Single-Cell Infrared Microspectroscopy Quantifies Dynamic Heterogeneity of Mesenchymal Stem Cells during Adipogenic Differentiation.

The central relevance of cellular heterogeneity to biological phenomena raises the rational needs for analytical techniques with single-cell resolution. Here, we developed a single-cell FTIR microspectroscopy-based method for the quantitative evaluation of cellular heterogeneity by calculating the cell-to-cell similarity distance of the infrared spectral data. Based on this method, we revealed the infrared phenotypes might reflect the dynamic heterogeneity changes in the cell population during the adipogenic differentiation of the human mesenchymal stem cells. These findings provide an alternative label-free optical approach for quantifying the cellular heterogeneity, and the combination with other single-cell analysis tools will be very helpful for understanding the genotype-to-phenotype relationship in cellular populations.

Study of the intracellular nanoparticle-based radiosensitization mechanisms in F98 glioma cells treated with charged particle therapy through synchrotron-based infrared microspectroscopy.

The use of nanoparticles (NP) as dose enhancers in radiotherapy (RT) is a growing research field. Recently, the use of NP has been extended to charged particle therapy in order to improve the performance in radioresistant tumors. However, the biological mechanisms underlying the synergistic effects involved in NP-RT approaches are not clearly understood. Here, we used the capabilities of synchrotron-based Fourier Transform Infrared Microspectroscopy (SR-FTIRM) as a bio-analytical tool to elucidate the NP-induced cellular damage at the molecular level and at a single-cell scale. F98 glioma cells doped with AuNP and GdNP were irradiated using several types of medical ion beams (proton, helium, carbon and oxygen). Differences in cell composition were analyzed in the nucleic acids, protein and lipid spectral regions using multivariate methods (Principal Component Analysis, PCA). Several NP-induced cellular modifications were detected, such as conformational changes in secondary protein structures, intensity variations in the lipid CHx stretching bands, as well as complex DNA rearrangements following charged particle therapy irradiations. These spectral features seem to be correlated with the already shown enhancement both in the DNA damage response and in the reactive oxygen species (ROS) production by the NP, which causes cell damage in the form of protein, lipid, and/or DNA oxidations. Vibrational features were NP-dependent due to the NP heterogeneous radiosensitization capability. Our results provided new insights into the molecular changes in response to NP-based RT treatments using ion beams, and highlighted the relevance of SR-FTIRM as a useful and precise technique for assessing cell response to innovative radiotherapy approaches.

Rejection of the biophoton hypothesis on the origin of photoreceptor dark noise.

Rod photoreceptors of the vertebrate retina produce, in darkness, spontaneous discrete current waves virtually identical to responses to single photons. The waves comprise an irreducible source of noise (discrete dark noise) that may limit the threshold sensitivity of vision. The waves obviously originate from acts of random activation of single rhodopsin molecules. Until recently, it was generally accepted that the activation occurs due to the rhodopsin thermal motion. Yet, a few years ago it was proposed that rhodopsin molecules are activated not by heat but rather by real photons generated within the retina by chemiluminescence. Using a high-sensitive photomultiplier, we measured intensities of biophoton emission from isolated retinas and eyecups of frogs (Rana ridibunda) and fish (sterlet, Acipenser ruthenus). Retinal samples were placed in a perfusion chamber and emitted photons collected by a high-aperture quartz lens. The collected light was sent to the photomultiplier cathode through a rotating chopper so that a long-lasting synchronous accumulation of the light signal was possible. The absolute intensity of bio-emission was estimated by the response of the measuring system to a calibrated light source. The intensity of the source, in turn, was quantified by measuring rhodopsin bleaching with single-rod microspectrophotometry. We also measured the frequency of discrete dark waves in rods of the two species with suction pipette recordings. Expressed as the rate constant of rhodopsin activation, it was 1.2 × 10-11/s in frogs and 7.6 × 10-11/s in sterlets. Approximately two thirds of retinal samples of each species produced reliably measurable biophoton emissions. However, its intensity was ≥100 times lower than necessary to produce the discrete dark noise. We argue that this is just a lower estimate of the discrepancy between the hypothesis and experiment. We conclude that the biophoton hypothesis on the origin of discrete dark noise in photoreceptors must be rejected.

