CONTENTS
CONTENTS
Cytometry refers to the measurement (-metry) of cells (cyto-). These measurements may be of the cell's physical properties (length, volume, etc.) or of its biochemical properties (protein content, lipid content etc.) Traditionally these measurements have been made by light or fluorescence microscopy. Microscopy is a labour-intensive task, prone to error and operator fatigue. It is also slow to perform and consequently conclusions are drawn from measurements of, at best, a few hundred cells. An alternative method of measuring protein content for example, would be to perform bulk biochemical measurements on a population of cells. However in doing this one makes the assumption that all of the cells in a population are behaving in a similar manner. The extent to which this assumption is true will vary with the cell type under investigation but in many cases the heterogeneity of a population of cells will be large, not least because of changes that occur as the cells progress through the cell cycle.
The term flow cytometry refers to these same physical
and biochemical measurements however, the measurements are made as the cells
flow past an array of detectors. In microscopy a sample is placed on a
microscope slide and the objective lens ("detector") is moved over the sample;
in contrast in the flow cytometer the detectors are fixed in position and the
sample is moving. Typically, the cell sample is introduced into the centre of a
stream of sheath fluid. The sheath fluid is pumped much more quickly than the
sample and so the cells are constrained to the centre of the sheath fluid. This
process, known as hydrodynamic focusing, allows the cells to be delivered
reproducibly to the centre of the measuring point. At the measuring point (Fig
1) the stream of cells intersects a beam of light. In most flow cytometers the
light source is a laser or an arc lamp. In the Microcyte a laser diode is used;
this reduces the size, weight and cost of the flow cytometer.
When cells interact with the light from the laser diode some of the light is scattered out of the beam. A detector in the Microcyte collects light scattered in the forward direction. The amount of scattered light in this region gives a measure of the size of the scattering particle. In addition to light scattering, the Microcyte also measures particle-associated fluorescence. Fluorescent stains that bind specifically to a cellular component (e.g. DNA) may be added before analysis. Alternatively stains that are excluded by living cells but taken up by dead cells may be used. In this way two parameters can be measured simultaneously.
Flow cytometry offers several advantages over conventional methods. The problem of sample heterogeneity was mentioned above. In the flow cytometer measurements are made on individual cells and so sample heterogeneity can be quantified. This means that under some circumstances it may be possible to identify subpopulations of cells or to detect contaminants. Flow cytometry is a rapid technique; measurements are typically made at rates of 1000 cells.s-1. This means that many thousands of cells can be measured in a realistic time scale.
Haemocytometer counts are laborious to perform, lead to operator fatigue and are prone to error. For a count of n organisms the percentage error may be represented as %, i.e. ±1% for a count of 10,000 counts. Rapid sample analysis by flow cytometry permits analysis of >10,000 cells per sample, in most situations it is unlikely that more than 1000 cells would be counted by haemocytometry (>3% error).
Plate counts, although the "gold standard" in microbiology, detect only those cells capable of growing under the conditions provided (pH, carbon source, incubation temperature etc.). As such they give an underestimate not only of the total count but also of the true viable count. The Microcyte detects all particles based on their light scattering properties. There is no requirement that the cells will grow on a designated medium or even that they be alive. This total count can be combined with a viability stain (see below) where a measure of the viable microbial load is required.
Most of the advantages here are related to those given under haemocytometer counts above. In addition, a suitable fluorescent stain may be added to the sample prior to analysis enabling simultaneous determination of both total and viable counts.
A common method of particle counting is to use systems based on the Coulter principle. However the Microcyte has been designed with microorganisms in mind and, unlike the Coulter counter, has a large dynamic range and in the same sample can count (and size) particles from as low as 0.4 um diameter to 15 um. The Coulter counter can determine the concentration of appropriately sized particles but unlike the Microcyte it cannot simultaneously monitor the viability of the sample. Where viability is not an issue the fluorescence parameter of the Microcyte may be used to make other physiological measurements such as determining the DNA content of the particles - this may be useful for distinguishing between biological and non- biological particles.
The majority of flow cytometers are designed primarily for the analysis of mammalian cells (~10 um diameter) and do not function optimally with bacteria (~1 um diameter); in some instruments bacteria are on the limit of detection and difficult to distinguish from background noise. The Microcyte has been designed to count and size particles between 0.4 and 15 um, and the background has been reduced to allow bacteria to be detected easily.
The Microcyte is much smaller than any other commercially available flow cytometer. With dimensions of just 330 x 430 x 160 mm and a weight of just 10 kg the instrument is fully portable. It can be powered from its own rechargeable internal batteries for several hours or by an external 12V D.C. source making it ideal for both field and laboratory applications.
The optical components of the Microcyte are enclosed in a solid aluminium block and so do not need to be aligned (daily alignment is necessary in most other flow cytometers). Internal feedback ensures the stability of both light source and detector - no photomultiplier voltages need to be adjusted and there are no lamp deterioration effects. This makes operation of the Microcyte much simpler, the operator simply switches on the instrument and it is ready for use.
