Anushree Saha

Application of Raman Spectroscopy to Identify Microcalcifications and Underlying Breast Lesions at Stereotactic Core Needle Biopsy

Microcalcifications are a feature of diagnostic significance on a mammogram and a target for stereotactic breast needle biopsy. Here, we report development of a Raman spectroscopy technique to simultaneously identify microcalcification status and diagnose the underlying breast lesion, in real-time, during stereotactic core needle biopsy procedures. Raman spectra were obtained ex vivo from 146 tissue sites from fresh stereotactic breast needle biopsy tissue cores from 33 patients, including 50 normal tissue sites, 77 lesions with microcalcifications, and 19 lesions without microcalcifications, using a compact clinical system. The Raman spectra were modeled on the basis of the breast tissue components, and a support vector machine framework was used to develop a single-step diagnostic algorithm to distinguish normal tissue, fibrocystic change (FCC), fibroadenoma, and breast cancer, in the absence and presence of microcalcifications. This algorithm was subjected to leave-one-site-out cross-validation, yielding a positive predictive value, negative predictive value, sensitivity, and specificity of 100%, 95.6%, 62.5%, and 100% for diagnosis of breast cancer (with or without microcalcifications) and an overall accuracy of 82.2% for classification into specific categories of normal tissue, FCC, fibroadenoma, or breast cancer (with and without microcalcifications). Notably, the majority of breast cancers diagnosed are ductal carcinoma in situ (DCIS), the most common lesion associated with microcalcifications, which could not be diagnosed using previous Raman algorithm(s). Our study shows the potential of Raman spectroscopy to concomitantly detect microcalcifications and diagnose associated lesions, including DCIS, and thus provide real-time feedback to radiologists during such biopsy procedures, reducing nondiagnostic and false-negative biopsies.

Raman spectroscopy: a real-time tool for identifying microcalcifications during stereotactic breast core needle biopsies

Microcalcifications are an early mammographic sign of breast cancer and a target for stereotactic breast needle biopsy. We present here a Raman spectroscopic tool for detecting microcalcifications in breast tissue based on their chemical composition. We collected ex vivo Raman spectra from 159 tissue sites in fresh stereotactic breast needle biopsies from 33 patients, including 54 normal sites, 75 lesions with microcalcifications and 30 lesions without microcalcifications. Application of our Raman technique resulted in a positive predictive value of 97% for detecting microcalcifications. This study shows that Raman spectroscopy has the potential to detect microcalcifications during stereotactic breast core biopsies and provide real-time feedback to radiologists, thus reducing non-diagnostic and false negative biopsies.

Development and comparative assessment of Raman spectroscopic classification algorithms for lesion discrimination in stereotactic breast biopsies with microcalcifications.

Microcalcifications are an early mammographic sign of breast cancer and a target for stereotactic breast needle biopsy. Here, we develop and compare different approaches for developing Raman classification algorithms to diagnose invasive and in situ breast cancer, fibrocystic change and fibroadenoma that can be associated with microcalcifications. In this study, Raman spectra were acquired from tissue cores obtained from fresh breast biopsies and analyzed using a constituent-based breast model. Diagnostic algorithms based on the breast model fit coefficients were devised using logistic regression, C4.5 decision tree classification, k-nearest neighbor (k -NN) and support vector machine (SVM) analysis, and subjected to leave-one-out cross validation. The best performing algorithm was based on SVM analysis (with radial basis function), which yielded a positive predictive value of 100% and negative predictive value of 96% for cancer diagnosis. Importantly, these results demonstrate that Raman spectroscopy provides adequate diagnostic information for lesion discrimination even in the presence of microcalcifications, which to the best of our knowledge has not been previously reported.

Structural changes of human serum albumin in response to a low concentration of heavy ions

Lead ions in solution interact strongly with human serum albumin and modify the properties and function of albumin molecules. In the present study, we used optical spectroscopic techniques to explore the binding sites of lead, present in albumin. Structural and chemical analysis of albumin molecules using fluorescence and Raman spectroscopy, predicted the modification of two major amino acids in albumin due to lead binding. No secondary structural changes are observed in the protein molecule, which is further confirmed using circular dichroism absorption measurements. The results indicate that loss of charge from the binding site of albumin by the charged lead ions, give rise to dipole interaction which acts as the major contributor to promote protein agglomeration.

Precision of Raman spectroscopy measurements in detection of microcalcifications in breast needle biopsies.