Synchrotron macro ATR-FTIR microspectroscopy for high-resolution chemical mapping of single cells.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy has been used widely for probing the molecular properties of materials. Coupling a synchrotron infrared (IR) beam to an ATR element using a high numerical aperture (NA) microscope objective enhances the spatial resolution, relative to transmission or transflectance microspectroscopy, by a factor proportional to the refractive index (n) of the ATR element. This work presents the development of the synchrotron macro ATR-FTIR microspectroscopy at Australian Synchrotron Infrared Microspectroscopy (IRM) Beamline, and demonstrates that high quality FTIR chemical maps of single cells and tissues can be achieved at an enhanced spatial resolution. The so-called "hybrid" macro ATR-FTIR device was developed by modifying the cantilever arm of a standard Bruker macro ATR-FTIR unit to accept germanium (Ge) ATR elements with different facet sizes (i.e. 1 mm, 250 µm and 100 µm in diameter) suitable for different types of sample surfaces. We demonstrated the capability of the technique for high-resolution single cell analysis of malaria-infected red blood cells, individual neurons in a brain tissue and cellular structures of a Eucalyptus leaf. The ability to measure a range of samples from soft membranes to hard cell wall structures demonstrates the potential of the technique for high-resolution chemical mapping across a broad range of applications in biology, medicine and environmental science.

A methodology study on chemical and molecular structure imaging in modified forage leaf tissue with cutting-edge synchrotron-powered technology (SR-IMS) as a potential research tool.

To date there is no any study on imaging molecular chemistry and chemical structure of biotech-modified plant tissue on a molecular basis. The objective of this methodology study was to apply a non-invasive and non-destructive synchrotron powered technology - SR-IMS to image molecular chemistry of the modified forage leaf tissue. The infrared molecular vibrational microspectroscopy powered with synchrotron light at Advanced Light Source (ALS, Lawrence Berkeley National Lab, Berkeley, California, Dept. of Energy, USA) were applied. The synchrotron beamline time was arranged by National Synchrotron Light Source (Scientist Dr. Lisa Miller, Brookhaven National Lab, Dept. of Energy, USA). The various molecular functional groups in the forage tissue included CH symmetric and asymmetric regions, amides I and II regions, structure and non-structure CHO regions, carbonyl ester region with peak areas at ca. 3644-3000 cm-1, ca 3005-2979 cm-1, ca. 1722-1483 cm-1, ca. 1488-1412 cm-1, ca. 1296-1189 cm-1, and ca. 1194-951 cm-1. The spectral peak area ratio imaging of chemical functional groups were also studied which included the ratio of peak area under ca. 1722-1483 cm-1 to peak area under ca. 3005-2979 cm-1 and the ratio of peak area under ca. 1722-1483 cm-1 to peak area under ca. 1194-951 cm-1. The results showed that the advanced synchrotron-based technology - SR-IMS was able to image the forage tissue at an ultra-highly resolution within intact tissue within cellular and subcellular dimension. It revealed the forage tissue in a molecular chemical sense and provided an insight on nutrient properties and their molecular structure as well as chemical features. In conclusion, the synchrotron-radiation SR-IMS is able to image molecular structure of the forage leaf tissue at an ultra-highly resolution. The advanced SR-IMS technique could provide leaf tissue four kinds of information simultaneously: tissue structure, tissue chemistry, tissue nutrients, and tissue environment of forage.

Monitoring Drug Crystallization in Percutaneous Penetration Using Localized Nanothermal Analysis and Photothermal Microspectroscopy.

