science

Microfluorimetry

Microfluorimetry is a science topic covered in the lgStudy science library. This page brings together a partial reference excerpt, illustrations, worked examples, real-world applications and a short study plan, so you can understand Microfluorimetry rather than just read about it. In short: Microfluorimetry is an adaption of fluorimetry for studying the biochemical and biophysical properties of cells by using microscopy to image cell components tagged with fluorescent molecules. It is a type of microphotometry that gives a quantitative measure of the qualitative nature of fluorescent measurement and therefore, allows for definitive results that would have been previously indiscernible to the naked eye.

Key takeaways

Microfluorimetry belongs to science; place it in that map before memorising details.
Learn the definition first, then one example that makes the definition concrete.
Connect Microfluorimetry to a quantity you can measure, compute or draw — that is where exam questions come from.
Reproduce the core statement of Microfluorimetry from memory before moving on to harder problems.

Reference excerpt

Microfluorimetry is an adaption of fluorimetry for studying the biochemical and biophysical properties of cells by using microscopy to image cell components tagged with fluorescent molecules. It is a type of microphotometry that gives a quantitative measure of the qualitative nature of fluorescent measurement and therefore, allows for definitive results that would have been previously indiscernible to the naked eye.

Uses Microfluorimetry has uses for many different fields including cell biology, microbiology, immunology, cell cycle analysis and "flow karyotyping" of cells. In flow karotyping, isolated metaphase chromosomes are stained and measured in a flow microfluorometer. Fluorescent staining of chromosomes can also give distribution about the relative frequency of occurrence and the chromosomal DNA content of the measured chromosomes. This technique allows for karyotyping at higher speeds than with previous methods and was shown to be accurate using Chinese hamster chromosomes. Flow microfluorimetry (FMF) can also be used to determine different populations of cells using fluorescent markers with small cell samples. The markers used for measurement in flow microfluorimetry are made up of fluorescent antigens or DNA binding agents. It allows for the accurate measure of an antibody reacting with an antigen. Flow microfluorimetry is also used in pharmaceutical research to determine cell type, protein and DNA expression, cell cycle, and other properties of a cell during drug treatment. For example, microfluorimetry is used in neurons to compare the effects of neurotoxins on both calcium ion concentration and mitochondrial membrane potential in individual cells. Microfluorimetry can also be used as a method to distinguish different microorganisms from one another by analyzing and comparing the DNA content of each cell. This same concept can also be applied to distinguish between cell types using a suitable fluorescent dye which varies depending on purpose and is a critical technique in modern cell biology and genomics. Another use of microfluorometry is flow cytometry which uses the emission of fluorochrome molecules and usually a laser as a light source to create data from particles and cells. It can be used to separate chromosomes at a very high rate and used easily with next-gen sequencing. This technique can simply results by separating only the relevant chromosomes at a very fast rate. For example, E. coli bacteriophages lambda and T4 were able to be separated by flow cytometry which allowed for genomic analysis which was previously difficult.

Concept Microfluorimetry is building upon the established method of fluorimetric measurement. Using a dye that fluoresces in the presence of a target compound, fluorimetry can detect the presence of the compound by determining the presence and intensity of fluorescence. Differences in the intensity can be used to determine concentration of the compound. Additionally, if the dye undergoes a spectral shift then you can determine the absolute concentration of the target regardless of knowledge of the concentration of the dye. Fura-2 is an example of a fluorescent dye used to measure calcium. Microfluorimetry expands on fluorimetry by adding a microscopic component to measurements to allow analysis of single cells and other microscopic interests.

Microfluorometer A microfluorometer is a fluorescence spectrophotometer combined with a microscope, designed to measure fluorescence spectra of microscopic samples or areas or can be configured to measure the transmission and reflectance spectra of microscopic sample areas. It can either be a complete microfluorometer built exclusively for fluorescence microspectroscopy or the fluorescence spectrometer unit which attaches to the optical port of a microscope. A microfluorometer can be used to estimate amounts and distributions of chemical components in individual cells or in chromosomes. In order to estimate the amount of chemical components, its fluorescent intensity is measured by photoelectrical photometry while distribution is found by measuring the intensities of photos of negative chromosomes' metaphase plates. A microspectrophotometer can measure transmission, absorbance, reflectance and emission spectra then using built in algorithms a spectra is produced that can be compared against previous data in order to determine composition, concentration, etc.

Limitations There are many sources of error in the process but biological errors such as an inability to prepare homogenous samples are more likely to be a limitation than technical errors.

See also Fluorescence spectroscopy, for a complete discussion of fluorescent measurement. Fluorometer, for a fuller description of the devices for fluorescent measurement.

References

External links http://microspectra.com/component/content/article/52-craictech/81-microfluorometer, Microfluorometer https://www.youtube.com/watch?v=_lmkEqTHj3Y, A video explanation of an application of microfluorimetry http://www5.pbrc.hawaii.edu/bln/ian/imaged_microfluorimetry.html, A readout from microfluorimetry

Illustrations

Microfluorimetry: Schizosaccharomyces pombe fluoresced with dihydroethidium — Schizosaccharomyces pombe fluoresced with dihydroethidium

Worked examples

Example 1 — a first encounter with Microfluorimetry

Start with the simplest possible case. Write down what Microfluorimetry claims or describes in one sentence, then invent the smallest concrete situation in which that sentence is true. In science, the smallest case is usually a single object, a single equation or a single measurement. Check that every symbol or term in your sentence has a meaning in that case.

Example 2 — changing one variable

Take the situation from Example 1 and change exactly one quantity: double it, halve it, or set it to zero. Predict what should happen to Microfluorimetry before you calculate. Comparing your prediction with the result is the fastest way to find out whether you understand the idea or only the words.

Example 3 — an exam-style question

Typical questions about Microfluorimetry ask you to (a) state it precisely, (b) apply it to given data, and (c) explain a limitation. Practise writing all three answers in under five minutes; the third part is what separates a full-mark answer from an average one.

Applications of Microfluorimetry

In research: Microfluorimetry appears in science research whenever the underlying quantities have to be modelled precisely. Papers usually cite it as a starting assumption and then explore where it breaks down.
In technology and industry: Engineering practice reuses Microfluorimetry in design rules, simulations and safety margins. Knowing the idea lets you read a specification sheet and understand why the numbers look the way they do.
In the classroom: Microfluorimetry is common in secondary-school and first-year university syllabi. It links to neighbouring topics Microscopy, so understanding it makes those chapters shorter.
In everyday life: Look for Microfluorimetry outside the textbook — in sport, cooking, traffic, electronics or the sky above you. An example you found yourself is remembered far longer than one you were given.

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How to study Microfluorimetry in 20 minutes

Read the reference excerpt below once, without taking notes.
Close the page and write down what Microfluorimetry means in your own words.
Compare your version with the excerpt and mark what you missed.
Work through the three examples above with pen and paper.
Explain Microfluorimetry out loud to somebody else — or to Teacher Smith in the lgStudy chat.

Frequently asked questions

What is Microfluorimetry in simple terms?

Why does Microfluorimetry matter?

Because it connects several science ideas at once: it gives you a definition you can apply, a quantity you can calculate, and a way to check whether a result is plausible.

How should I study Microfluorimetry?

Read the excerpt, restate it from memory, then work through the examples and applications listed on this page. The five-step study plan above takes about twenty minutes.

What does this page cover?

It gives you a compact reference excerpt plus original lgStudy explanations, examples, applications and study material on Microfluorimetry.