Identification Of Single Cells Research Articles

Raman sorting and identification of single living micro-organisms with optical tweezers

We report on a novel technique for sorting and identification of single biological cells and food-borne bacteria based on laser tweezers and Raman spectroscopy (LTRS). With this technique, biological cells of different physiological states in a sample chamber were identified by their Raman spectral signatures and then they were selectively manipulated into a clean collection chamber with optical tweezers through a microchannel. As an example, we sorted the live and dead yeast cells into the collection chamber and validated this with a standard staining technique. We also demonstrated that bacteria existing in spoiled foods could be discriminated from a variety of food particles based on their characteristic Raman spectra and then isolated with laser manipulation. This label-free LTRS sorting technique may find broad applications in microbiology and rapid examination of food-borne diseases.

Identification of Single Bacterial Cells in Aqueous Solution Using Confocal Laser Tweezers Raman Spectroscopy

We report on a rapid method for reagentless identification and discrimination of single bacterial cells in aqueous solutions using a combination of laser tweezers and confocal Raman spectroscopy (LTRS). The optical trapping enables capturing of individual bacteria in aqueous solution in the focus of the laser beam and levitating the captured cell well off the cover plate, thus maximizing the excitation and collection of Raman scattering from the cell and minimizing the unwanted background from the cover plate and environment. Raman spectral patterns excited by a near-infrared laser beam provide intrinsic molecular information for reagentless analysis of the optically isolated bacterium. In our experiments, six species of bacteria were used to demonstrate the capability of the confocal LTRS in the identification and discrimination between the diverse bacterial species at various growth conditions. We show that synchronized bacterial cells can be well-discriminated among the six species using principal component analyses (PCA). Unsynchronized bacterial cells that are cultured at stationary phases can also be well-discriminated by the PCA, as well as by a hierarchical cluster analysis (HCA) of their Raman spectra. We also show that unsynchronized bacteria selected from random growth phases can be classified with the help of a generalized discriminant analysis (GDA). These findings demonstrate that the LTRS may find valuable applications in rapid sensing of microbial cells in diverse aqueous media.

Raman spectroscopic identification of single yeast cells

AbstractEukaryotes (such as yeasts) and prokaryotes (such as bacteria) differ in size, molecular complexity, etc. By definition, eukaryotic cells store their DNA in a separate internal compartment, the so‐called nucleus. Owing to this molecular compartmentalization, the identification of yeast cells by vibrational spectroscopy at a single‐cell level is challenging. This contribution reports on first results of micro‐Raman analysis together with a hierarchical cluster analysis allowing for an online identification method for yeast cells at a single‐cell level. For the classification analysis, an average spectrum from 10 different measuring points within a single yeast cell was used to overcome the heterogeneity of the yeast cells. Copyright © 2005 John Wiley & Sons, Ltd.

Graphical representation of ribosomal RNA probe accessibility data using ARB software package

BackgroundTaxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ hybridization (FISH), besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments.ResultsThe PROBE Design tools of the ARB software package take into consideration several criteria such as number, position and quality of diagnostic sequence differences while designing oligonucleotide probes. Additionally, new visualization tools were developed to enable the user to easily examine further sequence associated criteria such as higher order structure, conservation, G+C content, transition-transversion profiles and in situ target accessibility patterns. The different types of sequence associated information (SAI) can be visualized by user defined background colors within the ARB primary and secondary structure editors as well as in the PROBE Match tool.ConclusionUsing this tool, in silico probe design and evaluation can be performed with respect to in situ probe accessibility data. The evaluation of proposed probe targets with respect to higher-order rRNA structure is of importance for successful design and performance of in situ hybridization experiments. The entire ARB software package along with the probe accessibility data is available from the ARB home page .

Functional identification and monitoring of individual ? and ? cells in cultured mouse islets of Langerhans

The aim of the present study was to evaluate, by use of fluorescence microscopy and immunofluorescence stainings, the use of a fluorescent membrane potential sensitive probe as a means to identify and monitor changes in membrane potential of individual cell types in whole islets of Langerhans over time. Our work supports the use of the fluorescent probe bis-(1,3 dibutylbarbituric acid) trimethine oxonol (diBAC(4)(3)), in identification of single alpha and beta cells in the periphery of mouse pancreatic islets cultured on extracellular matrix. At a low extracellular glucose concentration (3 mM), heterogeneous staining of the islets was observed. Approximately 97% of the peripheral cells that stained brightly with diBAC(4)(3) were glucagon positive. Additional diBAC(4)(3) studies, demonstrated that an increase in glucose concentration from 3 to 10 mM is paralleled by repolarization of alpha cells and depolarization of beta cells. This suggests that reciprocity of glucagon and insulin release upon glucose stimulation is coupled to divergent changes in membrane potential of these cell types and supports the use of diBAC(4)(3) as a means to detect changes in secretion in both cell types.