Drug crystallization on and in the skin has been reported following application of topical or transdermal formulations. This study explored novel probe-based approaches including localized nanothermal analysis (nano-TA) and photothermal microspectroscopy (PTMS) to investigate and locate drug crystals in the stratum corneum (SC) of porcine skin following application of simple ibuprofen (IBU) formulations. We also conducted in vitro skin permeation studies and tape stripping. The detection of drug crystals in the SC on tape strips was confirmed using localized nano-TA, based on the melting temperature of IBU. The melting of IBU was also evident as indicated by a double transition and confirmed the presence of drug crystals in the SC. The single point scans of PTMS on the tape strips allowed collection of the photothermal FTIR spectra of IBU, confirming the existence of drug crystals in the skin. The combined methods also indicated that drug crystallized in the SC at a depth of ∼4-7 µm. Future studies will examine the potential of these techniques to probe crystallization of other commonly used actives in topical and transdermal formulations.

Contributions to advances in blend pellet products (BPP) research on molecular structure and molecular nutrition interaction by advanced synchrotron and globar molecular (Micro)spectroscopy.

To date, advanced synchrotron-based and globar-sourced techniques are almost unknown to food and feed scientists. There has been little application of these advanced techniques to study blend pellet products at a molecular level. This article aims to provide recent research on advanced synchrotron and globar vibrational molecular spectroscopy contributions to advances in blend pellet products research on molecular structure and molecular nutrition interaction. How processing induced molecular structure changes in relation to nutrient availability and utilization of the blend pellet products. The study reviews Utilization of co-product components for blend pellet product in North America; Utilization and benefits of inclusion of pulse screenings; Utilization of additives in blend pellet products; Application of pellet processing in blend pellet products; Conventional evaluation techniques and methods for blend pellet products. The study focus on recent applications of cutting-edge vibrational molecular spectroscopy for molecular structure and molecular structure association with nutrient utilization in blend pellet products. The information described in this article gives better insight on how advanced molecular (micro)spectroscopy contributions to advances in blend pellet products research on molecular structure and molecular nutrition interaction.

Insight into GFPmut2 pH Dependence by Single Crystal Microspectrophotometry and X-ray Crystallography.

The fluorescence of Green Fluorescent Protein (wtGFP) and variants has been exploited in distinct applications in cellular and analytical biology. GFPs emission depends on the population of the protonated (A-state) and deprotonated (B-state) forms of the chromophore. Whereas wtGFP is pH-independent, mutants in which Ser65 is replaced by either threonine or alanine (as in GFPmut2) are pH-dependent, with a p Ka around 6. Given the wtGFP pH-independence, only the structure of the protonated form was determined. The deprotonated form was deduced on the basis of the crystal structure of the Ser65Thr mutant at basic pH, assuming that it corresponds to the conformation populated in solution. Here, we present an investigation where structures of the protonated and deprotonated forms of GFPmut2 were determined from crystals grown in either MPD at pH 6 or PEG at pH 8.5, and moved to either higher or lower pH. Both crystal forms of GFPmut2 were titrated monitoring the process via polarized absorption microspectrophotometry in order to precisely correlate the protonation process with the structures. We found that (i) in solution, chromophore titration is not thermodynamically coupled with any residue and Glu222 is always protonated independent of the protonation state of the chromophore; (ii) the lack of coupling is reflected in the structural behavior of the chromophore and Glu222 environments, with only the former showing variations with pH; (iii) titrations of low-pH and high-pH grown crystals exhibit a Hill coefficient of about 0.75, indicating an anticooperative behavior not observed in solution; (iv) structures where pH was changed in the crystal point to Glu222 as the ionizable group responsible for the outset of the anticooperative behavior; and (v) in GFPmut2 the canonical GFP proton wire involving the chromophore is not interrupted at the level of Ser205 and Glu222 at basic pH as in the Ser65Thr mutant. This allows proposing the structure of the deprotonated state of GFPmut2 as an alternative model for the analogous state of wtGFP.

