Analysis of a mutant exhibiting conditional sorting to dense core secretory granules in Tetrahymena thermophila.

The formation of dense core granules (DCGs) requires both the sorting of granule contents from other secretory proteins and a postsorting maturation process. The Tetrahymena thermophila strain SB281 fails to synthesize DCGs, and previous analysis suggested that the defect lay at or near the sorting step. Because this strain represents one of the very few mutants in this pathway, we have undertaken a more complete study of the phenotype. Genetic epistasis analysis places the defect upstream of those in two other characterized Tetrahymena mutants. Using immunofluorescent detection of granule content proteins, as well as GFP tagging, we describe a novel cytoplasmic compartment to which granule contents can be sorted in growing SB281 cells. Cell fusion experiments indicate that this compartment is not a biosynthetic intermediate in DCG synthesis. Sorting in SB281 is strongly conditional with respect to growth. When cells are starved, the storage compartment is degraded and de novo synthesized granule proteins are rapidly secreted. The mutation in SB281 therefore appears to affect DCG synthesis at the level of both sorting and maturation.     

Diversity of nitrite reductase genes in "Candidatus Accumulibacter phosphatis"-dominated cultures enriched by flow-cytometric sorting

"Candidatus Accumulibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumulibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.     

Improved sensitivity in comparative genomic hybridization analysis of DNA heteroploid cell mixtures after pre-enrichment of subpopulations by fluorescence activated cell sorting.

Cytogenetic analysis of solid tumors with comparative genomic hybridization (CGH) is hampered by the dilution of DNA from individual tumor subpopulations with DNA from other cells. We investigated to what extent this dilution effect can be alleviated using fluorescence activated cell sorting (flow sorting) of experimental DNA heteroploid cell mixtures prior to CGH. From mixtures of normal lymphocytes with triploid K-562 cells the individual components were sorted according to stemline DNA content and processed by CGH in comparison with pure K-562 samples and the original mixtures. Compared with 30 autosome copy number imbalances found in pure K-562 samples, a mixture with 32% K-562 cells showed 16 imbalances, and none were detected in mixtures with 13% or 5% K-562 cells. In contrast, 29, 22 and 23 imbalances were detected in K-562 nuclei sorted from the 32%, 13% and 5% mixtures, respectively. This indicate that CGH analysis of flow sorted DNA aneuploid subpopulations enables a specific cytogenetic analysis of the individual subclones in a DNA heteroploid cell population.     

Automatic cell identification and enrichment in lung cancer: V. Adenocarcinoma and large cell undifferentiated carcinoma

The aims of this study were to develop a protocol for the identification and enrichment of cancer cells from sputum obtained from patients with adenocarcinoma of the lung (n = 6) and large-cell undifferentiated carcinoma of the lung (n = 2), and to compare these findings with the results from our previous studies on other cell types from lung cancer. The hypotheses tested were: Cancer cells in sputum can be preserved following flow sorting. Enrichment for cancer cells from acridine orange (AO)-stained specimens can be achieved. Discrimination of cancer cells from noncancer cells is by AO green fluorescence and discrimination of lymphocytes from other cell types is by AO red fluorescence. Cancer cells are consistently enriched in the AO high green and red fluorescence region, although, for a given cell type, maximal enrichment is patient-dependent. Finally, cancer cell enrichment and lymphocyte exclusion can be done simultaneously. Cells from sputum were initially fixed, stained with AO, sorted on a dual parameter flow sorter, and classified into six groups corresponding to two ranges of green and three ranges of red fluorescence intensities. Cells of each region were stained by the method of Papanicolaou and differential counts were performed to determine the relative frequencies (i.e., purities) of leukocytes, macrophages, squamous cells, and cancer cells, in sorted and unsorted (i.e., control) samples. The average purity of leukocytes (81%), macrophages (6%), squamous cells (11%), and cancer cells (2%) varied markedly from sample to sample. However, the largest enrichment values (i.e., ratio of purity of a cell type in a sorted sample to its purity in the unsorted control sample) achieved for cancer cells consistently occurred for each patient sample in the region corresponding to high green and high red fluorescence intensities. Experimentally, a cancer cell average enrichment of sixteen-fold was obtained by this method. Additionally, fluorescence intensity ranges which increased the enrichment for macrophages by cell sorting typically excluded leukocytes and squamous cells, and vice versa. Finally, red fluorescence intensity was the primary discriminatory parameter for all cell types studied, although the additional use of green fluorescence intensity significantly increased cancer cell enrichment rates.(ABSTRACT TRUNCATED AT 400 WORDS)     

