New instrument and software: Is it ready for use?
Yes, it works really well. The software starts within the blink of an eye, is very intuitive and data analysis (gating, axis setting, advanced view options like point size and plot density coloring) is very easy. With the processing capacity that is now in the instrument, there is now less communication between instrument and computer, resulting in a smooth experience.
Can we use the new software (AmphaSoft 2.0) on our Ampha Z30 instrument?
The new software requires new hardware. It therefore only works with the new Ampha Z32. However, existing Ampha Z30 can indeed be upgraded for full compatibility with an Ampha Z32 instrument.
In the beginning 0.5 MHz was recommended for pollen viability assays. Now it seems to be 2 MHz Is this true for all species? What is it based on?
Initially we measured pollen at a carrier frequency of 0.5MHz and this was implemented as the default setting for the Ampha Z30. Now, with the new Ampha Z32 instrument we achieved a higher signal sensitivity at 2MHz by further tuning the electronics. Therefore, we recommend to use the default frequencies, which is 2 MHz for Ampha Z32 (and upgraded Ampha Z30 instruments). For Ampha Z30 it remains 0.5 MHz.
When changing the first frequency in the parameters we observe clear changes in the plots that are obtained. What happens?
The first frequency, called carrier frequency, should always be left at the default frequency (see above). Please note that when you change this carrier frequency, you may also have to change other settings due to the complex nature of the impedance signal. The second, third and fourth frequencies can be chosen freely. More frequencies are used if you are interested in more information. For small cells the sensitivity can be optimized by using only one or two freqeuncies. For pollen applications we recommend to always use the default settings (see pollen list).
Are there any markers needed to label the cells before the measurement?
Ampha Z32 works without labels, markers or dyes of any kind. It is based purely on electrical characteristics of the cell which vary dependent on the state of the cell (like viability or maturity stages of pollen/spores). Therefore, no dyes or pre-treatments are needed and no fading of fluorescent chemicals may occur. All you have to do is collect pollen, dilute them in aqueous buffer solution and measure.
How does it work? How can viable cells be discriminated from dead cells without labeling?
The measurement is purely electrical, there are no optical parts. Therefore, no dyes are used. The electrodes in the chip detect the electric characteristics of cells as a response to the applied high-frequency field. Cell size, cell membrane activity (ion pumps) and cell interior (e.g. vacuoles or lipid enclosures) influence the resulting impedance. The effects of these cell characteristics depend on the frequency of the applied alternate current field which in turn allows the detection of such characteristics at varying field frequencies. Ampha Z32 measures up to four frequencies at the same time. For more details see the technical description of impedance microflow cytometry.
How long does a measurement take and how many cells are needed for a good measurement?
In general, a measurement takes about 30 seconds to a minute (depending on the concentration of cells in the solution and the number of cells you wish to measure). A reasonable measurement rate of Ampha Z32 is about 1’000 cells per second. As a rule of thumb for pollen viability we have obtained best results with samples at a concentration of about 100 to 1'000 cells per µl. The optimal concentration depends on the size of the cells.
How shall I determine the concentration of cells in my sample?
Ampha Z32 contains a precise built-in flow sensor. Sample volume and cell concentration are indicated during the measurement.
How many measurements can be performed with one chip?
With proper care which includes a thorough rinsing with Amphasys cleaning solution after every series of experiments and prefiltering of samples with an adequate filter, a chip can be used for up to a few hundred measurements.
In our experiments we have seen a beneficial effect of adding Tween to the buffer. This particularly reduced the clogging of the tubes and chip. Has Tween an effect on the results of Ampha Z32?
To reduce aggregation of sticky or oily pollen we recommend to use Tween 20 or 80 in low concentration at a final concentration of 0.05%. Do not use high concentrations, as these will dissolve the cell membranes and cause cell death. There is no negative effect on the measurement from a technological point of view.
Do you think the procedure and equipment for pollen analysis is robust enough and portable enough to be used in or close to the field?
The new instrument was designed to be used as a mobile device. With a weight of about 8 kg and a size of 25 x 27 x 35 cm the instrument can easily be moved and be placed at virtually any place (see our brochure for more technical details). Because Ampha Z32 contains no optical or other sensitive parts it is very robust and does not need any calibration before starting the experiment. The setup of the equipment after transport only takes a few minutes. Equally important, it runs on 24 V DC which can also be supplied by a battery. Rechargeable batteries or connector cables to a truck battery are available as accessory parts.
Which cells can be measured?
As a precondition they have to be single cells in aqueous solution. The only other limit is the cell size which naturally needs to be smaller than the microfluidic channel in the chip. Currently, chips are available with a channel size from 15 to 250 μm. Therefore, virtually any kind of single cells from humans, animals or plants (including pollen), as well as yeasts (including spores), bacteria, algae, and other micro-particles with a size ranging from about 1 to 150 μm can be measured. See list of publications for examples.
Can you distinguish between bacteria, yeast and other cells?
We can distinguish between viable or dead bacteria, yeast, cells and also non biological particles. Discrimination of species with the same size and similar characteristics is usually not possible.
Can you discriminate between different development states of pollen?
We are able to detect different development stages of pollen (ranging from uni-nucleate to tricellular).
Is it possible to determine ploidy?
Our experiments indicate that under certain conditions differences in ploidy can be detected.