state of a tissue. Preliminary measurements on ex vivo samples differentiated tumor from normal regions of tissues. Fluorescence spectral ratio maps give more structural detail of tissue than direct white light viewing. Tissue structure was observed in the fluorescence images. These results were correlated with histology.
Native fluorescence spectroscopy of biomolecules has emerged as a new modality to the medical community in characterizing the various physiological conditions of tissues. In the past several years, many groups have been working to introduce the spectroscopic methods to diagnose cancer. Researchers have successfully used native fluorescence to distinguish cancerous from normal tissue samples in rat and human tissue. We have developed three generations of instruments, called the CD-scan, CD-ratiometer and CD-map, to allow the medical community to use optics for diagnosing tissue. Using ultraviolet excitation and emission spectral measurements on both normal and cancerous tissue of the breast, gynecology, colon, and aerodigestive tract can be separated. For example, from emission intensities at 340 nm to 440 nm (300 nm excitation), a statistically consistent difference between malignant tissue and normal or benign tissue is observed. In order to utilize optical biopsy techniques in a clinical setting, the CD-scan instrument was developed, which allows for rapid and reliable in-vitro and in-vivo florescence measurements of the aerodigestive tract with high accuracy. The instrumentation employs high sensitivity detection techniques which allows for lamp excitation, small diameter optical fiber probes; the higher spatial resolution afforded by the small diameter probes can increase the ability to detect smaller tumors. The fiber optic probes allow for usage in the aerodigestive tract, cervix and colon. Needle based fiber probes have been developed for in-vivo detection of breast cancer.
Phosphorescence and fluorescence spectra of human breast tissues excited at two wavelengths were measured and compared at low temperature. A blue shift of fluorescence spectral peak was observed at low temperature for the excitation wavelength at 300 or 340 nm. The phosphorescence spectra excited at 300 nm were found to be centered at 412 nm while the fluorescence spectra consisted of two bands located at about 340 nm and 440 nm. For 340 nm excitation, the phosphorescence spectral profile was found to be relatively smooth but shifted to the red centered at 480 nm while the fluorescence consisted of two bands at 375 nm and 440 nm.
Fluorescence spectra of tryptophan and bacteria were measured at different concentrations using a Mediscience CD-Scan unit. The emission spectra of tryptophan were obtained using an excitation wavelength at 280 nm. The excitation spectra were obtained at the emission of 340 nm. The minimum detectable concentration of tryptophan was found to be 10-8 M. The emission spectra for bacteria were probed at 340 nm. The minimum detectable number of bacteria in a beam of the excitation light was determined to be about 30. Assuming that the emission band at 340 nm of bacteria comes from tryptophan, the number of tryptophan per bacterium was estimated to be 108. This approach to determine the number is almost consistent with that obtained using a weight method.
Emission and excitation fluorescence spectra have been measured for gynecological (GYN) tissues. Statistics have been established to evaluate the state of tissues and to distinguish cancerous from normal specimens.
Fluorescence spectroscopy of the human thymus gland and surrounding mediastinal fat were measured to evaluate this approach in distinguishing between thymus and fat tissues during therapeutic surgery for myasthenia gravis disease.
We report on the fluorescence spectroscopy of a multicellular tumor spheroid treated and non- treated with (beta) -all-trans retinoic acid. Following ten days of treatment with RA (10-6M), reproducible fluorescent changes were measured using a Mediscience CD scanner. The most significant changes included an increase in emission at 520 nm in the RA treated spheroids when excited at 340 nm. When investigating fluorescence emission at 450 nm, a blue shift in the excitation profile was noted. Molecular alterations induced by RA and as measured by optical fluorescence spectroscopy may arise from qualitative and/or quantitative changes in a number of key molecules involved in cellular differentiation, proliferation and/or electron transfer, i.e. NADH at (450 nm emission), flavins (520 nm), or cytokeratins (520 nm). To further explore alterations of cytokeratins, immunohistochemical staining showed an increase in AE1 positive cells induced by RA which paralleled the increased 520 nm signal. Our results indicate that certain vitamin derivatives are capable of modulating the intrinsic fluorescence profile emitted by neoplastic mucosa. Tissue autofluorescence may represent a significant marker for the biologic effect of cancer preventing agents in clinical trials.
C. Liu, Bidyut Das, Wenling Glassman, Gui Tang, Han-Ru Zhu, Daniel Akins, Stephanie Lubicz, Joseph Cleary, R. Prudente, Edward Celmer, E. Caron, Robert Alfano
NIR Raman scattering and fluorescence were investigated from malignant and normal biomedical media. Raman spectra were obtained from human normal, benign and cancerous tissues of the gynecological (GYN) tracts. Comparing the differences in intensity for the different Raman modes as well as the difference between the number of Raman lines, the normal (GYN) tissues can be distinguished from the malignant tissues. The fluorescence spectra from human breast tissues that were obtained showed that the ratio of fluorescence intensities at 340 nm to 440 nm can be used to distinguish between malignant and non- malignant tissues. Separate studies from normal and malignant breast cell lines show spectral differences assigned to NADH and flavins. These studies show that various optical techniques have the potential to be useful in medical diagnostic applications.
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