Enhanced chemical and spatial recognition of fish bones in surimi by Tri-step infrared spectroscopy and infrared microspectroscopic imaging.

Surimi is an intermediate product with an increasing popularity worldwide. Discrimination of impurities like fish bones in surimi has become an urgent issue owing to the food safety and the improved requirements for assessment methods in identification of surimi quality and grades. A Tri-step infrared spectroscopy, including Fourier transform infrared spectroscopy (FT-IR), second derivative infrared spectroscopy (SD-IR) and two-dimensional correlation infrared spectroscopy (2DCOS-IR) has been applied to integrally discriminate different contents (1%-8%) of fish bones in surimi at macro-scale. Meanwhile, attenuated total reflection infrared spectroscopy (ATR-IR) microspectroscopic imaging has been employed to recognize and identify the location of fish bones (less than 1.0 mm in size) in micro-scale. Fishbone characteristic infrared absorption peak at 1011 cm-1 contributes to surimi peaks at 1045 cm-1 and 988 cm-1 confirmed by calculation of their peak heights and ratios of peak areas in original spectra. SD-IR spectra enhance the difference in range of 1440-500 cm-1, and specifically peak intensity at 599 cm-1 is significantly increased in surimi with 3%-8% fish bones. Moreover, 2DCOS-IR spectra reveal that surimi containing fish bones have increased intensity of auto-peaks at 525 cm-1, 519 cm-1, 512 cm-1 and 505 cm-1 mainly contributed by hydroxyapatite and collagen. In ATR-IR microspectroscopic images, a clear fishbone shape (800 × 200 µm) corresponding to its visible image is clearly observed in principal component (PC) score image, which is confirmed as a fish bone by corresponding pixel spectra. Furthermore, the single-wavenumber image shows the spatial chemical distribution of various components for both the fish bone and surimi. Consequently, fish bones can be integrally recognized by physical and chemical imaging manners. It has been demonstrated that the developed Tri-step infrared spectroscopy and ATR-IR microspectroscopic imaging could be applicable for rapidly recognizing impurities and adulterants in surimi.

A Generalized Approach to Forensic Dye Identification: Development and Utility of Reference Libraries.

While color is arguably the most important optical property of evidential fibers, the actual dyestuffs responsible for its expression in them are, in forensic trace evidence examinations, rarely analyzed and still less often identified. This is due, primarily, to the exceedingly small quantities of dye present in a single fiber as well as to the fact that dye identification is a challenging analytical problem, even when large quantities are available for analysis. Among the practical reasons for this are the wide range of dyestuffs available (and the even larger number of trade names), the low total concentration of dyes in the finished product, the limited amount of sample typically available for analysis in forensic cases, and the complexity of the dye mixtures that may exist within a single fiber. Literature on the topic of dye analysis is often limited to a specific method, subset of dyestuffs, or an approach that is not applicable given the constraints of a forensic analysis. Here, we present a generalized approach to dye identification that (1) combines several robust analytical methods, (2) is broadly applicable to a wide range of dye chemistries, application classes, and fiber types, and (3) can be scaled down to forensic casework-sized samples. The approach is based on the development of a reference collection of 300 commercially relevant textile dyes that have been characterized by a variety of microanalytical methods (HPTLC, Raman microspectroscopy, infrared microspectroscopy, UV-Vis spectroscopy, and visible microspectrophotometry). Although there is no single approach that is applicable to all dyes on every type of fiber, a combination of these analytical methods has been applied using a reproducible approach that permits the use of reference libraries to constrain the identity of and, in many cases, identify the dye (or dyes) present in a textile fiber sample.

Elucidation of penetration enhancement mechanism of Emu oil using FTIR microspectroscopy at EMIRA laboratory of SESAME synchrotron.