Laser microdissection and DNA typing of cells from single hair follicles

Isolation and identification of single cells from tissue samples or smears assume a great relevance in pathological and forensic applications; in this latter field, the possibility to identify a specific genetic profile can be obtained by short tandem repeat (STR) typing, allowing to achieve a scientific proof important in law courts. It is well known that DNA extraction may be performed from several tissue fragments, blood traces, spermatozoa as well as telogen hair. However, in the last case, few follicle cells are coupled to a great amount of keratin reducing the efficiency of DNA amplification. Recently, the introduction of laser microdissection technique has greatly improved the capability to select single cells without any cross-contamination. In the present report, we have performed a laser microdissection using a Leica AS LMD (Leica Microsystems, Germany), utilized on cutting the telogen hair in order to exclusively collect the lower part of the follicle and reduce keratin contamination. In this way we can accurately extract an adequate amount of DNA, successfully typed by STR profile.

Near-infrared Raman spectroscopy of single optically trapped biological cells

We report on the development and testing of a compact laser tweezers Raman spectroscopy (LTRS) system. The system combines optical trapping and near-infrared Raman spectroscopy for manipulation and identification of single biological cells in solution. A low-power diode laser at 785 nm was used for both trapping and excitation for Raman spectroscopy of the suspended microscopic particles. The design of the LTRS system provides high sensitivity and permits real-time spectroscopic measurements of the biological sample. The system was calibrated by use of polystyrene microbeads and tested on living blood cells and on both living and dead yeast cells. As expected, different images and Raman spectra were observed for the different cells. The LTRS system may provide a valuable tool for the study of fundamental cellular processes and the diagnosis of cellular disorders.

Occult micrometastasis: enrichment, identification and characterization of single disseminated tumour cells

The decision as to whether systemic adjuvant therapy should be applied in breast cancer patients for secondary prevention of metastatic relapse is based solely on the statistical prognosis. For this reason, the direct identification of minimal residual cancer in distant organs (e.g. bone marrow) is of particular importance. In breast cancer 25–43% of the patients exhibit micrometastatic disease in bone marrow, following resection of their primary tumours. Successful enrichment, reliable identification and molecular profiling of disseminated tumour cells at the single cell level are still key issues in ongoing and future studies. In addition, first attempts have been reported to evaluate the biology of disseminated tumour cells using in vitro and in vivo models. Taken together, the advancing characterization of disseminated tumour cells opens the avenue for the development of new therapeutic approaches aimed at preventing metastatic relapse.

Detection of micrometastasis through tissue-specific gene expression: its promise and problems.

The detection of micrometastasis holds great promise for earlier staging of patients with malignant diseases and may ultimately guide therapeutic decisions. So far, reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of genes expressed by the tumor in a tissue-specific manner is the method with the highest diagnostic sensitivity. It is well-established that the identification of single tumor cells is feasible in tissues and bodily fluids in both experimental and clinical samples. However, at present it is difficult to assign clinical significance to results obtained from such tests, primarily because their diagnostic specificity is disputed, both conceptionally and methodologically. For example, amplification of candidate mRNA targets is detectable in non-cancer patients using conditions that generally fail to generate such signals from healthy individuals. We have established that transcription of the tissue-specific genes can be affected by different means. Specifically, some target mRNA species are detectable in peripheral blood nuclear cells as low abundance constitutive-like expression, whereas others are induced through in vitro tissue culturing. In addition, mRNA expression may be distinctly upregulated by different cytokines or growth factors in vivo. Also, background transcription of target mRNAs can occur in different lineages of peripheral blood cells. Finally, expression may be substantially different in tissues such as peripheral blood, bone marrow, or lymph nodes. As a consequence, cancer patients in unrelated clinical situations may present with different levels of background expression, making the diagnostic specificity of test results difficult to assess. To add to this complexity, an increasing body of literature is being generated using various targets for a multitude of malignant diseases. There is a great variety of methods for sampling, specimen processing, nucleic acids recovery, test conditions, and readout formats, making it impossible to compare data. In summary, modalities of quantitative RT-PCR methods and standardization issues should be discussed to address these questions.