牛分离精子对其IVF胚胎染色体影响的研究

本研究采用传统的细胞遗传学方法,研究了由流式细胞仪分离的、染色未分离的及作为对照用的未染色未分离的分别来自于3头公牛的精子IVF（in vitro fertilization, IVF)后产生的6~8 d囊胚的染色体异常情况,以确定流式细胞仪分离精子的过程及染色对胚胎染色体异常的影响。结果显示,分离精子、染色未分离精子和未染色未分离精子的胚胎中,染色体组成为异常,即嵌合体的胚胎分别占40.7%（59/145）、35.8%（38/106）和37.0%（37/100）,三者染色体异常的比例无显著差异。胚胎染色体异常的频率在不同公牛之间存在差异（33.0% 比 44.6%）（p<0.05）。本研究的结果证明,染料和分离过程没有影响精子的DNA进而影响胚胎的染色体组成;胚胎染色体异常的频率在不同公牛之间存在差异。     

Selection using the alpha-1 integrin (CD49a) enhances the multipotentiality of the mesenchymal stem cell population from heterogeneous bone marrow stromal cells

Bone marrow-derived mesenchymal stem cells consist of a developmentally heterogeneous population of cells obtained from colony forming progenitors. As these colonies express the alpha-1 integrin (CD49a), here we single-cell FACS sorted CD49a+ cells from bone marrow in order to create clones and then compared their colony forming efficiency and multilineage differentiation capacity to the unsorted cells. Following selection, 40% of the sorted CD49a+ cells formed colonies, whereas parental cells failed to form colonies following limited dilution plating at 1 cell/well. Following ex vivo expansion, clones shared a similar morphology to the parental cell line, and also demonstrated enhanced proliferation. Further analysis by flow cytometry using a panel of multilineage markers demonstrated that the CD49a+ clones had enhanced expression of CD90 and CD105 compared to unsorted cells. Culturing cells in adipogenic, osteogenic or chondrogenic medium for 7, 10 and 15 days respectively and then analysing them by quantitative PCR demonstrated that CD49a+ clones readily underwent multlineage differentiation into fat, bone and cartilage compared to unsorted cells. These results thus support the use of CD49a selection for the enrichment of mesenchymal stem cells, and describes a strategy for selecting the most multipotential cells from a heterogeneous pool of bone marrow mononuclear stem cells.     

Influence of storage time on functional capacity of flow cytometrically sex-sorted boar spermatozoa

Sex-sorting of boar spermatozoa is an emerging biotechnology, still considered suboptimal owing to the slowness of the process, which requires long sorting periods to obtain an adequate number of spermatozoa to perform a non-surgical insemination. This period involves storage of sorted cells that could impair their functional capacity. Here, we have studied how the storage of sex-sorted boar spermatozoa affects their functional capacity. Sorted spermatozoa were assessed at various times (0, 2, 5h or 10h) during storage after sorting and compared with diluted and unsorted spermatozoa for sperm motility patterns, plasma membrane and acrosomal integrity and their ability to penetrate homologous IVM oocytes. Sex-sorted sperm motility and membrane integrity only decreased significantly (p<0.05) by the end of the storage period (10h) compared to unsorted spermatozoa. Sperm velocity, ALH and Dance increased significantly (p<0.05), immediately post-sorting, returning to unsorted sperm values during storage. Acrosome integrity was not seriously affected by the sorting process, but decreased (p<0.05) during storage after sorting. Sorted spermatozoa stored 2h after sorting did not differ from unsorted in penetration rates and numbers of spermatozoa per oocyte, reaching the highest (p<0.05) penetration rates and sperm numbers per oocyte, when co-cultured for 6 or more hours. Non-storage or storage for 5h or 10h negatively (p<0.05) affected sperm penetration ability. In conclusion, although flow cytometrically sex-sorted spermatozoa are able to maintain motility, viability and acrosomal integrity at optimal levels until 10h of storage after sorting, fertilizing ability is maintained only over shorter storage times (<5h).     