It has been proposed that Emu oil possesses skin permeation-enhancing effect. This study aimed to address its possible penetration enhancement mechanism(s) using IR microscopy, in accordance with LPP theory. The penetration of Emu oil through the layers of human skin was accomplished by monitoring oil-IR characteristic feature at 3006cm-1. The unsaturated components of Emu oil accumulated at about 270µm depth of skin surface. The interaction of Emu oil with lipid and protein constituents of SC was investigated in comparison with a commonly used enhancer, IPM. Inter-sample spectral differences were identified using PCA and linked with possible enhancement mechanisms. Emu oil treatment caused a change in the slope of the right contour of amide I band of the protein spectral range. This was also clear in the second derivative spectra where the emergence of a new shoulder at higher frequency was evident, suggesting disorganization of keratin α-helix structure. This effect could be a result of disruption of some hydrogen bonds in which amide CO and NH groups of keratin are involved. The low intensity of the emerged shoulder is also in agreement with formation of weaker hydrogen bonds. IPM did not affect the protein component. No conclusions regarding the effect of penetration enhancers on the SC lipids were obtained. This was due to the overlap of the endogenous (skin) and exogenous (oil) CH stretching and scissoring frequencies. The SC carbonyl stretching peak disappeared as a result of IPM treatment which may reflect some degree of lipid extraction.

Collecting Quality Infrared Spectra from Microscopic Samples of Suspicious Powders in a Sealed Cell.

The infrared (IR) microspectroscopical analysis of samples within a sealed-cell containing barium fluoride is a critical need when identifying toxic agents or suspicious powders of unidentified composition. The dispersive nature of barium fluoride is well understood and experimental conditions can be easily adjusted during reflection-absorption measurements to account for differences in focus between the visible and IR regions of the spectrum. In most instances, the ability to collect a viable spectrum is possible when using the sealed cell regardless of whether visible or IR focus is optimized. However, when IR focus is optimized, it is possible to collect useful data from even smaller samples. This is important when a minimal sample is available for analysis or the desire to minimize risk of sample exposure is important. While the use of barium fluoride introduces dispersion effects that are unavoidable, it is possible to adjust instrument settings when collecting IR spectra in the reflection-absorption mode to compensate for dispersion and minimize impact on the quality of the sample spectrum.

Chemical morphology of Areca nut characterized directly by Fourier transform near-infrared and mid-infrared microspectroscopic imaging in reflection modes.

Fourier transform near-infrared (NIR) and mid-infrared (MIR) imaging techniques are essential tools to characterize the chemical morphology of plant. The transmission imaging mode is mostly used to obtain easy-to-interpret spectra with high signal-to-noise ratio. However, the native chemical compositions and physical structures of plant samples may be altered when they are microtomed for the transmission tests. For the direct characterization of thick plant samples, the combination of the reflection NIR imaging and the attenuated total reflection (ATR) MIR imaging is proposed in this research. First, the reflection NIR imaging method can explore the whole sample quickly to find out typical regions in small sizes. Next, each small typical region can be measured by the ATR-MIR imaging method to reveal the molecular structures and spatial distributions of compounds of interest. As an example, the chemical morphology of Areca nut section is characterized directly by the above approach.

In vivo Raman Microspectroscopy: Intra- and Intersubject Variability of Stratum Corneum Spectral Markers.

BACKGROUND: In vivo Raman spectroscopy is a powerful tool for real-time analysis and in situ evaluation of tissues such as the skin. The efficiency of this technique has been widely demonstrated as a label-free method for in vivo evaluation of the skin. The aim of this study is to gather information about inter- and intra-individual variations in the spectral descriptors of water content and structure, organization of the lipid barrier and structure of proteins in the stratum corneum (SC). METHODS: In vivo SC measurements were performed on 17 female volunteers aged 20-30 years (phototypes I and II). For intra-individual variability, spectral collection was performed on 5 successive days per volunteer. Shapiro-Wilk and Cochran tests were applied to check the normality and the homoscedasticity of variances. ANOVA was then applied to evaluate intra- and intergroup variability. RESULTS: ANOVA was performed on the spectral descriptors of water content and structure, organization of the lipid barrier and secondary structure of proteins in the SC. No significant intra- and interday variability was observed for all volunteers. Despite the low value of the total relative standard deviation, a highly significant variation was observed between volunteers. CONCLUSION: Interindividual variability for Raman measurements is significant for a set of volunteers with normal nondiseased SC and close phototypes. This variability should be taken into consideration as a threshold for significant variance when working in vivo.