New concepts for staging and therapy of cervix cancer by endoscopic surgery

Non-invasive staging of cervical cancer according to FIGO is out-dated. In contrast to clinical staging exact histopathologic evaluation of tumor extension and tumor biology can be achieved via laparoscopic evaluation. Invasive endoscopic staging is advantageous and should be integrated into the diagnostic armamentarium of gynecologic oncologists and radiotherapists. Through magnification and blood-less surgery minute anatomical structures can be identified laparoscopically. Thus, fibers of autonomic nerves can be visualized and parasympathetic fibres can be preserved. Conservation of these neural structures leads to significant reduction of postoperative morbidity of bladder and rectum. The concept of the sentinel lymph node is also valid for the uterine cervix. Selected biopsy of the sentinel node and identification of single tumor cells leads potentially to higher oncologic safety and may reduce morbidity associated with radical lymphadenectomy. In early stage cervical cancer fertility may be preserved by radical trachelectomy which is combined with laparoscopic parametric and pelvic lymphadenectomy. Following radical trachelectomy a pregnancy rate of 40% is achieved, the recurrence rate following conventional radical hysterectomy is identical.

Determination of cellular thiol levels in individual viable lymphocytes by means of fluorescence intensity and polarization

Cellular thiol levels regulate lymphocyte proliferation and death and play a significant role in the immune response. Therefore, the ability to analyze the total protein and non-protein thiol compounds and their distribution among individual living lymphocytes is of great importance. A quantitative measurement of intracellular sulphydryl groups in living lymphocytes using the Cellscan mark F (CS- F) cytometer, in conjunction with the probe CMFDA, is described. This technique permits the detection, identification, and study of sub-populations and single cells in a sample of heterogeneous lymphocytes. The Cellscan apparatus is a laser based scanning cytometer incorporating a unique cell carrier which allows repeated, high-precision measurements of fluorescence intensity (FI) and fluorescence polarization (FP) to be made on intact individual living cells under controlled physiological conditions. The discernible effect of fluorophore molecules bound to thiols having a higher FP than free molecules was used to estimate their relative fractions in living lymphocytes. The results were more conspicuous when the ratio between FP measured at two wavelengths (FPR) of the fluorogenic molecules was used for analysis. In addition, the intracellular dynamic changes in the FI, FP and FPR of the fluorescent probe were also monitored. The cellular sulphydryl content of each lymphocyte within a population was recorded by the CS- F, and sub-populations or individual cells were classified according to their thiol levels and their metabolic rates. Changes in thiol concentration were observed following mitogenic activation of peripheral lymphocytes.

DNA injection into single cells of intact mice.

We describe a technique wherein single muscle fibers of intact mice are made transgenic by intracellular injection of DNA expression vectors for the reporter genes lacZ and green fluorescent protein (GFP). Application of in vivo imaging techniques allowed identification of single cells during the injections, and of the same single cells when the muscle was reexposed days or weeks later. DNA concentration by itself had little effect on fiber survival or expression efficacy, but it seemed crucial to exceed a threshold of about 10(6) injected plasmid molecules in order to obtain expression. On the other hand, experiments with coinjection of two different plasmids suggested that relatively few individual molecules were eventually transcribed in expressing cells. Plasmid DNA remained localized to the injection site, and expression was confined to nuclei in the vicinity. Expression was stable, as reporter was detected 2-61 days after injection.

Sequential in situ hybridization on tissue sections: a novel approach for analysing co-expression of different mRNAs in a single cell

▼Non-radioactive in situ hybridization has been widely used as a high-resolution method for detecting specific mRNAs in tissue sections. However, it has been difficult to combine two colour reactions to detect the presence of different transcripts at the single cell level. Our method, which involves simultaneous hybridization with two different riboprobes followed by sequential colour reactions for each of them, relies on the removal of the first signal before recording the second one. Thus, this protocol avoids signal interference and permits the clear identification of single cells expressing more than one transcript. Moreover, the ability to use a single optics apparatus to take photographs of each expression pattern allows the direct analysis of the relative mRNA levels at the level of single cells in the section. We were interested in studying the co-expression of transcripts for different neurotrophic factor receptors (here cRet and Lifrβ) in single motoneurons of the facial motor nucleus. For a variety of reasons, however, none of the existing double in situ hybridization (dISH) protocols permits the required resolution of two signals at the single cell level. Those based on colocalization of a colorimetric signal with a radioisotopic signal (Ref. 1) do not have a cellular resolution and, therefore, can only be used to detect common expression ‘domains’. Those using colorimetric and fluorescent signals (Ref. 2, 3, 4, 5, 6) require a different detection system for each signal, making it difficult to compare expression levels directly. Alternatively, with

New insights into the biology of Philadelphia-chromosome-positive acute lymphoblastic leukaemia using a combination of May-Grünwald-Giemsa staining and fluorescence in situ hybridization techniques at the single cell level.

