The CytoSense focuses on properties of individual particles, in large numbers. Bulk analysis technologies on the other hand analyze a whole volume of sample directly. This is sufficient if you want total particulate matter or chlorophyll, or a crude size distribution. Things change if you want to determine the composition of a mixed particle suspension or microbial sample accurately. In aquatic ecosystems you might even want to detect and analyze less abundant, emerging, or rare species amongst the dominant groups. Now you will need to analyze a large number of single, individual particles - other methods will fail. Just like traditional microscopy basically, but if you want your results fast and frequently you will need an automated single particle analysis instrument: our CytoSense flow cytometer!
In flow cytometry, a stream of particles and/or cells in water is directed at high speed through a powerful laser beam. The stream is so narrow that the particles and cells pass in single file, one by one. By measuring the scattering of the laser light while each particle passes, and detecting possible fluorescence emitted by intracellular pigments, the particles can be identified and counted. This is an extremely fast and extremely accurate method. Multiple thousands of particles per second can be analyzed, and each particle leaves a footprint characteristic for its shape and internal structure.
A traditional flow cytometer produces a small set of numbers for each analyzed particle. Such a set consists of the output values of each detector, typically one forward scatter and one sideward scatter detector and three fluorescence detectors for the 'green', 'yellow' and 'red' parts of the spectrum. In this case these 5 numbers are captured for each particle, generating a listmode data file: long lists of numbers representing particles. This is socalled multivariate data because each data set represents information from five different detected optical entities or 'variables'.
Not so much the CytoSense. In stead of single values per particle, the machine records a whole footprint of data per particle, a silico image.
Particles of a certain kind of species have more or less similar optical properties. Big particles generally scatter more light than small particles, so for finding a group of a big cells in the data we need to find particle sets having at least a high forward scatter number and presumably with a certain similarity. Because the chlorophyll in phytoplankton cells emits red light when passing the blue laser focus, we can use it to separate phytoplankton cells from zooplankton and sediment particles. Suppose we found a group of big cells and/or particles using their light scatter properties, the next step would be then to see what fluorescence numbers we find in their data sets: particles with a minimal red fluorescence number in their data set represent the phytoplankton cells. This process can be repeated for further discrimination of subgroups. With thousands of particles measured, a plain list of all these sets of numbers is cumbersome. These numbers are visualized therefore in multiple scatter plots and distributions. Selection of a 'cluster' or group in one plot immediately shows their position in all the other plots for further discrimination. More important: this type of analysis can also be automated!
The CytoBuoy instruments utilize a type of scanning flow cytometry (silico-imaging). Per particle complete pulse shapes are recorded, as opposed to single datapoints in conventional flow cytometers. This allows for better classification of particles, and detection of e.g. the location of specific internal cell structures.
Furthermore, the size range of the CytoSense is extremely large compared to other flow cytometers. Anything will be detected from roughly 1 micrometer or less, up to anything that will enter the 1 mm tubing. Such a range makes for a versatile and exceptional instrument. We designed CytoSense to allow autonomous and in-situ operation, in a shipboard suspension frame or in the special 90 cm spherical buoy (CytoBuoy) for instance, using radio or WiFi telemetry. A CytoSense equipped with a high pressure sample loop system and a heavy aluminium housing we call the CytoSub, for submerged use down to the full photonic zone of 200 meter - with powering and data solutions.
The process of scanning a particle by direct acquisition and digitizing multiple detector output courses while it traverses the laser beam focus we call silico imaging. It results in a set of complete pulse shapes for each detector output channel available in the instrument, for each analyzed particle. These pulse shapes are characteristic for the shape and functional components e.g. 'body parts' of the particle. Although this CytoSense data format yields much more data than the regular FCM listmode data, over 1000 scans (particles) per second can be acquired. Just imagine how much information this gets you about a sample!
Whilst capable of analyzing thousands of particles per second, flow cytometers only measure a few single optical entities per cell. Flow cytometric listmode data rates are vast and the CytoSense silico images are captured at rates over 1000/s. Capturing of real photo's at the same rate would easily flood a hard disc per day. However, photographs are very useful for visual identification of data groups. Therefore CytoSense can be upgraded with a so-called targeted imaging-in-flow module. This module takes a limited amount of pictures from preselected target groups in the data space. That target group can then be identified. This maximizes the information depth in a limited data transfer situation such as moored systems using data acquisition telemetry. Each photo comes with the the matching silico-image coupled to it.
The standard CytoSense instrument measures 31 cm in diameter, and 55 cm in height. When placed in its benchtop frame it has a footprint of 37cmx37cm, and height is increased by roughly 20 cm. The instrument weighs approximately 20 kg. These figures may vary with upgrades applied. The hull of a CytoSub does not increase the size of the instrument, but makes it somewhat heavier (it can still be carried around by a person though). In water it is approximately neutrally buoyant.
Yes, the CytoSub can be taken out of the high pressure hull and placed on the benchtop in a regular lab-hull with an adapter ring. The high pressure sample loop can be easily uncoupled and the instrument can be used as a normal benchtop CytoSense.
This page is never finished, so if you have questions about the CytoBuoy instruments which are not on this page, or want to inquire about the possibilities in your specific situation, don't hesitate to contact us.