How Confocal Is Confocal Raman Microspectroscopy on the Skin? Impact of Microscope Configuration and Sample Preparation on Penetration Depth Profiles.

BACKGROUND/AIMS: The aim of the study was to elucidate the effect of sample preparation and microscope configuration on the results of confocal Raman microspectroscopic evaluation of the penetration of a pharmaceutical active into the skin (depth profiling). METHODS: Pig ear skin and a hydrophilic formulation containing procaine HCl were used as a model system. The formulation was either left on the skin during the measurement, or was wiped off or washed off prior to the analysis. The microscope configuration was varied with respect to objectives and pinholes used. RESULTS: Sample preparation and microscope configuration had a tremendous effect on the results of depth profiling. Regarding sample preparation, the best results could be observed when the formulation was washed off the skin prior to the analysis. Concerning microscope configuration, the use of a 40 × 0.6 numerical aperture (NA) objective in combination with a 25-µm pinhole or a 100 × 1.25 NA objective in combination with a 50-µm pinhole was found to be advantageous. CONCLUSION: Complete removal of the sample from the skin before the analysis was found to be crucial. A thorough analysis of the suitability of the chosen microscope configuration should be performed before acquiring concentration depth profiles.

Biochemical, biophysical, and genetic changes of porcine trophoblast-derived stem-like cells during differentiation as evaluated using Raman microspectroscopy, Atomic force microscopy, and quantitative polymerase chain reaction.

Porcine trophoblast-derived stem-like cells grown into serum medium start to differentiate and become senescent within 30 days. However, trophoblast-derived cells, cultured in vitro in a defined and non-serum medium, have the regenerative properties, such as indefinite passage and foreign DNA receptivity, similar to stem cells. To evaluate the biochemical, biophysical, and genetic changes of the terminal differentiation of trophoblast derived cells, Raman microspectroscopy, atomic force microscopy, and qPCR were applied. It was found that Raman spectral intensities of characteristic peaks, cell morphology, and Young's modulus can be used to distinguish differentiated and undifferentiated trophoblast cells. In addition, 17 cytoskeleton and extracellular matrix-related genes were significantly impacted by medium type (non-serum versus serum). Our findings suggest that Raman microspectroscopy and atomic force microscopy-both considered as label-free, non-invasive techniques-can be applied to distinguish differentiated trophoblast cells, and cellular biochemical information and biophysical properties can be indicative of cellular differences during cell differentiation. In addition, most of cytoskeleton-related genes exhibit similar pattern to that of Young's modulus during trophoblast cell differentiation, indicating the potential connection between cytoskeleton-related genes and cellular stiffness.

Optics of cone photoreceptors in the chicken (Gallus gallus domesticus).

Vision is the primary sensory modality of birds, and its importance is evident in the sophistication of their visual systems. Coloured oil droplets in the cone photoreceptors represent an adaptation in the avian retina, acting as long-pass colour filters. However, we currently lack understanding of how the optical properties and morphology of component structures (e.g. oil droplet, mitochondrial ellipsoid and outer segment) of the cone photoreceptor influence the transmission of light into the outer segment and the ultimate effect they have on receptor sensitivity. In this study, we use data from microspectrophotometry, digital holographic microscopy and electron microscopy to inform electromagnetic models of avian cone photoreceptors to quantitatively investigate the integrated optical function of the cell. We find that pigmented oil droplets primarily function as spectral filters, not light collection devices, although the mitochondrial ellipsoid improves optical coupling between the inner segment and oil droplet. In contrast, unpigmented droplets found in violet-sensitive cones double sensitivity at its peak relative to other cone types. Oil droplets and ellipsoids both narrow the angular sensitivity of single cone photoreceptors, but not as strongly as those in human cones.

