Optical transfection
Encyclopedia
Optical transfection is the process of introducing nucleic acids into cells using light. Typically, a laser is focussed to a diffraction limited spot (~1 µm diameter) using a high numerical aperture
microscope objective. The plasma membrane of a cell is then exposed to this highly focussed light for a small amount of time (typically tens of milliseconds to seconds), generating a transient pore on the membrane. The generation of a photopore allows exogenous plasmid DNA
, RNA
, organic fluorophore
s, or larger objects such as semiconductor quantum nanodots
to enter the cell. In this technique, one cell at a time is treated, making it particularly useful for single cell analysis.
To put the above simply, cells do not usually allow certain types of substances in to their interior space. Lasers can be used to burn a tiny hole on the cell surface, allowing those substances to enter. This is tremendously useful to biologists who are studying disease, as a common experimental requirement is to put things (such as DNA) into cells.
This technique was first demonstrated in 1984 by Tsukakoshi et al., who used a frequency tripled Nd:YAG to generate stable and transient transfection
of normal rat kidney cells. Since this time, the optical transfection of a host of mammalian cell types has been demonstrated using a variety of laser sources, including the 405 nm continuous wave (cw) , 488 nm cw , or pulsed
sources such as the 800 nm femtosecond pulsed Ti:Sapphire or 1064 nanosecod pulsed Nd:YAG.
has evolved. The original meaning of transfection was "infection by transformation", i.e. introduction of DNA (or RNA) from a eukaryote-infecting virus or bacteriophage
into cells, resulting in an infection. Because the term transformation had another sense in animal cell biology (a genetic change allowing long-term propagation in culture, or acquisition of properties typical of cancer cells), the term transfection acquired, for animal cells, its present meaning of a change in cell properties caused by introduction of DNA
(or other nucleic acid species such as RNA
or SiRNA
).
Because of this strict definition of transfection
, optical transfection also refers only to the introduction of nucleic acid species. The introduction of other impermeable compounds into a cell, such as organic fluorophore
s or semiconductor quantum nanodots
is not strictly speaking "transfection," and is therefore referred to as "optical injection" or one of the other many terms now outlined.
The lack of a unified name for this technology makes reviewing the literature on the subject very difficult. Optical injection has been described using over a dozen different names or phrases (see bulleted lists below). Some trends in the literature are clear. The first term of the technique is invariably a derivation of word laser, optical, or photo, and the second term is usually in reference to injection, transfection, poration, perforation or puncture. Like many cellular perturbations, when a single cell or group of cells is treated with a laser, three things can happen: the cell dies (overdose), the cell membrane is permeabilised, substances enter, and the cell recovers (therapeutic dose), or nothing happens (underdose). There have been suggestions in the literature to reserve the term optoinjection for when a therapeutic dose is delivered upon a single cell , and the term optoporation for when a laser generated shockwave treats a cluster of many (10s to 100s) cells . The first definition of optoinjection is uncontroversial. The definition of optoporation, however, has failed to be adopted, with a similar number of references using the term to denote the dosing of single cells as those using the term to denote the simultaneous dosing of clusters of many cells
As the field stands, it is the opinion of the authors of a review article on the subject that the term optoinjection always be included as a keyword in future publications, regardless of their own naming preferences.
Terms agreed by consensus
Terms under deliberation
Some of the above was reproduced with permission from.
1) Build an optical tweezers
system with a high NA objective
2) Culture cells to 50-60% confluency
3) Expose cells to at least 10 µg/ml of plasmid DNA
4) Dose the plasma membrane of each cell with 10-40 ms of focussed laser, at a power of <100 mW at focus
5) Observe transient trasfection 24-96h later
6) Add selective medium
if the generation of stable colonies is desired
Numerical aperture
In optics, the numerical aperture of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. By incorporating index of refraction in its definition, NA has the property that it is constant for a beam as it goes from one...
microscope objective. The plasma membrane of a cell is then exposed to this highly focussed light for a small amount of time (typically tens of milliseconds to seconds), generating a transient pore on the membrane. The generation of a photopore allows exogenous plasmid DNA
DNA supercoil
DNA supercoiling refers to the over- or under-winding of a DNA strand, and is an expression of the strain on the polymer. Supercoiling is important in a number of biological processes, such as compacting DNA. Additionally, certain enzymes such as topoisomerases are able to change DNA topology to...
, RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
, organic fluorophore
Fluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
s, or larger objects such as semiconductor quantum nanodots
Quantum dot
A quantum dot is a portion of matter whose excitons are confined in all three spatial dimensions. Consequently, such materials have electronic properties intermediate between those of bulk semiconductors and those of discrete molecules. They were discovered at the beginning of the 1980s by Alexei...
to enter the cell. In this technique, one cell at a time is treated, making it particularly useful for single cell analysis.
To put the above simply, cells do not usually allow certain types of substances in to their interior space. Lasers can be used to burn a tiny hole on the cell surface, allowing those substances to enter. This is tremendously useful to biologists who are studying disease, as a common experimental requirement is to put things (such as DNA) into cells.
This technique was first demonstrated in 1984 by Tsukakoshi et al., who used a frequency tripled Nd:YAG to generate stable and transient transfection
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
of normal rat kidney cells. Since this time, the optical transfection of a host of mammalian cell types has been demonstrated using a variety of laser sources, including the 405 nm continuous wave (cw) , 488 nm cw , or pulsed
Modelocking
Mode-locking is a technique in optics by which a laser can be made to produce pulses of light of extremely short duration, on the order of picoseconds or femtoseconds ....
sources such as the 800 nm femtosecond pulsed Ti:Sapphire or 1064 nanosecod pulsed Nd:YAG.
Terminology
The meaning of the term transfectionTransfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
has evolved. The original meaning of transfection was "infection by transformation", i.e. introduction of DNA (or RNA) from a eukaryote-infecting virus or bacteriophage
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
into cells, resulting in an infection. Because the term transformation had another sense in animal cell biology (a genetic change allowing long-term propagation in culture, or acquisition of properties typical of cancer cells), the term transfection acquired, for animal cells, its present meaning of a change in cell properties caused by introduction of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
(or other nucleic acid species such as RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
or SiRNA
Sírna
Sírna Sáeglach , son of Dian mac Demal, son of Demal mac Rothechtaid, son of Rothechtaid mac Main, was, according to medieval Irish legend and historical tradition, a High King of Ireland...
).
Because of this strict definition of transfection
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
, optical transfection also refers only to the introduction of nucleic acid species. The introduction of other impermeable compounds into a cell, such as organic fluorophore
Fluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
s or semiconductor quantum nanodots
Quantum dot
A quantum dot is a portion of matter whose excitons are confined in all three spatial dimensions. Consequently, such materials have electronic properties intermediate between those of bulk semiconductors and those of discrete molecules. They were discovered at the beginning of the 1980s by Alexei...
is not strictly speaking "transfection," and is therefore referred to as "optical injection" or one of the other many terms now outlined.
The lack of a unified name for this technology makes reviewing the literature on the subject very difficult. Optical injection has been described using over a dozen different names or phrases (see bulleted lists below). Some trends in the literature are clear. The first term of the technique is invariably a derivation of word laser, optical, or photo, and the second term is usually in reference to injection, transfection, poration, perforation or puncture. Like many cellular perturbations, when a single cell or group of cells is treated with a laser, three things can happen: the cell dies (overdose), the cell membrane is permeabilised, substances enter, and the cell recovers (therapeutic dose), or nothing happens (underdose). There have been suggestions in the literature to reserve the term optoinjection for when a therapeutic dose is delivered upon a single cell , and the term optoporation for when a laser generated shockwave treats a cluster of many (10s to 100s) cells . The first definition of optoinjection is uncontroversial. The definition of optoporation, however, has failed to be adopted, with a similar number of references using the term to denote the dosing of single cells as those using the term to denote the simultaneous dosing of clusters of many cells
As the field stands, it is the opinion of the authors of a review article on the subject that the term optoinjection always be included as a keyword in future publications, regardless of their own naming preferences.