Incidence of chromosome aberrations in mammalian sperm stained with Hoechst 33342 and UV-laser irradiated during flow sorting

The separation of two sperm populations is possible using the technique of flow sorting, provided that a significant difference exists in the DNA content of X- and Y-bearing sperm. In order to ascertain whether or not chromosome damage was induced in sorted sperm, chromosome preparations were made from isolated sperm that had been microinjected into hamster eggs. While egg chromosomes exhibited a low frequency of chromosome aberrations, ranging from 4 to 7%, a large proportion of sperm cells exhibited chromosome damage. Between 29% of unstained and unsorted sperm and 38% of stained and unsorted sperm exhibited some type of chromosomal abnormality and this proportion increased to 50% in sorted sperm. If only damaged sperm nuclei are considered, the two unsorted sperm groups had a mean of 0.6 breaks, 0.8 triradial exchanges, and 0.2 quadriradial exchanges per nucleus. However, sorted sperm, which were stained with a fluorochrome and exposed to UV-laser irradiation, exhibited a mean of 2.9 breaks, 2.6 triradial, and 1.9 quadriradial exchanges per nucleus in which damage occurred. These observations indicate that the treatments and manipulations to which sperm nuclei are subjected during flow sorting cause chromosomal aberrations, and that exposure of the cells to UV-laser irradiation contributes substantially to the chromosome damage observed.     

Boar sperm changes after sorting and encapsulation in barium alginate membranes

A routine use of boar-sexed semen is limited by the long sorting time necessary to obtain an adequate number of sexed spermatozoa for artificial insemination and by the high susceptibility of spermatozoa of this species to damages induced by sorting procedure and subsequent cryopreservation. The aim of this work was to study the impact of encapsulation in barium alginate membrane on sorted boar spermatozoa by evaluating membrane integrity, chlortetracycline staining patterns, protein tyrosine phosphorylation, and Hsp70 immunolocalization during storage over 72 hours in liquid or encapsulated form. The encapsulation procedure significantly (P < 0.05) decreased the overall membrane integrity of control unsorted semen (81.8 vs. 57.4, CTR vs. CPS), but did not negatively affect the overall viability and the chlortetracycline staining patterns of sorted encapsulated cells. Moreover, encapsulation significantly decreased (P < 0.05) the overall phosphotyrosin A pattern cell percentage in unsorted (98.4 vs. 92.6, CTR vs. CPS) but not in sorted semen (64.0 vs. 74.2; SORT CTR vs. SORT CPS). As for Hsp70, the overall percentage of cells displaying the different patterns was significantly influenced (P < 0.05) by treatment but not by storage time. The sorting procedure seems to induce the major changes, whereas encapsulation tends to exert a protective effect on sorted semen by increasing the percentage of spermatozoa displaying the T pattern (2.8 vs. 24.3; SORT CTR vs. SORT CPS). In conclusion, our data confirm that the damaging impact of the encapsulation in barium alginate capsules seems to be limited when compared with that of the sorting procedure and, moreover, the association of the two procedures does not result in an algebraic sum of the negative effects. These results suggest the possibility of a future utilization of the encapsulation technology in order to store sorted spermatozoa and permit their controlled release in the female genital tract.     

In vitro fertilization with flow-cytometrically-sorted bovine sperm

An attractive feature of IVF is that fewer sexed sperm are needed than for artificial insemination. However, sperm sexed by flow cytometry/cell sorting are probably pre-capacitated, necessitating modifications to standard IVF systems for optimal success. With current procedures, the percentages of oocytes fertilized with sorted and unsorted frozen bovine sperm are similar, and events during the first cell cycle are timed similarly for sorted and unsorted sperm. However, in most cases, blastocyst production with sorted sperm was approximately 70% of controls produced with unsorted sperm. In some early studies, there appeared to be an unexplained delay of about half a day in blastocyst development. Nevertheless, some dozens of apparently normal calves, pre-sexed with 90% accuracy, have resulted from frozen embryos produced via IVF with sexed sperm. IVF also has proven useful as a bioassay for improving sperm-sorting procedures such as determining potential detrimental effects of laser power. It is likely that use of IVF in cattle breeding programs will increase considerably when sexed, frozen sperm become commercially available.     

