Raman spectroscopy for cytopathology of exfoliated cervical cells.

Cervical cancer is the fourth most common cancer affecting women worldwide but mortality can be decreased by early detection of pre-malignant lesions. The Pap smear test is the most commonly used method in cervical cancer screening programmes. Although specificity is high for this test, it is widely acknowledged that sensitivity can be poor mainly due to the subjective nature of the test. There is a need for new objective tests for the early detection of pre-malignant cervical lesions. Over the past two decades, Raman spectroscopy has emerged as a promising new technology for cancer screening and diagnosis. The aim of this study was to evaluate the potential of Raman spectroscopy for cervical cancer screening using both Cervical Intraepithelial Neoplasia (CIN) and Squamous Intraepithelial Lesion (SIL) classification terminology. ThinPrep® Pap samples were recruited from a cervical screening population. Raman spectra were recorded from single cell nuclei and subjected to multivariate statistical analysis. Normal and abnormal ThinPrep® samples were discriminated based on the biochemical fingerprint of the cells using Principal Component Analysis (PCA). Principal Component Analysis - Linear Discriminant Analysis (PCA-LDA) was employed to build classification models based on either CIN or SIL terminology. This study has shown that Raman spectroscopy can be successfully applied to the study of routine cervical cytology samples from a cervical screening programme and that the use of CIN terminology resulted in improved sensitivity for high grade cases.

Analyses of ionizing radiation effects in vitro in peripheral blood lymphocytes with Raman spectroscopy.

The use of Raman spectroscopy to measure the biochemical profile of healthy and diseased cells and tissues may be a potential solution to many diagnostic problems in the clinic. Although extensively used to identify changes in the biochemical profiles of cancerous cells and tissue, Raman spectroscopy has been used less often for analyzing changes to the cellular environment by external factors such as ionizing radiation. In tandem with this, the biological impact of low doses of ionizing radiation remains poorly understood. Extensive studies have been performed on the radiobiological effects associated with radiation doses above 0.1 Gy, and are well characterized, but recent studies on low-dose radiation exposure have revealed complex and highly variable responses. We report here the novel finding that demonstrate the capability of Raman spectroscopy to detect radiation-induced damage responses in isolated lymphocytes irradiated with doses of 0.05 and 0.5 Gy. Lymphocytes were isolated from peripheral blood in a cohort of volunteers, cultured ex vivo and then irradiated. Within 1 h after irradiation spectral effects were observed with Raman microspectroscopy and principal component analysis and linear discriminant analysis at both doses relative to the sham-irradiated control (0 Gy). Cellular DNA damage was confirmed using parallel γ-H2AX fluorescence measurements on the extracted lymphocytes per donor and per dose. DNA damage measurements exhibited interindividual variability among both donors and dose, which matched that seen in the spectral variability in the lymphocyte cohort. Further evidence of links between spectral features and DNA damage was also observed, which may potentially allow noninvasive insight into the DNA remodeling that occurs after exposure to ionizing radiation.

Competitive evaluation of data mining algorithms for use in classification of leukocyte subtypes with Raman microspectroscopy.

Raman microspectroscopy has been investigated for some time for use in label-free cell sorting devices. These approaches require coupling of the Raman spectrometer to complex data mining algorithms for identification of cellular subtypes such as the leukocyte subpopulations of lymphocytes and monocytes. In this study, three distinct multivariate classification approaches, (PCA-LDA, SVMs and Random Forests) are developed and tested on their ability to classify the cellular subtype in extracted peripheral blood mononuclear cells (T-cell lymphocytes from myeloid cells), and are evaluated in terms of their respective classification performance. A strategy for optimisation of each of the classification algorithm is presented with emphasis on reduction of model complexity in each of the algorithms. The relative classification performance and performance characteristics are highlighted, overall suggesting the radial basis function SVM as a robust option for classification of leukocytes with Raman microspectroscopy.

Cell survival and DNA damage in normal prostate cells irradiated out-of-field.