The price of the Microcyte compares extremely favourably with that of other flow cytometers.
A wide variety of samples are amenable to flow cytometry. However, the operator must ensure that there are no large clumps of cells present in the sample as these could block the flow cell. If reliable measurements are to be made then the sample must be presented as monodisperse cells, rather than as clumps. Two cells that are stuck together will appear as one large cell to the flow cytometer.
For reliable counting the particle concentration should be between 1 x 103 and 1 x 107 / ml. With the higher concentrations the count is done on 1 ul, while for lower concentrations the measurement time can be increased so that the number of particles in 5 or more microlitres are counted. For more information on counting particles with the Microcyte see Application sheet 1
The operator must ensure that no large clumps of particles are present for the reasons described above. The sample must not be too concentrated nor too dilute to allow reliable counting without coincident events. If the sample is too concentrated the Microcyte will display a warning message on the screen, while if it is too dilute then no particles will be detected. If a fluorescent stain is required then the operator must add this before analysing the sample. To run the sample on the Microcyte an Eppendorf-type tube containing at least 0.2 ml is simply placed into the sample holder and the run button is pressed.
Particles in the size range 0.4 to 15 um can be counted. The Microcyte has been tested with bacteria, yeast, animal cells and latex beads.
Light scattering measurements have found many applications in biology, ranging from the assessment of bacterial concentration in a suspension to resolution of the fine structure of the cell. Optical density measurement is a common method for the estimation of microbial biomass. When light interacts with a cell suspension some of that light is scattered out of the incident beam, while some is absorbed. The more concentrated the suspension, the more light is absorbed and scattered and thus biomass concentration can be estimated by measuring the amount of light that is transmitted through the suspension. As mentioned earlier, however, the special power of flow cytometry is its ability to make measurements on single cells rather than on populations of cells. When a single cell intersects the light beam of a flow cytometer some of the light is scattered out of the beam. The amount of light that is scattered by a cell is a complex function of its size, shape and refractive index. The sensitivity of light scattering to each of these factors is dependent upon the range of angles over which the scattered light is collected. Light scattered at small angles (i.e. forward light scatter as detected in the Microcyte) is most dependent upon the size of the scattering particle.
When a compound absorbs light electrons are raised from the ground state to an excited state. The electrons return to the ground state via a variety of routes; some processes such as the loss of the energy by heat do not result in fluorescence, but certain molecules also lose energy by a process of radiative transition (fluorescence). Fluorescence is always of a lower energy, and hence longer wavelength, than the exciting light, and this separation in wavelength is known as the Stokes shift. The Stokes shift enables the exciting and emitted light to be separated by optical filters and thus the amount of fluorescence can be quantified. While every cell in a population will scatter light not all cells will necessarily fluoresce significantly; therefore light scattering, together with fluorescence measurements are useful to give a measure of the percentage of fluorescent cells in a population.
A range of stains that can be excited at 635 nm can be found in the following table. Those shown in bold type in the table below have been tested successfully.
| Fluorescent Stain | Excitation (nm) | Emission (nm) | Reference(s) (see notes) | Determinand |
|---|---|---|---|---|
| TOTO-3 iodide | 642 | 660 | MP 17 / T-3604 | Nucleic acid |
| TO-PRO-3 iodide | 642 | 661 | MP 25 / T-3605 | Nucleic acid / viability |
| Oxazine 750 | 673 | 691 | 1, 2 | DNA |
| LD700 / rhodamine 700 | 1 | DNA | ||
| rhodamine 800 | 685 | 700 | 1, 2 | DNA |
| Cy5 | 3 | |||
| Napthofluorescein | 594 | 663 | MP 16 / N-650 | Enzyme substrate |
| Allophycocyanin | 650 | 660 | MP 14 / A-803 | Antibody label |
| polymethine cyanine dyes | ||||
| 1,1'-diethyl-3,3,3',3'-indodicarbocyanine iodide (DilC2(5)) | 636 | 657 | MP 23/D-1124 | Membrane / membrane energisation |
| 3,3'-diethyl-thiadicarbo-cyanine iodide (DiSC2(5)) | 649 | 671 | MP 23 / D-304, see also D-324, D-306, H-380 | Membrane energisation |
| Oxonol-V | 610 | 639 | MP 23 / O-266 | Membrane energisation |
| 5-(and-6)-carboxy-napthofluorescein, succinimidyl ester | 600 | 672 | MP 5 / C-653 | |
| 1,9-dimethylmethylene blue | 650 | 674 | MP 29 / D-665 | |
| DMOTC | 682 | 718 | 2 | |
| Oxazine 720 | 618 | 640 | 2 | |
| Nile blue | 640 | 672 | 2 | Lipid |
This page was written by Hazel Davey hlr@aber.ac.uk
Author: Hazel Davey
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