Making thousands of photo’s of microorganisms in minutes with nanosecond exposure time is a challenge. CytoSense offers an unconventional combination of high resolution imaging in a wide flowcell. No changing of flow cells or lenses to switch from small to larger particles is required. Special aplanatic18 x magnification (0.4NA) lenses yield an in-focus aberration free optical resolution (≤1µm) better than alternative 20x systems, as shown by doublets of microspheres of 0.93 µm.
Details of cells, such as spines of Desmodesmium, or groove and pore patterns in diatoms are visible, with good resolution over the whole field-of-view. This allows imaging of larger particles like diatoms, chains, colonies and microzooplankton with fine detail of their in-focus parts.
The flow cell is wide enough (> 1 mm2) to allow unhampered entry of larger particles, whith a camera sensor covering a large area of the flowcell.
Unmanipulated bright field imaging yields high resolution images. The intrisically narrow depth-of-focus reveals the 3D spatial structure of larger cells and organisms, like a cutaway picture. The CytoSense photo of the Tinitinnid shows the cell outline and peduncle clearly inside the cross section of the lorica. With images of multiple individuals collected, a more complete picture can be constructed, as shown with the Volvox colonies photo’s.
Plankton particles are low refractive index particles, meaning low contrast. The pigmented areas show up dark but other parts of the cells are difficult to see in normal bright field microscopy. CytoSense uses Köhler type oblique illumination to enhance contrast, as shown by the image of a Dolychospermium trichome with a surrounding mucilage sheath clearly visible.
Benefits of flow modulated laser scanning
Normal flow cytometer electronics can’t digest the scattering and fluorescence signals of long and big particles. CytoSense electronics differ; they continuously record the signals from each particle while passing through a thin sheet of laser light in flow controlled direction and velocity. This gives a projection or scan along the length axis of the particles. Data output is provided in standard flow cytometry format (scatter plots) but each data point expands to the full recorded signals.
a more simple pulse shape of a 29µm particle (on background photo)
a more complex pulse shape of 199µm particle (on background photo)
Quality 1: high sensitivity
Small and dim particles are detected by the sharp laser focus. Mathematical background reduction in the scans allows better separation from background noise (100 nanometer polystyrene beads detected by sideward scatter). All particles above sensitivity level are detected – none left out, with less coincidence by true zero dead time electronics. Concentrations of up to 1011 per liter can be analyzed at an acquisition rate of 7,000 particles per second with coincidence less than ca. 10%.
Quality 2: large dynamic range
Scanning electronics provide linear acquisition of the light scattering and fluorescence signals – from small (0.1 µm) to big and long particles, filaments and organisms, up to >2500 µm in length, provides a very large dynamic range. This allows reliable determination of aggregates size and length, and the amount of single cells per colony for instance for Microcystis sp. Colonies of thousands of single cells are recorded reliably.
CytoSense analysis of aggregate formation of Scenedesmus cells by a poly-electrolytic starch derivatie
Quality 3: morphological information
Particle morphology is increasingly clear for particles over ca. 10 µm, giving robust 1-dimensional information such as localisation of pigments. Particle lengths are directly determined with high accuracy (R² = 0,99 scans versus photo’s). Note the large dynamic range in fluorescence levels, whereas these signals can be recorded up to a particle length of 2500 µm.
Signal scan (with photo) of a 0.5mm long Thalassiosira sp. chainform colony.
Quality 4: facilitates targeted imaging
The scan data allows highly complex decisions to be made in a millisecond for each particle, basing on multiple scattering and fluorescence properties of the particle, and its detected position in the flowcell. This allows realtime decisions to target any specific user defined particle group for taking photographs.
Quality 5: fast and precise
The highly condensed informative data format allows fast analysis (thousands of particles per second), fast processing, high discrimination clustering and classification, and basic biovolume determination.
The quality of cell and particle counting
The overall counting quality is determined by an unbiased particle size range, high sensitivity, absence of redundancy (coincidence) and the prevention of particle loss.
Size dependency by detection limit
The bias effect of flow cell/orifice dimensions on the counting of particles is a less known phenomenon. Particles smaller than the limiting orifice are expected to enter the device freely, but are partially blocked in practice except for the very small particles. Blocked particles remain uncounted, limiting the accurate determination of particle size distributions (PSD) to ca. 1/3 of the flowcell size or inlet mesh, as demonstrated (graph). Therefore the CytoSense has a Ø 1mm inlet for reliable counting of all particles up to 300µm. Particles up to ca. 800 µm may enter but access is increasingly selective above ca. 300 µm.