Interest in out-of-field radiation dose has been increasing with the introduction of new techniques, such as volumetric modulated arc therapy (VMAT). These new techniques offer superior conformity of high-dose regions to the target compared to conventional techniques, however more normal tissue is exposed to low-dose radiation with VMAT. There is a potential increase in radiobiological effectiveness associated with lower energy photons delivered during VMAT as normal cells are exposed to a temporal change in incident photon energy spectrum. During VMAT deliveries, normal cells can be exposed to the primary radiation beam, as well as to transmission and scatter radiation. The impact of low-dose radiation, radiation-induced bystander effect and change in energy spectrum on normal cells is not well understood. The current study examined cell survival and DNA damage in normal prostate cells after exposure to out-of-field radiation both with and without the transfer of bystander factors. The effect of a change in energy spectrum out-of-field compared to in-field was also investigated. Prostate cancer (LNCaP) and normal prostate (PNT1A) cells were placed in-field and out-of-field, respectively, with the PNT1A cells being located 1 cm from the field edge when in-field cells were being irradiated with 2 Gy. Clonogenic and γ-H2AX assays were performed postirradiation to examine cell survival and DNA damage. The assays were repeated when bystander factors from the LNCaP cells were transferred to the PNT1A cells and also when the PNT1A cells were irradiated in-field to a different energy spectrum. An average out-of-field dose of 10.8 ± 4.2 cGy produced a significant reduction in colony volume and increase in the number of γ-H2AX foci/cell in the PNT1A cells compared to the sham-irradiated control cells. An adaptive response was observed in the PNT1A cells having first received a low out-of-field dose and then the bystander factors. The PNT1A cells showed a significant increase in γ-H2AX foci formation when irradiated to 20 cGy in-field in comparison to out-of-field. However, no significant difference in cell survival or colony volume was observed whether the PNT1A cells were irradiated in-field or out-of-field. Out-of-field radiation dose alone can have a damaging effect on the proliferation of PNT1A cells when a clinically relevant dose of 2 Gy is delivered in in-field. Out-of-field radiation with the transfer of bystander factors induces an adaptive response in the PNT1A cells.

Exosomes are involved in mediating radiation induced bystander signaling in human keratinocyte cells.

There is much evidence supporting the existence of bystander effects in cells that were never exposed to radiation. Directly irradiated cells and bystander cells can communicate with each other using gap junctional intercellular communication or by releasing soluble factors into the surrounding medium. Exosomes and microvesicles are also known to mediate communication between cells. The main aim of this study is to establish whether exosomes and microvesicles are involved in radiation induced bystander signaling. Human keratinocytes, HaCaT cells, were irradiated (0.005, 0.05 and 0.5 Gy) using γ rays produced from a cobalt 60 teletherapy unit. After irradiation, the cells were incubated for 1 h and the irradiated cell conditioned medium (ICCM) was harvested. Exosomes were isolated from the ICCM using ultracentrifugation. Exosomes were characterized using light scattering analysis (LSA) and scanning transmission electron microscopy (STEM). Cytotoxicity and reactive oxygen species assays and real time calcium imaging were performed either with ICCM from which exosomes and microvesicles were removed or with the exosome fraction resuspended in cell culture media. The characterization data showed a particle size distribution indicative of both exosomes (30-100 nm) and microvesicles (>100 nm) and the light scattering analysis showed increased concentration of both exosomes and microvesicles with increasing dose. Western blotting confirmed the presence of an exosomal protein marker, TSG 101. Treatment of unirradiated cells with ICCM in which exosomes and microvesicles were removed resulted in abrogation of ICCM induced effects such as reduction in viability, calcium influx and production of reactive oxygen species. Addition of exosomes to fresh media produced similar effects to complete ICCM. These results suggest a role for exosomes and microvesicles in radiation induced bystander signaling.

Raman spectroscopic mapping for the analysis of solar radiation induced skin damage.

The effects of simulated solar irradiation of an artificial skin model have been examined using Raman spectroscopy and the results are compared with cytotoxicological and histological profiling. Samples exposed for times varying between 30 minutes and 240 minutes were incubated post exposure for a period of 96 hours. The cytotoxicological response as measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay demonstrated a ~50% loss of viability of the artificial tissue after 120 minutes exposure. Histological staining of tissue sections showed considerable loss of cellular content in the epidermal layer at this endpoint. Raman spectroscopic mapping of tissue sections, coupled with K-means cluster analysis (KMCA) clearly identified the dermal and stratum corneum layers and differentiated further substructures of the epidermis. Post irradiation, a significant loss of DNA features in the basal layer was apparent in the results of the KMCA. Principal Components Analysis (PCA) of layers identified by the KMCA post exposure compared with controls indicated a significant increase in the lipidic signatures of the stratum corneum. In the dermal layer, little photodamage was observed, but a similar increase in lipidic signatures in the basal layer was accompanied by a decrease in DNA and protein contributions. The spectral profiles of the photodamage to the basal layer as identified by PCA are consistent over the exposure periods of 30-240 minutes, but an examination of the evolution of features associated with specific biochemical components indicated DNA damage and loss of lipidic signatures at the early exposure times, whereas changes in protein signatures appeared to evolve over longer periods. In comparison to the cytotoxicological responses, the study demonstrates that Raman spectroscopy can identify biochemical changes as a result of solar exposure at time points significantly earlier than changes in tissue viability are observed.

Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy.

Three dimensional collagen gels have been used as matrices for the imaging of live cells by Raman spectroscopy. The study is conducted on a human lung adenocarcinoma (A549) and a spontaneously immortalized human epithelial keratinocyte (HaCaT) cell line. The lateral resolution of the system has been estimated to be <1.5 µm making it possible to access the subcellular organization. Using K-means clustering analysis, it is shown that the different subcellular compartments of individual cells can be identified and differentiated. The biochemical specificity of the information contained in the Raman spectra allows the visualization of differences in the molecular signature of the different sub-cellular structures. Furthermore, to enhance the chemical information obtained from the spectra, principal component analysis has been employed, allowing the identification of spectral windows with a high variability. The comparison between the loadings calculated and spectra from pure biochemical compounds enables the correlation of the variations observed with the molecular content of the different cellular compartments.

Three dimensional collagen gels as a cell culture matrix for the study of live cells by Raman spectroscopy.

Three dimensional collagen gels are evaluated as matrices for the study of live cells by Raman spectroscopy. The study is conducted on a human lung adenocarcinoma (A549) and a spontaneously immortalized human epithelial keratinocyte (HaCaT) cell line. It is demonstrated, using the Alamar Blue assay, that both cell models exhibit enhanced viability in collagen matrices compared to quartz substrates, commonly used for Raman spectroscopy. Using principal component analysis, it is shown that the Raman spectral analysis of cells in collagen matrices is minimally contaminated by substrate contributions and the cell to cell spectral variations are greatly reduced compared to those measured on quartz substrates. Furthermore, the spectral measurements are seen to have little contribution from the cell culture medium, implying that cultures can be kept viable over prolonged measurement or mapping procedures.

An investigation of the RWPE prostate derived family of cell lines using FTIR spectroscopy.

Interest in developing robust, quicker and easier diagnostic tests for cancer has lead to an increased use of Fourier transform infrared (FTIR) spectroscopy to meet that need. In this study we present the use of different experimental modes of infrared spectroscopy to investigate the RWPE human prostate epithelial cell line family which are derived from the same source but differ in their mode of transformation and their mode of invasive phenotype. Importantly, analysis of the infrared spectra obtained using different experimental modes of infrared spectroscopy produces similar results. The RWPE family of cell lines can be separated into groups based upon the method of cell transformation rather than the resulting invasiveness/aggressiveness of the cell line. The study also demonstrates the possibility of using a genetic algorithm as a possible standardised pre-processing step and raises the important question of the usefulness of cell lines to create a biochemical model of prostate cancer progression.

Spectroscopic and chemometric approaches to radiobiological analyses.

Vibrational spectroscopy is an attractive modality for the analysis of biological samples, providing a complete non-invasive acquisition of the biochemical fingerprint of the sample. It has been demonstrated that this data provides the means to assay multiple functional responses of a biological system at a spatial resolution as low as a micron within the sample. As the interaction of ionizing radiation with biological systems involves chemical reactions between the products of radiation-induced damage and various structural and functional units within the cell, the vibrational spectroscopic modalities have received attention as potential measurement platforms for the in situ examination of the chemistry of biological species in radiobiology. This presents challenges in relation to sample preparation and the construction of suitable analytical methodologies. In this work protocols for sample preparation and approaches to multivariate analysis of vibrational spectra in radiobiological analysis are detailed and the utility of the methodology in analyzing the evolution of biochemical responses to radiobiological damage are highlighted.

Fourier transform infrared microspectroscopy and multivariate methods for radiobiological dosimetry.

