Terms list

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Fluorescent proteins and dyes:

GFP – green fluorescent protein

RFP – red fluorescent protein

BFP – blue fluorescent protein

YFP – yellow fluorescent protein

CFP – cyan fluorescent protein

OFPorange fluorescent protein

GCaMP – GFP-calcium sensor (also available in other colors)

GECI – genetically encoded calcium indicator. A general term for fluorescent-protein based calcium sensors (e.g. GCaMP).

Initial “e” or “E” – enhanced

Initial “y” – Yeast, refers to protein adapted for expression in yeast.

Initial “m” – monomeric (e.g. mCherry)

Initial “d” – dimeric (e.g. dTomato)

Initial “td” – tandem (two proteins as a tandem repeat).

Initial “cp” – circularly permutated.

The Fruit series – usually RFP with fruit names (mCherry, dtomato, mPlum etc…)

FabLEM – Fab-based live endogenous modification labelling (FabLEM) are the antigen binding fragments (Fab) of a specific monoclonal antibody, conjugated to a fluorescent dye. The Fabs are small enough to enter the cell and the nucleus.

FlAsH – Fluorescein-arsenical helix binder. It is a small fluorescent molecule that binds to tetra-cysteine structures in proteins.

Fluorescent timers – fluorescent proteins or dyes that change their emission peak after a certain amount of time (minutes to hours).

Fluorescent sensors – fluorescent proteins or dyes that change their excitation/emission behavior upon environmental changes (e.g. pH, Ca2+ flux etc…).

HaloTagA protein fusion tag derived from the enzyme DhaA from Rhodococcus rhodochrous. The protein can covalently bind to a synthetic chemical ligand that can be labelled with a fluorescent dye.

LSS – large Stokes shift, a difference of >100nm between excitation and emission peaks.

Photoactivatable/photoconvertible – fluorescent proteins or dyes that require pre-activation with a certain waveband in order to emit light upon excitation (photoactivation) or convert to a different emission color (photoconversion).

QY – quantum yield, ratio between the number of photons absorbed to the number of photons emitted.

EC (ε) – Extinction coefficient, in M-1cm-1 units, is a measurement of the capability of a certain fluorescent protein to absorb light at a certain wavelength

Brightness – the multiplication of ε*QY. Can also be relative measure (usually relative to EGFP; EGFP=1).

Photostability – a measure (in seconds) of how long it takes for half of the number of proteins to bleach at the maximum excitation

Maturation rate – a measure (in minutes or hours)  for how long does it take a fluorescent protein to create the chromophore. Sometimes may include also time it takes the protein to fold.

Phototoxicity – toxic effects of light (commonly excitation light) on living cells or molecules in cells.

Photobleaching – the rate at which the excitation light destroys the fluorescent protein or dye. Depends on power and duration of excitation light.

pH stability – the effect of pH on the excitation/emission, brightness or stability of the fluorescent protein or dye.

SNAP-TagA protein fusion tag derived from the human enzyme O6-methylguanine DNA methyltransferase. The protein can covalently bind to a synthetic chemical ligand that can be labelled with a fluorescent dye.

Optics and spectroscopy:

Airy discs – point sources of light in the specimen (e.g. fluorescent proteins) appear as bright disks, surrounded by bright concentric rings.

NA – numerical aperture

SNR – Signal to Noise Ratio

Methods:

FISH – fluorescent in situ hybridization is a method to detect localization of nucleic acids (DNA, RNA) of specific sequences.

IF – immunofluorescence is a method that uses fluorescent dyes-conjugated antibodies to detect localization of specific epitopes in the sample.

FCS – fluorescent correlation spectroscopy, a method for high-resolution spatial and temporal analysis of low concentrated fluorescent bio-molecules.

FACS – fluorescence activated cell sorting, a method that uses flow cytometry, combined with fluorescence detection, to sort cells based on different properties.

FRET – Fluorescence resonance energy transfer, excitation of a donor fluorophore leads to emission of light in a waveband that excites another fluorophores. Close proximity (<10nm) is required.

FRAP – fluorescence recovery after photobleaching, following complete photobleaching of fluorophores at the studied region, the re-appearance of fluorophores in that region is measured.

Uncaging – Certain biologicaly active molecules can be “caged” by covalently bonding with another molecule. By uncaging, the caged molecule is released (the covalent bond is broken) by a source of light (usually UV). By using laser light, the molecule can be uncaged at a specific, small volume of choice (e.g. near the end of an axon).

TIRF – Total Internal Reflection Fluorescence.  An imaging method that relies on light reflected from the bottom part of the sample. Allows imaging to a depth of only ~100nm.

Image analysis:

ROI – region of interest

Voxel– volume pixel (a three dimensional pixel)

Z-sections – thin optical sections of the sample. Z-sections can be stacked into a projection of the sample, or into 3D models.

Pseudo-colors – colors that are assigned to each channel by the image analysis program. Do not necessarily represent the actual color (i.e. emission wavelength).

 Gausian fitting algorithm(s) – common algorithms in high and super-resolution microscopy, intended to determine the exact location of a point light source.

Mask– an image of the “shadow” of an ROI, created by specific programs from the original image. Is used for further image analysis to determine localization of objects (e.g. spots) in or out of the ROI.

Up: Image of cells destined for analysis. Down: Mask image of the cells of interest.

 

 

 

 

 

 

 

 

 

 

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