Photoactivated Localization Microscopy (PALM)
Photoactivated Localization Microscopy, or PALM imaging (1) exploits the unique behavior of special fluorophores (PALM-FPs), whose spectral properties change upon exposure to specific wavelengths of light.
PALM PRINCIPLE
In the right conditions, upon illumination with the activation, light only a random and sparse number of PALM-FPs are activated and emit light for a short period time. The light emission from individual fluorophores can be recorded and used to localize and track single molecules with sub-diffraction precision, as in other SMLM super-resolution techniques. By concatenation of activation/detection/deactivation steps all the fluorophores in the sample can be accurately counted, localized, and tracked over time.
What are the advantages of PALM?
More than for other SMLM techniques, in PALM the characteristics of the fluorophores are of paramount importance. On the basis of the spectral change induced upon activation we can distinguish:
- Photoactivatable (PA) fluorophores, activated from low fluorescent states (OFF) to high fluorescence states (ON).
- Photoconvertible (PC) fluorophores, that upon activation change from one fluorescent color to another.
The spectral change can be irreversible, or it can be switched on and off reversibly (switchable fluorophores).
The most used PALM-FPs are fluorescent proteins: these can be appended to other proteins via genetic engineering and thus allow super resolution imaging of virtually any protein-of-interest within the cell, independently of the availability of antibodies or other affinity reagents.
However, also certain organic fluorophores can be activated only by illumination at specific wavelengths, without photoswitching buffers and at physiological oxygen levels (as opposed to dSTORM); these have been used in combination with genetically encoded tags in hybrid systems that have expanded the traditional fluorescent toolbox available for PALM imaging (SNAP, CLIP, Halo-tags).
Photoactivatable (PA) fluorophores Irreversible
| Fluorophore | OFF | Activation light | ON
Excitation/Emission |
|
| PA-GFP 2 | dark | UV-Violet | Blue –
(504 nm – |
– Green
– 517 nm) |
| PA-TagRFP 3 | dark | UV-Violet | Orange-
(563nm – |
– Red
– 594nm) |
| PA-mCherry 4 | dark | UV-Violet | Orange-
(570nm – |
– Red
– 596nm) |
| PA-mKate2 5 | dark | UV-Violet | Red-
(586nm – |
– Far Red
– 628nm) |
Photoactivatable (PA) fluorophores Reversible
| Fluorophore | OFF | Activation/deactivation light | ON
Excitation/Emission |
||
| Dronpa 6 | dark | UV- | -Blue | Blue-
(503 nm- |
–Green
-518nm) |
| Padron 7 | dark | Blue- | -UV | Blue-
(503 nm- |
-Green
-518nm) |
| Dreiklang 7 | dark | UV- | –Violet | Blue-
(515nm- |
-Green
-529nm) |
| rsCherry 8 | dark | Green- | -Blue | Green-
(570 nm- |
-Red
-596 nm) |
| rsCherry-rev 8 | dark | Blue- | -Green | Green-
(570 nm- |
-Red
-596 nm) |
| FP595 9 | dark | Green- | -Red | Orange-
(590 nm- |
-Red
-600 nm) |
| PA-JF549 10,* | dark | UV- | -Violet | Green-
(549 nm- |
-Red
-573 nm) |
| PA-JF646 10, * | dark | UV- | –Violet | Red-
(646 nm- |
-Far Red
-665 nm) |
* organic fluorophores
Photoconvertible (PC) fluorophores Irreversible
| Fluorophore | Pre-conversion
Excitation/Emission |
Photoconversion light | Post-conversion
Excitation/Emission |
|||
| PS-CFP2 11 | UV-
(400 nm- |
-Cyan
-468nm) |
UV- | -Violet | Blue-
(490 nm- |
–Green
-511 nm) |
| Kaede 12 | Blue-
(508 nm- |
-Green
-518nm) |
UV- | -Violet | Orange-
(572 nm- |
–Red
-582 nm) |
| mMaple3 13 | Blue-
(489 nm- |
-Green
-505nm) |
UV- | -Violet | Orange-
(566 nm- |
–Red
-583 nm) |
| Dendra2 14,15 | Blue-
(490 nm- |
-Green
-507nm) |
UV- | -Violet | Green-
(553 nm- |
–Red
–573 nm) |
|
Blue |
||||||