Terms agreed by consensus
- Optoinjection (or any derivations of laser injection, optical injection, photoinjection): The transfer of any membrane impermeable substance into a cell using light. A general term that also encompasses optical transfection.
- Optical transfection (or any derivations of laser transfection, optotransfection, phototransfection): A specific type of optical transfection - the transfer of nucleic acids into a cell using light for the purposes of eliciting protein translation from those acids. To be in line with the current definition of transfection in the biological community, non-nucleic acids (such as fluorophores) cannot, by definition, be optically transfected (only optically injected).
- Photoporation (or any derivations of [laser-] or [optical-] or [opto-] or [photo-] AND [ poration] or [-permeabilisation] or [-puncture] or [-perforation]): The generation of a transient hole or holes on the plasma membrane (or cell wall) of a cell usually for the purpose of optical injection. See possible exception: Optoporation
- -surgery (such as cell nanosurgery, laser nanosurgery, laser surgery): A general term that incorporates all of the above definitions, but also includes the concepts of the ablation or optical manipulation of cell material for other purposes besides pore generation. Examples include selective cell ablation to purify cell populations, chromosome dissection, cytoskeleton disruption, organelle ablation, axotomy{ , or the optical tweezing or isolation of intracellular material.
Terms under deliberation
- Optoporation: Has been suggested to mean the dosing of a cluster of cells with a shockwave mediated mechanism, which usually results in a doughnut shaped therapeutic zone . On the contrary, has also been synonymously used with the term photoporation
- Laserfection: Has been suggested to mean the dosing of a cluster of cells with a circularly shaped therapeutic zone. Term reserved for Cyntellect’s laser-enabled analysis and processing (LEAP) system.
- Light-induced convective transmembrane transport: A newly coined term for optoinjection.
Some of the above was reproduced with permission from.
Methods
A typical optical transfection protocol is as follows:1) Build an optical tweezers
Optical tweezers
Optical tweezers are scientific instruments that use a highly focused laser beam to provide an attractive or repulsive force , depending on the refractive index mismatch to physically hold and move microscopic dielectric objects...
system with a high NA objective
2) Culture cells to 50-60% confluency
3) Expose cells to at least 10 µg/ml of plasmid DNA
4) Dose the plasma membrane of each cell with 10-40 ms of focussed laser, at a power of <100 mW at focus
5) Observe transient trasfection 24-96h later
6) Add selective medium
Growth medium
A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens.There are different types of media for growing different types of cells....
if the generation of stable colonies is desired
See also
- TransfectionTransfectionTransfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
- TransformationTransformation (genetics)In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
- TransductionTransduction (genetics)Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
- Cationic liposomeCationic liposome- life :Cationic liposomes are structures that are made of positively charged lipids and are increasingly being researched for use in gene therapy due to their favourable interactions with negatively charged DNA and cell membranes. Cationic liposomes are also known as cationic lipoplexes....
- NucleofectionNucleofectionNucleofection refers to electroporation, a transfection method which enables transfer of nucleic acids such as DNA, RNA, Small interfering RNA into cells. Nucleofection, also referred to as Nucleofector Technology, was invented by the biotechnology company amaxa...
- Magnet assisted transfectionMagnet assisted transfectionMagnet Assisted Transfection is a transfection method, which uses magnetic force to deliver DNA into target cells. Therefore, nucleic acids are first associated with magnetic nanoparticles...
External links
- Research in optical transfection at the University of St Andrews
- Overview of transfection methods in Nature Methods 2, 875 - 883 (2005)