Invariable biomass-specific primary production of taxonomically discrete picoeukaryote groups across the Atlantic Ocean

Oceanic photosynthetic picoeukaryotes (< 3 μm) are responsible for > 40% of total primary production at low latitudes such as the North-Eastern tropical Atlantic. In the world ocean, warmed by climate changes, the expected gradual shift towards smaller primary producers could render the role of photosynthetic picoeukaryotes even more important than they are today. Little is still known, however, about how the taxonomic composition of this highly diverse group affects primary production at the basin scale. Here, we combined flow cytometric cell sorting, NaH1?CO? radiotracer incubations and class-specific fluorescence in situ hybridization (FISH) probes to determine cell- and biomass-specific inorganic carbon fixation rates and taxonomic composition of two major photosynthetic picoeukaryote groups on a ～7500-km-long latitudinal transect across the Atlantic Ocean (Atlantic Meridional Transect, AMT19). We show that even though larger cells have, on average, cell-specific CO? uptake rates ～5 times higher than the smaller ones, the average biomass-specific uptake is statistically similar for both groups. On the other hand, even at a high taxonomic level, i.e. class, the contributions to both groups by Prymnesiophyceae, Chrysophyceae and Pelagophyceae are significantly different (P < 0.001 in all cases). We therefore conclude that these group's carbon fixation rates are independent of the taxonomic composition of photosynthetic picoeukaryotes across the Atlantic Ocean. Because the above applies across different oceanic regions the diversity changes seem to be a secondary factor determining primary production.     

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

In this work we analyzed the effects of three culture systems on developmental ability of bovine embryos in vitro produced with sexed sperm, the survival to vitrification (cryologic vitrification method) of such blastocysts, and their pregnancy rates after embryo transfer to recipients, both as fresh and after vitrification/warming. Finally, we measured the accuracy of the sorting protocol by a polymerase chain reaction-based method to validate the embryo sex at blastocyst stages. We confirmed an individual effect of the bull as well as development rates of embryos produced with sorted sperm lower than embryos with unsorted sperm, independent of the culture system used. The cryoresistance to vitrification of embryos produced with sexed sperm did not differ from that of conventionally produced embryos (re-expansion rates at 24 and 48 h: 74.6% vs. 75.5%, and 64.5% vs. 68.1% for embryos produced with conventional and sorted sperm, respectively; hatching rates at 48 h: 63.55% vs. 55.5% for embryos produced with conventional and sorted sperm, respectively). Finally, no significant differences were found in pregnancy rates after the embryo transfer of fresh and vitrified/warmed blastocysts (52.8% vs. 42.0%, respectively; P > 0.05). Male and female embryos produced with sorted sperm showed the same quality in terms of developmental ability, cryoresistance, and pregnancy rates after transfer. Our culture system, coupled with the vitrification in fiber plugs, provides good quality sex-known embryos which survive vitrification at similar rates than embryos produced with conventional unsorted sperm; also it produces good pregnancy rates after transfer of sexed embryos both fresh and after vitrification and warming.     

Cell sorting and microchemistry of cultured human fibroblasts: applications in genetics and aging research

Cell sorting is a way to isolate viable subpopulations of cells present in a mixture. The drawback of the isolation method is the shortage of material for subsequent biochemical determinations. We have employed a combination of (ultra-) microchemistry and cell sorting to overcome this problem. The methods enable determinations of protein and several enzyme activities on triton extracts of 5,000-10,000 sorted cells. In addition, using ultramicromethods we could determine enzyme activity in single sorted cells. This combination of methods is used for clinical genetic studies on heterozygote detection in Fabry's disease, an X-linked genetic disease. Moreover, microchemistry is used to study enzyme activities in sorted autofluorescent "aged" fibroblasts.     

Physiology of spermatozoa at high dilution rates: the influence of seminal plasma

Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar.     

Groups without Cultured Representatives Dominate Eukaryotic Picophytoplankton in the Oligotrophic South East Pacific Ocean

Background

Photosynthetic picoeukaryotes (PPE) with a cell size less than 3 µm play a critical role in oceanic primary production. In recent years, the composition of marine picoeukaryote communities has been intensively investigated by molecular approaches, but their photosynthetic fraction remains poorly characterized. This is largely because the classical approach that relies on constructing 18S rRNA gene clone libraries from filtered seawater samples using universal eukaryotic primers is heavily biased toward heterotrophs, especially alveolates and stramenopiles, despite the fact that autotrophic cells in general outnumber heterotrophic ones in the euphotic zone.

Methodology/Principal Findings

In order to better assess the composition of the eukaryotic picophytoplankton in the South East Pacific Ocean, encompassing the most oligotrophic oceanic regions on earth, we used a novel approach based on flow cytometry sorting followed by construction of 18S rRNA gene clone libraries. This strategy dramatically increased the recovery of sequences from putative autotrophic groups. The composition of the PPE community appeared highly variable both vertically down the water column and horizontally across the South East Pacific Ocean. In the central gyre, uncultivated lineages dominated: a recently discovered clade of Prasinophyceae (IX), clades of marine Chrysophyceae and Haptophyta, the latter division containing a potentially new class besides Prymnesiophyceae and Pavlophyceae. In contrast, on the edge of the gyre and in the coastal Chilean upwelling, groups with cultivated representatives (Prasinophyceae clade VII and Mamiellales) dominated.

