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Different techniques are needed to measure various aspects of a cell and its processes. For example, researchers may want to measure cell confluency, which describes the percentage of your culture dish the cells occupy or the volume of red blood cells, representing the ratio of red blood cells to the volume of whole blood. These require entirely different protocols, which we will explore here.
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Jetzt kostenlos anmeldenDifferent techniques are needed to measure various aspects of a cell and its processes. For example, researchers may want to measure cell confluency, which describes the percentage of your culture dish the cells occupy or the volume of red blood cells, representing the ratio of red blood cells to the volume of whole blood. These require entirely different protocols, which we will explore here.
This article is advanced, so you might find you only need to know one or just a few of the following measurements
As its name suggests, cell mass refers to the mass of a single cell or group of cells. Cell mass measurement is not as simple as placing your sample on a normal weighing scale because living cells lie within the picogram range. Instead, researchers use a specialised picobalance that uses a cantilever, a structure containing a horizontal beam or element supported only on one end. By attaching your cells to one end, you can quantify the mass change and calculate the cell mass.
Using this picobalance technology, researchers have shown that the mass of murine fibroblast cells ranges from 12 to 15 picograms.
Cell confluency is a percentage of a given area covered by cells. This method evaluates the growth of your cultured cells over time, and this property can be measured via visual or automated methods.
Visual estimations prove subjective and will inherently introduce variability. This method includes taking an image of your cell sample and estimating how many cells cover a given area. However, measuring confluency by automated means is the preferred option by scientists, as this method returns accurate data and is quicker than visual estimates. Machines such as Solentim Cell Metric can determine confluence by taking an image of your sample and analysing the area of adherent cells.
Fig. 1 - Four different confluency of cells under the microscope
Cell size measurement is often performed using a graticule (also called a reticule), which contains a scale with 100 divisions on the eyepiece of a light microscope. A stage micrometre, a slide containing a scale of a known length, must be used to calculate how many micrometres are contained within one division on the graticule. Once you calculate this, you can measure the size of any cell.
A graticule is an apparatus that contains a scale with 100 divisions and is placed on the microscope eyepiece. A stage micrometre is a slide containing a known length scale to calibrate a graticule.
As mentioned above, you need a stage micrometre to calibrate your graticule. Outlined below are the steps:
Fig. 2 - Three fields of view are seen in this figure. A) An eyepiece graticule B) Superimposed cells and an eyepiece graticule C) A micrometre and an eyepiece graticule
Suppose you use a graticule that contains 100 divisions, a stage micrometre that contains 100 divisions of 10 μm and a magnification of 4x. Suppose the full length of the graticule spans just 20 divisions of the stage micrometre. Thus, the full length of the graticule is:
20 divisions x 10 μm = 200 μm
I.e., 100 graticule divisions represent 200 μm and therefore:
1 graticule division = 2 μm.
This value represents your magnification factor, and as long as you use the same magnification on your microscope, you can measure the length of your specimens in your sample. If you measured your cell length to be 2 graticule divisions, then we can calculate the true cell length by using the following equation:
True size (μm) = number of graticule divisions x magnification factor.
In this case, the true cell size is 2 x 2 = 4 μm.
Your blood contains many other cells and components alongside red blood cells. These components include platelets, leukocytes and plasma. Red blood cell volume describes the ratio or percentage of red blood cells to the volume of whole blood; this is most commonly known as haematocrit. In healthy adults, this value should range from 40 to 48%.
Fig. 3 - Separated content of a blood sample
Patients will have their blood drawn in clinics and hospitals, and automated haematology analysers will return the haematocrit count.
A low haematocrit count indicates an abnormally low percentage of red blood cells and that the patient may be experiencing:
A high haematocrit indicates an abnormally high percentage of red blood cells and that the patient may be experiencing:
Dehydration can cause an increase in the haematocrit count as it causes plasma levels to decrease. As the percentage of plasma decreases but the red blood cell count remains the same, the haematocrit ratio will increase.
Polycythaemia vera is a blood disorder in which an individual's bone marrow makes too many red blood cells. This disorder is caused by mutations in the JAK2 gene and leads to the individual's blood becoming too thick, making them prone to blood clots and strokes.
Fig. 4 - Those with Polycythaemia Vera have too many red blood cells in their blood
Cell migration describes the rate of cell motility. This measurement is key as motility is involved in important processes such as tissue development and immune responses. There are different types of assays that researchers use to measure cell migration, such as:
Here, we will focus on the pipette scratch assay and transwell assay.
The pipette scratch assay involves manually creating a wound with a pipette in cell culture. The cells found on the edge of the new wound will migrate towards the cell-free gap. Images are taken at regular intervals throughout the assay duration to calculate the rate of cell migration.
The transwell invasion assay involves a chamber containing an upper and lower layer separated by a permeable layer coated with extracellular matrix proteins, such as collagen. Cells are seeded ("planted") onto the upper layer while the lower layer is filled with media containing a chemoattractant. Over time, the cells will migrate through the membrane, and the rate of cell migration is calculated by counting the migrated cells with a plate reader.