| KikGr, mKikGr 16,17 | Blue-
(507 nm- |
-Green
–517nm) |
UV- | -Violet | Orange-
(566 nm- |
–Red
-583 nm) |
| mEOS, mEOS2 18, * | Blue-
(506 nm- |
-Green
–519nm) |
UV- | -Violet | Orange-
(573 nm- |
–Red
-584 nm) |
| mEOS3.1, mEOS3.2 19, * | Blue-
(507 nm- |
-Green
–516nm) |
UV- | -Violet | Orange-
(572 nm- |
–Red
-580 nm) |
| mEOS 4b 20, * | Blue-
(505 nm- |
-Green
–516nm) |
UV- | -Violet | Orange-
(572 nm- |
–Red
-580 nm) |
*Reversible with intermediate dark state
Photoconvertible (PC) fluorophores Reversible
| Fluorophore | Pre-conversion
Excitation/Emission |
Photoconversion light | Post-conversion
Excitation/Emission |
||
| mIrisGFP 21 | UV-
(400 nm- |
-Cyan
-468nm) |
UV-Violet | Blue-
(490 nm- |
-Green
–511 nm) |
| NijiGFP 22 | Blue-
(508 nm- |
-Green
-518 nm) |
UV-Violet | Orange-
(572 nm- |
-Red
–582 nm) |
What are the advantages of PALM?
Versatility and specificity
Through gene tagging PALM-FP can be appended to practically any protein in a cell or organism, with very high labelling specifity. Thus, PALM is the most effective super-resolution technique to image “difficult” targets:
- when antibodies or other affinity tags are not available or are unable to discriminate close isoforms;
- when the target is difficult to label, because in a crowded intracellular compartment, or surrounded by a difficult-to-penetrate envelope.
Compatible with live imaging
PALM-FPs require relatively mild conditions to photoswitch, differently from most STORM fluorophores; thus, PALM is the technique of choice to perform live super-resolution imaging. In particular, when combined with tracking algorithms, PALM allows to do single particle tracking (SPT-PALM) in living cells and organisms, with nanometric spatial resolution and millisecond temporal resolution.
Quantitative imaging
PALM high labelling specificity and low overcounting ((most irreversible PALM-FPs are activated only once) make it a highly useful tool for quantitative experiments (23) and precise single molecule counting. Homogeneous illumination and precise control of activation and excitation light, such as permitted by Abbelight ASTER technology is essential for quantitative PALM.
What are the drawbacks of PALM and how to overcome them?
Compared to other SMLM techniques (PAINT, dSTORM), PALM has generally shown lower resolutive power, because fluorescent proteins are in general less bright than organic fluorophores: less light translates into lower precision of localization and thus resolution. Recently developed brighter variants of fluorescent proteins, and even more photoswitchable organic fluorophores have greatly improved the resolutive power of PALM.
PALM multicolor workflows are often difficult, because of large and overlapping excitation/emission spectra of fluorescent proteins, and their simultaneous co-activation with UV light. Also in this case, photoswitchable organic fluorophores increase the multiplexing flexibility of PALM experiments. Moreover, the combination with appraches such as Abbelight spectral demixing can greatly simplify simultaneous multicolor PALM.
Structural PALM imaging of RNA polymerase (rpoC-Dendra2) in fixed Escherichia coli cells
Courtesy of Bartosz Turkowyd & Ulrike Endesfelder, SYNMIKRO MPI Marburg / CMU-Pittsburgh
Single-particle tracking (spt)PALM imaging of RNA polymerase (rpoC-Dendra2) in living Escherichia coli cells
Courtesy of Bartosz Turkowyd & Ulrike Endesfelder, SYNMIKRO MPI-Marburg / CMU-Pittsburgh
If you want to know more about cutting-edge PALM techniques, including primed conversion, check out the Webinar of Pr. Ulrike Endesfelder:
Visualizing cellular life: from single cell imaging to in vivo single-molecule biochemistry
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