The scientific literature contains an ever-growing number of reports of applications of vibrational spectroscopy as a multivariate non-invasive tool for analysis of biological effects at the molecular level. Recently, Fourier transform infrared microspectroscopy (FTIRM) has been demonstrated to be sensitive to molecular events occurring in cells and tissue after exposure to ionizing radiation. In this work the application of FTIRM in the examination of dose-dependent molecular effects occurring in skin cells after exposure to ionizing radiation with the use of partial least-squares regression (PLSR) and generalized regression neural networks (GRNN) was studied. The methodology is shown to be sensitive to molecular events occurring with radiation dose and time after exposure. The variation in molecular species with dose and time after irradiation is shown to be non-linear by virtue of the higher modeling efficiency yielded from the non-linear algorithms. Dose prediction efficiencies of approximately +/-10 mGy were achieved at 96 h after irradiation, highlighting the potential applications of the methodology in radiobiological dosimetry.

Bystander effect induced changes in apoptosis related proteins and terminal differentiation in in vitro murine bladder cultures.

PURPOSE: Radiation-induced bystander effects are now an established phenomenon seen in numerous cell and tissue culture models. The aim of this investigation was to examine the bystander signal and response in a multicellular primary tissue culture system in vitro. METHODS AND MATERIALS: Murine bladder samples were explanted and directly exposed to gamma radiation, or treated with irradiated tissue conditioned medium (ITCM) generated from the directly irradiated cultures. RESULTS: Results indicated that there was a strong bystander signal produced by the tissue that caused both dose-dependent and -independent changes in the ITCM treated tissue. Significantly increased B-cell lymphoma 2 (Bcl2) expression was noted after treatment with 0.5Gy and 5Gy ITCM (approximately 80%), while dose-dependent changes were observed in c-myelocytomatosis (cMyc) (39.48% at 0.5 Gy ITCM, 81.28% at 5 Gy ITCM) and the terminal differentiation marker uroplakin III (17.88% at 0.5 Gy). Nuclear fragmentation was also significantly increased at both doses of ITCM. CONCLUSION: These data suggest that the bystander signal produced in a multicellular environment induces complex changes in the ITCM-treated culture, and that these changes are reflective of a coordinated response to maintain integrity throughout the tissue.

Single walled carbon nanotubes induce indirect cytotoxicity by medium depletion in A549 lung cells.

The ability of two types of single walled carbon nanotubes (SWCNT), namely Arc Discharge (AD) and HiPco single walled carbon nanotubes, to induce an indirect cytotoxicity in A549 lung cells by means of medium depletion was investigated. The nanotubes were dispersed in a commercial cell culture medium and subsequently removed by centrifugation and filtration. Spectroscopic analysis confirmed the removal of the nanotubes and showed differing degrees of alteration of the composition of the medium upon the removal of the nanotubes. The ability to induce an indirect cytotoxic effect by altering the medium was evaluated using two endpoints, namely the Alamar Blue (AB) and the Clonogenic assay. Exposure of the A549 cells to the depleted medium which had previously contained carbonaceous nanoparticles, revealed significant cytotoxicity for both endpoints employed. The results presented demonstrate that single walled carbon nanotubes can induce an indirect cytotoxicity by alteration of cell culture medium (in which they have previously been dispersed) which potentially results in a false positive toxic effect being observed in cytotoxicity studies.

Vibrational spectroscopy for cervical cancer pathology, from biochemical analysis to diagnostic tool.

Cervical cancer is the second most common cancer in women worldwide with 80% of cases arising in the developing world. The mortality associated with cervical cancer can be reduced if this disease is detected at the early stages of development or at the pre-malignant state (cervical intraepithelial neoplasia, CIN). The aim of this study was to investigate the potential of Raman spectroscopy as a diagnostic tool to detect biochemical changes accompanying cervical cancer progression. Raman spectra were acquired from proteins, nucleic acids, lipids and carbohydrates in order to gain an insight into the biochemical composition of cells and tissues. Spectra were also obtained from histological samples of normal, CIN and invasive carcinoma tissue from 40 patients. Multivariate analysis of the spectra was carried out to develop a classification model to discriminate normal from abnormal tissue. The results show that Raman spectroscopy displays a high sensitivity to biochemical changes in tissue during disease progression resulting in an exceptional prediction accuracy when discriminating between normal cervical tissue, invasive carcinoma and cervical intraepithelial neoplasia (CIN). Raman spectroscopy shows enormous clinical potential as a rapid non-invasive diagnostic tool for cervical and other cancers.