It is sometimes difficult to discriminate chronic myeloid leukaemia (CML) in lymphatic blast crisis from Ph-chromosome positive acute lymphoblastic leukaemia (ALL). Previous studies have suggested that ALL is restricted to the lymphatic lineage only, whereas CML involves all cell lineages. In four cases of Ph-positive ALL we combined the standard May-Grünwald-Giemsa staining with FISH at the single cell level and were able to demonstrate that > or = 98% of lymphatic blasts carried the Philadelphia chromosome. Erythropoiesis was not involved when this technique was applied. Using immunological identification of single cells (FICTION), we detected the t(9;22) in 100% of CD19-positive B lymphoblasts in all four cases, in some CD3-positive T cells in two patients, and in > or = 98% of CD34-positive precursor cells. However, in two out of four patients the myeloid cell compartment was involved in the malignant transformation, unequivocally demonstrated not only by the combination of MGG and FISH but also by FICTION using the antibodies CD13 and CD33. The observation that both patients with myeloid cell lineage involvement had a myeloid co-expression on their blasts and a better survival supports the concept of a separate, biologically determined subgroup in Ph-positive ALL, leading to further investigations, and individually tailored treatment strategies.

Increase in Fluorescence Intensity of 16S rRNA In Situ Hybridization in Natural Samples Treated with Chloramphenicol

Despite the numerous advantages of fluorescent in situ hybridization for the identification of single prokaryotic cells with 16S rRNA probes, use of the technique with natural samples, especially those from the marine environment, is still problematic. The low percentage of fluorescently labeled cells constitutes the primary problem for in situ hybridization of natural samples, probably due to low cellular rRNA content. This study represents an attempt to improve detection of marine prokaryotes by increasing cellular rRNA content without changing the species composition. Cells from three California coastal sites were treated with chloramphenicol, an inhibitor of protein synthesis and rRNA degradation, at 100 (mu)g/ml and then were probed with a "universal" 16S rRNA fluorescent probe and viewed by image-intensified video microscopy. Counts of fluorescent cells increased from ca. 75% for untreated samples to ca. 93 to 99% for chloramphenicol-treated samples, compared to counts produced by DAPI (4(prm1),6-diamidino-2-phenylindole) staining, after at least 45 min of exposure to the drug (these percentages include autofluorescent cells, which averaged 6%). This suggests that most cells in these samples were active. We hypothesize that the low fluorescent-cell counts previously reported were probably often due to the fluorescence intensity of labeled cells being below the detection level rather than to high levels of dead cells in marine environments. This method may aid in the characterization of bacterioplankton with fluorescent probes.

Detection of K- ras point mutation by in situ PCR in cell suspensions: Comparison of the indirect and direct methods

In situ PCR is a new technique for the localization of low copy number sequences. We report here a method for the in situ visualization of a point mutation in K- ras codon 12 by indirect in situ PCR. Twenty-five primers were examined to select mutant-specific primers. Harvested cell lines were fixed and suspended in PCR mixture. Forty cycles of PCR in cell suspension was performed in a thermal cycler using a hot start method. Cells were cytocentrifuged onto slides, and post-fixation was performed. The specimens on the slides were then hybridized with a digoxigenin-labeled probe, followed by color reaction. Both Calu-1 (mutated: TGT) and NCI-H460 (wild type: GGT) cells had strong hybridization signals in the nuclei with general primers. But with mutant-specific primers, only Calu-1 cells had hybridization signals. No signal was observed without primers or Taq DNA polymerase. Southern blotting of the same preparation confirmed desired amplification. We also applied direct in situ PCR, but this method failed to detect the point mutation. We conclude that our indirect in situ PCR method shows the feasibility of in situ identification of single cells carrying point mutations.

Improved microscopic identification of Clavibacter michiganensis subsp. sepedonicus cells by combining in situ hybridization with immunofluorescence.