Dependency of the detection
Small particles may remain uncounted if their scatter or fluorescence signal is too low to surpass the detection threshold of the instrument. CytoSense main particle detection mechanism is light scattering, common to all particles, and it is sensitive enough to detect and count individual particles of 0.1 µm (polystyrene). This implies another benefit: when counting particles basing on their fluorescence, those with very dim or almost no fluorescence are detected and not overlooked!
Comparison with other devices
Various comparisons showed that CytoSenses/Subs counting is equally accurate as compared to other good working (conventional) flow cytometers or other instruments like a ‘Coulter Counter’ (graph shown) for eligible particles.
Courtesy HH Jakobsen, Univ. Arhus.
Very high or low particle concentrations may limit accuracy. CytoSense sample dosing rate is highly adjustable which facilitates reliable counting from low to very high concentrations (up to 6 orders of magntude).
Comparison of CytoSense counting with microscopy
The basically different techniques of counting are well documented and reliable, however important prerequisites of their value are 1) cell integrity for recognition and 2) the statistics of the counting method in practice.
Traditional Uthermöhl inverted microscopy is most widely used for the counting of plankton and lugol’s fixative is mostly used to preserve the sample. This destroys pigment fluorescent properties of cells and morphology of various cells or parts of cells like fibrils of diatoms, dissolving calcified structures etc. This hampers recognition and therewith counting of these cells by such methods. CytoSense analyses fresh unpreserved samples without physical constraints to the cells, keeping them 100% intact. For example abundant Rhabdosphaera cells were seen in CytoSense images of Marseille and Crete coastal water whereas these were overlooked by inverted microscopy; in fact photo’s of such fresh cells are virtually non-existent.
CytoSense images of Rhabdosphaera cells (Crete and Marseille coastal water) with rhabdolith spines, haptonema and double flagella.
Counting statistics in practice
Large volumes of sample (like 1 liter) are typically used for inverted microscopy. In practice only a few hundreds of individual cells are actually being counted which limits the statistical significance for non-dominant groups severely. CytoSense analyses only a few milliliters but all individuals in the whole volume are counted. A comparison with river water (Afgedamde Maas) analysed both ways shows that the statistical significance of CytoSense counting for the less abundant species was roughly one to two orders of magnitude better (a few groups shown in the table).
Data approach and examples of data output
Getting level 1 data and level 2 data
CytoSense records multi-variate data from individual particles, cells or organisms (viz. fig. 14.). This level 1 data consist of the optical properties of the particle: its forward and sideward laser light scattering and its fluorescence in typicall three spectral bands. These signals are recorded during the passage of each particle, giving one-dimensional morphological and localization information as well as the totals and particle length. In addition a bright-field monochrome photo is captured. These data is captured for many individual particles and is subsequently clustered into several groups of more or less similar particles, e.g. certain size classes, shapes (for example filamentous) or fluorescence properties (for example blue-green algae) or even to the species level if the discrimination is sufficient.
Dotplot (left) with level 1 and 2 data and overview diagram (right) with level 3 data
The left diagram in fig.15 represents measuring results for a single run (sample nr. 12). It contains level 1 (green arrow) data: each dot represents an individual particle with its fluorescence values. Multiple correlated diagrams for the various measured entities can be shown. The values for selected groups (such as the red and blue ellipses) give the totals and averages for these groups representing level 2 (blue arrow) data. The right side diagram shows level 3 data: one of various entities (in this case the cell concentration) for two groups as found in a series of successive samples.
The interactive proprietary software program CytoClus can be used for this type of data analysis as shown in figs. 16. and 17.
CytoClus is an interactive data analysis program with multiple functions andf export features built-in, including some functions of EasyClus which allows automated clustering and classification.
CytoClus analysis with some example photo collections shown representing groups of particles in the data.
EasyClus is also available, based on years of experience with handling large amounts of flow cytometric monitoring data. It helps to process multivariate flow cytometric datasets automatically and efficiently. Each particle is characterized by signal profiles, like a finger print. EasyClus groups these fingerprints belonging to specific algae types, viz. fig. 18. Results can be validated by images. Groups of similar fingerprints are stored in a database. Cell sizes, biovolumes, cell concentrations, biomass, chlorophyll and biodiversity indicators are extracted from the cytometric data by EasyClus.