The involvement of calcium and MAP kinase signaling pathways in the production of radiation-induced bystander effects.

Much evidence now exists regarding radiation-induced bystander effects, but the mechanisms involved in the transduction of the signal are still unclear. The mitogen-activated protein kinase (MAPK) pathways have been linked to growth factor-mediated regulation of cellular events such as proliferation, senescence, differentiation and apoptosis. Activation of multiple MAPK pathways such as the ERK, JNK and p38 pathways have been shown to occur after exposure of cells to radiation and a variety of other toxic stresses. Previous studies have shown oxidative stress and calcium signaling to be important in radiation-induced bystander effects. The aim of the present study was to investigate MAPK signaling pathways in bystander cells exposed to irradiated cell conditioned medium (ICCM) and the role of oxidative metabolism and calcium signaling in the induction of bystander responses. Human keratinocytes (HPV-G cell line) were irradiated (0.005-5 Gy) using a cobalt-60 teletherapy unit. The medium was harvested 1 h postirradiation and transferred to recipient HPV-G cells. Phosphorylated forms of p38, JNK and ERK were studied by immunofluorescence 30 min-24 h after exposure to ICCM. Inhibitors of the ERK pathway (PD98059 and U0126), the JNK pathway (SP600125), and the p38 pathway (SB203580) were used to investigate whether bystander-induced cell death could be blocked. Cells were also incubated with ICCM in the presence of superoxide dismutase, catalase, EGTA, verapamil, nifedipine and thapsigargin to investigate whether bystander effects could be inhibited because of the known effects on calcium homeostasis. Activated forms of JNK and ERK proteins were observed after exposure to ICCM. Inhibition of the ERK pathway appeared to increase bystander-induced apoptosis, while inhibition of the JNK pathway appeared to decrease apoptosis. In addition, reactive oxygen species, such as superoxide and hydrogen peroxide, and calcium signaling were found to be important modulators of bystander responses. Further investigations of these signaling pathways may aid in the identification of novel therapeutic targets.

Temperature-induced nucleation of poly(p-phenylene vinylene-co-2,5-dioctyloxy-m-phenylene vinylene) crystallization by HiPco single-walled carbon nanotubes.

Hybrid systems of the conjugated organic polymer poly(p-phenylene vinylene-co-2,5-dioctyloxy-m-phenylene vinylene)(PmPV) and HiPco single-walled carbon nanotubes (SWNTs) are explored using spectroscopic and thermal techniques to determine specific interactions. Vibrational spectroscopy indicates a weak interaction, and this is further elucidated using differential scanning calorimetry (DSC), confocal laser scanning microscopy, temperature-dependent Raman spectroscopy, and temperature-dependent infrared spectroscopy of the raw materials and the composite. An endothermic transition is observed in the DSC of both the polymer and the 0.1% HiPco composite in the region of 50 degrees C. Also observed in the DSC of the composite is a double-peaked endotherm at -39 and -49 degrees C, which does not appear in the polymer. The Raman spectroscopy of the polymer upon increasing the temperature to 60 degrees C shows a diminished cis-vinylene mode at 1575 cm(-1), with an increase in relative intensity of the trans-vinylene mode at 1630 cm(-1). Partially irreversible change in isomerization suggests increased order in the polymer. This change in the polymer is also manifest in the Raman composite spectrum upon increase of the temperature to 60 degrees C, where the spectrum becomes abruptly dominated by nanotubes. Raman spectroscopy of the composite shows no change at -35 degrees C; however, infrared absorption measurements suggest that the transition at -35 degrees C derives from the polymer side chains. Here the composite at -35 degrees C shows a change in the absorbance of the polymer side chain aryl-oxide linkage at 1250 cm(-1) and alkyl-oxide stretch at 1050 cm(-1). Infrared spectra thus suggest that the transitions in the lower temperature region around -35 degrees C are side chain-induced, while Raman spectra suggest that the transition at 60 degrees C is backbone-induced. Furthermore, temperature cycling induces an irreversible decrease in the mean fluorescence intensity of the polymer, coupled with a further reduction in the mean fluorescence intensity of the composite. This suggests that an increase in crystallization of the composite is supported and enhanced by an increase in ordering of the polymer. Implications are discussed.