Microcalcifications are an early mammographic sign of breast cancer and a target for stereotactic breast needle biopsy. We developed Raman spectroscopy decision algorithms to detect breast microcalcifications, based on fit coefficients (FC) derived by modeling tissue Raman spectra as a linear combination of the Raman spectra of 9 chemical and morphologic components of breast tissue. However, little or no information is available on the precision of such measurements and its effect on the ability of Raman spectroscopy to make predictions for breast microcalcification detection. Here we report the precision, that is, the closeness of agreement between replicate Raman spectral measurements--and the model FC derived from them--obtained ex vivo from fresh breast biopsies from patients undergoing stereotactic breast needle biopsy, using a compact clinical Raman system. The coefficients of variation of the model FC averaged 0.03 for normal breast tissue sites, 0.12 for breast lesions without, and 0.22 for breast lesions with microcalcifications. Imprecision in the FC resulted in diagnostic discordance among replicates only for line-sitters, that is, tissue sites with FC values near the decision line or plane. The source of this imprecision and their implications for the use of Raman spectroscopy for guidance of stereotactic breast biopsies for microcalcifications are also discussed. In summary, we conclude that the precision of Raman spectroscopy measurements in breast tissue obtained using our compact clinical system is more than adequate to make accurate and repeatable predictions of microcalcifications in breast tissue using decision algorithms based on model FC. This provides strong evidence of the potential of Raman spectroscopy guidance of stereotactic breast needle biopsies for microcalcifications.

Raman spectroscopic sensing of carbonate intercalation in breast microcalcifications at stereotactic biopsy

Microcalcifications are an early mammographic sign of breast cancer and frequent target for stereotactic biopsy. Despite their indisputable value, microcalcifications, particularly of the type II variety that are comprised of calcium hydroxyapatite deposits, remain one of the least understood disease markers. Here we employed Raman spectroscopy to elucidate the relationship between pathogenicity of breast lesions in fresh biopsy cores and composition of type II microcalcifications. Using a chemometric model of chemical-morphological constituents, acquired Raman spectra were translated to characterize chemical makeup of the lesions. We find that increase in carbonate intercalation in the hydroxyapatite lattice can be reliably employed to differentiate benign from malignant lesions, with algorithms based only on carbonate and cytoplasmic protein content exhibiting excellent negative predictive value (93–98%). Our findings highlight the importance of calcium carbonate, an underrated constituent of microcalcifications, as a spectroscopic marker in breast pathology evaluation and pave the way for improved biopsy guidance.

Raman microspectroscopy of melanosomes: the effect of long term light irradiation

Melanosomes are long lived organelles in retinal pigment epithelium cells and are primarily responsible for photoprotection. However, with aging or prolong light radiation, the function of melanosomes diminishes, which may be due to photobleaching of melanin pigments present in melanosomes. In this study, melanosomes were isolated from the retinal pigment epithelium cells and exposed to green light (532 nm), and the chemical changes were monitored using Raman microspectroscopy. Photochemical changes were recorded for different power levels and exposure times. The threshold power and the rate for irreversible photobleaching of melanosomes were calculated by fitting the experimental data with a proposed model.

Detection of picomolar concentrations of lead in water using albumin-based fluorescence sensor

Comprehensive analysis of fluorescence of albumin shows a weak fluorescence band at 430 nm, whose intensity exhibits a remarkable sensitivity to the presence of heavy ions in water. Using this fluorescence as a marker, as low as 10 pM concentration of lead can be routinely detected. Such a great sensitivity is explained in terms of electrostatic interactions in solution, which promote protein agglomeration. The latter is independently confirmed using dynamic light scattering measurements.

Raman versus CARS microscopy: when one is better than the other

Since the first introduction of Raman microscope in 1973, optical and laser technology has made a tremendous step forward. However, despite of the obvious advantages of being a very informative and nondestructive method of studying biological samples, spontaneous Raman scattering suffers from a series of limitations such as a fluorescent background and a low signal level. Nonlinear Raman spectroscopy and, in particular, spectroscopy of coherent anti-Stokes Raman scattering (CARS) can resolve most of the problems associated with conventional Raman spectroscopy. In this report, the most critical issues of the CARS microspectroscopy setup design are reviewed and several exciting potential applications of the broadband CARS microspectroscopy, where the CARS microscopy has an advantage with respect to Raman microscopy, are outlined.

Raman microspectroscopy of retinal pigment epithelium cells: real-time imaging the effects of photooxidative stress

Raman microspectroscopy is applied for the first time to study the effect of high-power light irradiation on isolated melanosomes and melanosomes in retinal pigmented epithelium cells.