Single Cell - Viability and Analysis - Ampha Z32 Impedance Flow Cytometry
i.e. Pollen Count and Viability Analysis
Single Cell Viability Analysis by Impedance Flow Cytometry - Ampha Z32
Amphasys’ new-generation flow cytometer is a plug & play device designed to blend simplicity with flexibility. This ‘next gen’ pollen analyzer offers technological advances that not only saves time (and therefore money) in the lab, but also enables higher quality pollen for greater yields. The absence of optical components minimizes set-up times and maintenance to a minimum. Time-consuming sample preparation procedures and expensive fluorescent dyes do not belong in Amphasys’ world, because they are exclusively founded on electrical measurements. Latest generation digital signal processing electronics are specifically tailored to Amphasys’ semi-disposable chips so that best-of-class impedance analysis on single cells is achieved.
The small dimensions of Ampha Z32 and the robustness of the electrical measurement provide a mobile and portable solution for any kind of cell analysis, be it in your laboratory or outside in the field. Designed initially for research purposes, Ampha Z32 is now particularly suitable for routine pollen analysis applications, where detailed, high-quality cell analyses and ease-of-use, are major requirements.
The heart of the cytometer consists of a microfluidic chip equipped with microelectrodes. This high-sensitivity sensor measures changes of the electrical resistance of the fluidic medium when cells or other particles pass through the applied AC electric field.
In order to ensure highest possible sensitivity, Amphasys has developed a number of chips with different channel dimensions ranging from 15 to 250 μm. The recent production of the new 250 μm chip marks a milestone in chip design and is a breakthrough for the application to large pollen. The whole range, from very small cells like bacteria, to very large cells such as certain pollen grains like pumpkin, maize or cotton can now be analyzed. The pollen analyzer’s chips can be used for up to several hundred measurements when treated with care.
Reagents and Disposables
Amphasys provides specialized reagents and consumables for many kinds of cells and applications, as well as cleaning solutions for the care of the fluidic system.
The electric signal recorded by the highly sensitive electrodes is influenced by the cell itself as well as the surrounding liquid. The contents of the buffers may have an effect on the biochemical behavior of the cells as well as the conductivity of the liquid, and therefore, on the signal. For these reasons, the conditions of the buffers including solvents, additives, salts, pH etc. have been optimized to keep cells viable and to simultaneously obtain the highest measurement.
As the pollen analyzer designed for flexibility, Ampha Z32 bridges the gap between the lab and the field. Its weight of about 8 kg and dimensions of 25 x 27 x 35 cm make it perfectly suited as a table-top lab instrument as well as a mobile field instrument. With the optionally available mobility package, including a 24 V rechargeable battery and a 24 V truck adapter cable, it can be used independent from a power outlet.
The instrument is of a robust build and can easily be transported in the trunk or on the back seat of a car. However, we also provide heavy duty aluminum transport casing if you plan to move the instrument on a regular basis.
To view tutorial videoes press here>>
Temperature, humidity and other factors like the application of pesticides or inbreeding depression, have a direct impact on pollen viability, quantity and seed yield.
Now, with the Ampha Z32, you can get a precise measure of the pollen viability along your pollen supply chain in the lab or greenhouse, while eliminating time-consuming and inaccurate germination and staining assays. Pollen viability staining is a traditional process that introduces a coloring agent to the substance, then applying heat. Viable pollen will turn various shades of warm colors, while ‘aborted’ pollen assumes a cooler tone. Historically, while pollen viability staining tests are quicker and easier than its predecessor, often germination tests are required in addition to staining to observe the actual viability of the pollen tested. The Ampha Z32 supercedes both those testing methods, providing fast, accurate results and improved workflows.
Increase breeding efficiency and get to market earlier through:
- Improving crossing success rates
- Maximizing the window for master lines
- Breeding for heat tolerance by selecting for pollen viability under heat stress
- Monitoring the sterility of cytoplasmic male sterile (CMS) lines
Hybrid Seed Production
Control over pollen quality and pollination along the pollen supply chain is a key success factor to ensure high quality seed and yield.