Early events in the apoptotic cascade initiated in cells treated with medium from the progeny of irradiated cells.

Genomic instability and the bystander effect have recently been linked experimentally. It has previously been shown that medium from irradiated cells can induce early events in the apoptotic cascade, such as mobilisation of intracellular calcium, loss of mitochondrial membrane potential and an increase in reactive oxygen species, in unhit cells. The aim of this study was to determine if medium from the progeny of irradiated cells could also initiate apoptosis in unhit cells. Human keratinocytes were irradiated (0.5 and 5 Gy) and medium was harvested up to the 7th passage post-irradiation and transferred to unirradiated keratinocytes. Intracellular calcium levels, mitochondrial membrane potential and the level of reactive oxygen species were all monitored for a period of 24 h following medium transfer. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential and increases in reactive oxygen species (from 6 h after medium transfer) were observed. There was no significant difference between medium generated by cells irradiated at the different doses. The data suggest that initiating events in the apoptotic cascade were induced in unhit cells by a signal produced by irradiated cells and that this signal can still be produced in the progeny of irradiated cells.

Initiation of apoptosis in cells exposed to medium from the progeny of irradiated cells: a possible mechanism for bystander-induced genomic instability?

Genomic instability and bystander effects have recently been linked experimentally both in vivo and in vitro. The aim of the present study was to determine if medium from irradiated cells several passages distant from the original exposure could initiate apoptosis in unirradiated cells. Human keratinocytes (from the HPV-G cell line) were irradiated with 0.5 Gy or 5 Gy gamma rays. Medium was harvested at each passage up to the 7th passage (approximately 35 population doublings) postirradiation and transferred to unirradiated keratinocytes. Intracellular calcium levels, mitochondrial membrane potential, and the level of reactive oxygen species were all monitored for 24 h after medium transfer. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential, and increases in reactive oxygen species (from 6 h after medium transfer) were observed in the recipient cells. There was no significant difference between medium conditioned by cells irradiated with 0.5 or 5 Gy. The effect of medium from progeny was the same as the initial effect reported previously and did not diminish with increasing passage number. The data suggest that initiating events in the cascade that leads to apoptosis are induced in unirradiated cells by a signal produced by irradiated cells and that this signal can still be produced by the progeny of irradiated cells for several generations.

Oxidative stress in cells exposed to low levels of ionizing radiation.

The ability of medium from gamma-irradiated cells to induce early events in the apoptotic cascade, such as the mobilization of intracellular calcium, loss of mitochondrial membrane potential and increased levels of reactive oxygen species, in unirradiated cells was investigated. Medium from irradiated human keratinocytes was harvested and transferred to unirradiated keratinocytes. Intracellular calcium levels, mitochondrial membrane potential and the level of reactive oxygen species were all monitored for a period of 24 h following medium transfer. Rapid calcium fluxes (within 30 s), loss of mitochondrial membrane potential and increases in reactive oxygen species (from 6 h after medium transfer) were observed. There was no significant difference between the effects of medium generated by cells irradiated at 0.5 Gy or 5 Gy. The data suggest that a signal that leads to apoptosis is released from cells undergoing radiation-induced oxidative stress.

Calcium confusion--is the variability in calcium response by Sertoli cells to specific hormones meaningful or simply redundant?

When results of more than ten different studies on hormone-induced calcium signals in Sertoli cells are taken together, a wide variety of responses emerges. The reported changes range from increased concentrations, via no response at all, to decreased calcium concentrations. Minor variations in cell isolation techniques, culture conditions, or techniques for measuring the intracellular calcium could explain some of these differences. However, erratic variations in response are also observed within research groups under very similar experimental conditions. Such 'negative' findings are mainly reported orally and do not further penetrate the scientific community. As hormone-dependent calcium responses evidently may depend very much on the context of the cells, calcium transients would appear to be unreliable bioassay principles with which to detect the primary actions of FSH and effectors such as androgens on Sertoli cells. A more important biological question is whether these sometimes opposed calcium transients are connected with a particular cellular response. To date there is no evidence for such a tight coupling in Sertoli cells, implying that, at least under in vitro conditions, calcium signals might even be redundant altogether. Such calcium variability is probably not unique to Sertoli cells, and the aim of this commentary is to promote an open debate that may help to transform the current state of 'calcium confusion' into a better understanding of the intracellular calcium language.