Conclusions/Significance

Our data demonstrate that a very large fraction of the eukaryotic picophytoplankton still escapes cultivation. The use of flow cytometry sorting should prove very useful to better characterize specific plankton populations by molecular approaches such as gene cloning or metagenomics, and also to obtain into culture strains representative of these novel groups.     

Sorting things out: Assessing effects of unequal specimen biomass on DNA metabarcoding

Environmental bulk samples often contain many different taxa that vary several orders of magnitude in biomass. This can be problematic in DNA metabarcoding and metagenomic high‐throughput sequencing approaches, as large specimens contribute disproportionately high amounts of DNA template. Thus, a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples by specimen size (as a proxy for biomass) and balancing the amounts of tissue used per size fraction should improve detection rates, but this approach has not been systematically tested. Here, we explored the effects of size sorting on taxa detection using two freshwater macroinvertebrate bulk samples, collected from a low‐mountain stream in Germany. Specimens were morphologically identified and sorted into three size classes (body size < 2.5 × 5, 5 × 10, and up to 10 × 20 mm). Tissue powder from each size category was extracted individually and pooled based on tissue weight to simulate samples that were not sorted by biomass (“Unsorted”). Additionally, size fractions were pooled so that each specimen contributed approximately equal amounts of biomass (“Sorted”). Mock samples were amplified using four different DNA metabarcoding primer sets targeting the Cytochrome c oxidase I (COI) gene. Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification of taxa compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples at the same sequencing depth. Our results imply that sequencing depth can be decreased approximately fivefold when sorting the samples into three size classes and pooling by specimen abundance. Even coarse size sorting can substantially improve taxa detection using DNA metabarcoding. While high‐throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass or size is a simple yet efficient method to reduce current sequencing costs.     

Effects of deflected droplet electrostatic cell sorting on the viability and exoproteolytic activity of bacterial cultures and marine bacterioplankton

The cell-sorting capability of flow cytometers makes it possible to isolate specific populations of cells with pre-defined cytometric characteristics. A better knowledge of the biological effects of the sorting process is necessary for the future cell sorting applications. In this paper we report the effects of flow cytometric sorting on bacterial viability and exoproteolytic activity (EPA) of bacterial cultures and marine bacterioplankton. Sorting bacterial cultures and bacterioplankton samples reduce viability as assessed by plate counts and produce variations in the exoproteolytic activity. These effects indicate that deflected electrostatic sorting may significantly alter the biological properties of the sorted bacteria.     

Cloning of Plasmodium falciparum by single-cell sorting

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples.     

Cell autonomous sorting and surface positioning in the formation of primitive endoderm in embryoid bodies

The differentiation and formation of the primitive endoderm in early embryos can be mimicked in vitro by the aggregation of embryonic stem cells to form embryoid bodies. We present morphological evidence that primitive endoderm cells often first locate in the interior of embryoid bodies and subsequently migrate to the surface. Cell mixing experiments indicate that surface positioning is an intrinsic property of endoderm epithelial cells. Moreover, Disabled-2 (Dab2) is required for surface sorting and positioning of the endoderm cells: when Dab2 expression was eliminated, the differentiated endoderm epithelial cells distributed throughout the interior of the embryoid bodies. Surprisingly, E-cadherin is dispensable for primitive endoderm differentiation and surface sorting in embryoid bodies. These results support the model that primitive endoderm cells first emerge in the interior of the inner cell mass and are subsequently sorted to the surface to form the primitive endoderm.     

Automated single-cell analysis of aryl hydrocarbon hydroxylase in human lymphocytes

A method of rapidly analyzing hydroxybenzo (a) pyrene in single human lymphocytes with simultaneous sorting is reported. Cells with various levels of hydroxybenzo (a) pyrene were characterized and sorted by means of a Fluorescence Activated Cell Sorter into three groups with low, intermediate and high amounts of benzo (a) pyrene metabolite. The mean amount per cell was calculated after measuring the fluorescence of the cell suspensions in a fluorometer. Hydroxybenzo (a) pyrene may be detected and quantitated in individual cells at a level of sensitivity less than 10⁶ molecules per cell. The sorting capability allows for retrieval of precisely selected samples of cells for further studies of similar or other parameters.