A chemoattractant is a chemical that induces cell motility according to its chemical gradient. Cells can migrate away or towards the chemotactic gradient.
The cell density of a single cell refers to its mass to volume ratio. Researchers often use this measurement to monitor the progress of processes such as cell proliferation and apoptosis.
A microfluidic mass sensor (MMS) measures cell density, also termed a suspended microchannel resonator (SMR). This machine can hold two fluids of different densities; one fluid being the cells under investigation and the other fluid being denser. The fluid density and buoyant cell mass are calculated, which allows the machine to calculate single-cell density.
Fluid density describes the mass of fluid in a given volume, while the cell buoyant mass refers to the change in cantilever mass as a cell travels across a channel. These measurements are beyond your specification, so you do not need to know these definitions in detail!
Cellular metabolism refers to the sum of all biochemical processes that occur in a cell, including catabolic and anabolic reactions. These processes include cellular respiration, protein synthesis and cellular trafficking. In particular, metabolic reactions change and deviate from the norm in disease states. As you can perhaps imagine, many reactions occur simultaneously; we will take a look at a couple of examples.
Catabolic reactions describe the breakdown of complex molecules into smaller subunits and the release of energy. On the other hand, anabolic reactions involve building complex molecules from smaller subunits and the consumption of energy.
Proinsulin is the precursor molecule of the more well-known hormone, insulin. Proinsulin is split at two sites to give rise to insulin, which regulates the cellular uptake of glucose. Only trace amounts of unsplit proinsulin are released into the bloodstream in healthy individuals. However, researchers have found that high levels of circulating unsplit proinsulin indicate insulin resistance, specifically in type-II diabetes.
Enzyme-linked immunosorbent assay (ELISA) kits measure the levels of unsplit proinsulin in clinical samples. These assays use antibodies and fluorescence-based technologies to quantify the level of proteins in a sample. By using these kits, you can measure the levels of circulating proinsulin to monitor proinsulin metabolism.
Oxygen is a key substrate in aerobic respiration, and the rate of oxygen consumption is a great indicator of a cell's state. For example, a low oxygen consumption rate may indicate mitochondrial dysfunction. In laboratories, researchers use automated machines to calculate the oxygen consumption rate.
Machines, such as the Seahorse Extracellular Flux Analyser, quantify mitochondrial respiration by reporting the oxygen consumption rate. By using fluorophores sensitive to pH, any changes in oxygen concentration are detected, and the oxygen consumption rate is monitored over time.
Cell mass can be quantified using picobalance technology.
Cell confluency can be measured by automated means whereby a machine takes images of your adherent cells and calculates how many cells are present in a given area.
Cell size can be measured using light microscopy and graticule calibration.
Red blood cell volume, otherwise known as haematocrit, can be measured using haematology analysers and indicates patient health.
Other cell measurements, such as cell migration, density and metabolism, can be measured and monitored using assay kits and specialised machines.
Cell migration is measured using assays such as the pipette scratch assay, transwell invasion assay and culture insert assy.
Cell density can be measured by using machine technologies such as microfluidic mass sensors. These machines calculate single-cell density by using fluid density and cell buoyant mass measurements.
Cellular metabolism refers to the sum of all catabolic and anabolic reactions that occur simultaneously in a cell.
Oxygen consumption, for example, can be monitored by using machines that use pH-sensitive fluorophores. Any change in oxygen levels will indicate the oxygen consumption rate.
Different cell properties, such as cell length and cell confluency can be measured and quantified.
For example, to measure cell length under a light microscope, you must first calibrate a graticule using a stage micrometer to calculate your magnification factor. Using this magnification factor, you can measure the true length of a cell.
You must first use a stage micrometer to calibrate a graticule. By aligning both pieces of apparatuses, you can calculate your magnification factor. This magnification factor can be used to calculate the true cell length by using the following equation:
true cell size (um) = number of graticule divisions x magnification factor.
Flashcards in Measuring Cells24
Start learningHow can cell mass be measured?
By weight or by density
What is cell confluency
The percentage of cells in a defined area
Why do we measure cell confluency?
To determine the rate of cell growth
How is cell confleuncy measured?
Visually by counting or with specific software
What do we need to measure cell size?
Graticule and stage micrometer
How do you calibrate a graticule?
This calibration only works for one lens/ magnification
Line up scale on the graticule and stage micrometer
The length of the devision needs to be calculated based on the micrometer
The units are compared e.g. 10 units on the micrometer are equivalent to 50 units on the graticule
If one unit on the micrometer is 10µm then one unit on the graticule is 2µm
when changing the magnification of the lense it needs to be divided by how many times the new magnification is greater so the measurement on the graticule needs to be divided by 10 if the calibration was done at 40x but is now at 400x
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