An oligonucleotide probe was selected from the 16S rRNA gene of Clavibacter michiganensis subsp. sepedonicus for specific in situ hybridization. The rhodamine-labelled oligonucleotide probe was used in conjunction with an indirect immunofluorescence procedure based on a specific monoclonal antibody detected with a fluorescein-labelled conjugate. Simultaneous labelling of bacterial cells with the oligonucleotide and antibody probes allows accurate microscopic identification of single cells when isolation and other methods of confirming bacterial identity are not possible.

Which compartments are involved in Philadelphia-chromosome positive chronic myeloid leukaemia? An answer at the single cell level by combining May-Grünwald-Giemsa staining and fluorescence in situ hybridization techniques.

Chronic myeloid leukaemia (CML) is believed to represent a stem cell disorder involving all three cell lineages. The typical chromosomal aberration, the Philadelphia chromosome, is the translocation (9;22)(q34;q11). Several studies with cytogenetics, fluorescence in situ hybridization (FISH), or polymerase chain reaction have investigated the presence of the t(9;22) in different cell compartments. However, questions still remain. In six cases of CML we combined the standard May-Grünwald-Giemsa staining with FISH at the single-cell level and were able to demonstrate that not only all maturation stages of granulopoiesis, erythropoiesis, and megakaryocytes, but also plasma cells, eosinophils, basophils and monocytes carried the Philadelphia chromosome in 53-98% of samples. Using immunological identification of single cells we were able to demonstrate that the t(9;22) is detectable in 34% of CD3-positive T lymphocytes, in 32% of CD19-positive B lymphocytes, and in 82% of CD34-positive precursor cells. The results give new insight into the biology of CML and may have implications for future therapeutic strategies.

Inhibition of endotoxin-induced nitric oxide synthase production in microglial cells by the presence of astroglial cells: a role for transforming growth factor beta.

In mixed glial cell cultures from cerebral cortices of newborn rats, endotoxin induces inducible nitric oxide (iNOS), nitric oxide (NO), and interleukin-1 beta (IL-1 beta) production in microglial cells. Earlier we demonstrated that endotoxin induced iNOS but not IL-1 beta expression in microglial cells is inhibited by the presence of astroglial cells. In the present paper we describe studies on the mechanism by which astroglial cells exert selective suppressive action on iNOS expression by microglial cells. Expression of iNOS and IL-1 beta was studied by single or double label immunocytochemical techniques and cell identification was performed with GSA-I-B4-isolectin and an antibody against GFAP. Production of IL-1 beta and NO was determined by measurement of IL-1 beta and nitrite concentrations in cell lysates and the culture medium, respectively. TGF beta, a cytokine known to inhibit NO production by endotoxin challenged macrophages, was measured in culture medium of mixed glial cell cultures using a bioassay. Microglial, astroglial, and mixed glial cell cultures produced similar concentrations of TGF beta. The potential effect of TGF beta was studied by using immunoneutralizing antibodies against TGF beta 1 and TGF beta 2 on the induction of iNOS in microglial cells in the presence of astroglial cells. Incubation of the mixed glial cell culture with these TGF beta antibodies (3 micrograms/ml) markedly increased endotoxin-induced NO production and iNOS expression in microglial cells, whereas the production of IL-1 beta was not affected. The antibodies against TGF beta 1 and TGF beta 2 marginally increased NO production in pure microglial cell cultures, nonetheless in cultures of purified microglial cells recombinant TGF beta 1 and TGF beta 2 together with endotoxin inhibited NO production. We conclude that the presence of astroglial cells is essential for the inhibitory effect of TGF beta on NO production by microglial cells (possibly) by activation of TGF beta or by increasing the sensitivity of microglial cells for TGF beta.

On stage single cell identification of rat spermatogenic cells

The study of spermatogenic cell physiology has been hindered by the absence of unbiased methods of identification of cells upon which single cell techniques are being applied. In this work, we have used histochemical techniques, digital videoimaging, quantification of chromatin-bound DNA probes, and measurements of cell diameter to identify single spermatogenic cells at different periods of development. Our criteria of identification permit the definition of four developmental stages of spermatogenesis on which to perform single cell analyses: spermatogonia B/preleptotene spermatocytes, leptotene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids. The use of voltage-sensitive dyes and Ca 2+-sensitive dyes does not interfere with the estimations of DNA content. The estimations of DNA content of spermatogenic cells can be performed both with near-UV exciged dyes (H 33342) and long wavelength-excited dyes (ethidium bromide), allowing the use of a wide range of physiological and immunocytochemical fluorescent probes to study the spermatogenic process.