Scan-Free Absorbance Spectral Imaging A(x, y, λ) of Single Live Algal Cells for Quantifying Absorbance of Cell Suspensions.

Label-free, non-invasive, rapid absorbance spectral imaging A(x,y,λ) microscopy of single live cells at 1.2 µm × 1.2 µm resolution with an NA = 0.85 objective was developed and applied to unicellular green algae Chlamydomonas reinhardtii. By introducing the fiber assembly to rearrange a two-dimensional image to the one-dimensional array to fit the slit of an imaging spectrograph equipped with a CCD detector, scan-free acquisition of three-dimensional information of A(x,y,λ) was realized. The space-resolved absorbance spectra of the eyespot, an orange organelle about 1 µm, were extracted from the green-color background in a chlorophyll-rich single live cell absorbance image. Characteristic absorbance change in the cell suspension after hydrogen photoproduction in C. reinhardtii was investigated to find a single 715-nm absorption peak was locally distributed within single cells. The formula to calculate the absorbance of cell suspensions from that of single cells was presented to obtain a quantitative, parameter-free agreement with the experiment. It is quantitatively shown that the average number of chlorophylls per cell is significantly underestimated when it is evaluated from the absorbance of the cell suspensions due to the package effect.

Characterising cytotoxic agent action as a function of the cell cycle using Fourier transform infrared microspectroscopy.

Fourier Transform Infrared (FTIR) micro-spectroscopy measurements were acquired to study infrared signatures of chemotherapeutic response as a function of the cell cycle. Renal carcinoma Caki-2 cells were exposed to IC50 doses of 5-fluorouracil and Paclitaxel for a period of 24 hours. The inherent cell cycle infrared signatures from untreated and drug-treated cells were successfully retrieved by the construction of a robust SVM able to discriminate the cell cycle phases of this cell line with an average accuracy of 83.7%. The overriding infrared signature observed relates to an apoptotic biochemical response that does not appear to be correlated with the events affected by the drugs' mode of action or the cell cycle. Since apoptosis is a well conserved mechanism among living species, these results suggest that both the stages of proliferation as well as the absence/presence of apoptosis need to be taken into account in order to elucidate the fine biochemical details revealing the immediate cellular response to the drug in order to assign reliable spectral patterns of drug action.

Diagnosis approach of chronic lymphocytic leukemia on unstained blood smears using Raman microspectroscopy and supervised classification.

We have investigated the potential of Raman microspectroscopy combined with supervised classification algorithms to diagnose a blood lymphoproliferative disease, namely chronic lymphocytic leukemia (CLL). This study was conducted directly on human blood smears (27 volunteers and 49 CLL patients) spread on standard glass slides according to a cytological protocol before the staining step. Visible excitation at 532 nm was chosen, instead of near infrared, in order to minimize the glass contribution in the Raman spectra. After Raman measurements, blood smears were stained using the May-Grünwald Giemsa procedure to correlate spectroscopic data classifications with cytological analysis. A first prediction model was built using support vector machines to discriminate between the two main leukocyte subpopulations (lymphocytes and polymorphonuclears) with sensitivity and specificity over 98.5%. The spectral differences between these two classes were associated to higher nucleic acid content in lymphocytes compared to polymorphonuclears. Then, we developed a classification model to discriminate between neoplastic and healthy lymphocyte spectra, with a mean sensitivity and specificity of 88% and 91% respectively. The main molecular differences between healthy and CLL cells were associated with DNA and protein changes. These spectroscopic markers could lead, in the future, to the development of a helpful medical tool for CLL diagnosis.