Excessive warm nights, droughts, rain, and pesticide applications negatively affect the pollen viability, quantity, and timing of pollen shed, leading to a reduced seed set and yield loss. Consequently, measuring and understanding pollen quality in hybrid seed production allows maximization of yield, improvement of the female to male ratio and forecasting of your yield. Further, the Ampha Z32 pollen analyzer optimizes the cross pollination process with the use of preserved pollen. Harvested pollen is analyzed by the Ampha Z32 to determine its quality. It is preserved, then prior to pollination, its quality can again be analyzed using the Ampha Z32 to identify the highest quality viable pollen to produce the highest yields.
Fruit and Nut Production
Effective pollination is indispensable to ensure high seed and fruit yield. Asynchronous flowering of pollinator trees in the orchard, rising temperatures and droughts, as well as the decline of natural insect pollinators severely affect this biological process and cause significant losses in tree nut and fruit production.
Challenges in Pollen Analysis
Traditional methods to determine pollen viability often rely on tedious sample preparation steps and labor intensive microscopy analysis.
With the Ampha Z32, sample preparation is just a simple resuspension and filtration that takes only minutes and works for all species (currently over 200 species tested).
Pollen Viability & Seed Set
Seed set in tomato as a result of pollination with pollen of varying quality.
Pollen viability measured on the Ampha Z32 before pollination: A = 31%, B = 3.5%, and C = 0.3%. The size of fruits resulting from pollinations with these pollen samples (pictured), as well as the seed set (graph) correlate with the pollen viability.
Other species show similar behavior, although each species has individual correlation patterns.
Pollen Viability & Germination
Pollen viability analysis with IFC and in vitro germination show an excellent correlation ( R2 = 0.96). While over 10,000 pollen per minute were analyzed with the Ampha Z32, only 100 pollen could be counted in the same time with the germination assay. This high count results in a standard deviation of <1% for IFC
Counting pollen as part of a pollen dispersion, pollen potential or pollen emission analysis is the standard method for calculating the number of males needed for pollination.
An accepted method relies on collecting the pollen in Isoton media for preservation and counting with a Coulter Counter. The comparison between the Ampha Z32 and the Coulter Counter shows that besides providing viability data, the Ampha Z32 can at the same time also be used to rapidly count large amounts of pollen.
Pollen Ploidy Analysis
Analysis of ploidy in Watermelon Pollen (Citrullus lanatus).
Diploid and tetraploid samples can be distinguished by analyzing their average size. Similar results were obtained for many other species like kiwi, tomatoes, potatoes, wheat, and cyclamen.
No time-consuming DAPI staining is required to determine the stage in microscope development anymore! IFC on the Ampha Z32 is so sensitive, it allows to distinguish between unpolarized and polarized microspores, as well as bi-cellular and tri-cellular pollen.
Besides staging, the Ampha Z32 allows early detection of induction success, allowing earlier selection of cultures. Optimize your DH induction protocols and monitor the induction success
Pollen Expert Blog
Double Haploid Plant Production - March 23, 2021
Overcoming the Pollination Gamble with Science - November 19, 2020
Measuring Wheat Pollen Viability - A Delicate Procedure?! - October 20, 2020
How Pollen Quality Affects Seed Set - September 22, 2020
Unravelling Hidden Secrets Behind Dead Tomato Pollen - June 4, 2020
Impact of Pollen Quality on Tomato Seed Production - February 28, 2020
|Available sensing channel dimensions||15 x 15 μm, 30 x 30 μm, 50 x 50 μm, 80 x 80 um, 120 x 120 μm or 250 x 250 μm
(depending on application requirements)
|Frequency range||0.1 MHz – 30 MHz|
|Frequency selection||Up to 4 different frequencies simultaneously|
|Sample volume||50-2000 μl (typically)|
|Concentration range||1 x 10(3) to 1 x 10(7)cells/ml|
|Particle size||1 - 60 μm (up to 150 μm with 250 μm chip)|
|Sample flow rate||5 – 2500 μl/min (depending on channel dimension)|
|Pump||Peristaltic pump with disposable pump head|
|Labware compatibility||Standard 5 ml polystyrene round - bottom tubes (Falcon® 352058)|
Operating System (min requirement)
|Laptop with Windows® 7 or 10|
|15” TFT LCD display|
|i5 or i7 QuadCore processor|
|128 GB SSD|
Dimensions & Weight
|Dimensions (W x D x H)||255 x 275 x 353 mm|
|Temperature||16° - 32°C|
|Humidity||10% - 90% relative non - condensing|
|Power||24V DC ± 10% , max. 3A, < 90 W|