Photoporation

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The ability to load membrane impermeable substances into cells is of great interest to cell biologists. Optical transfection is one of the few technologies that allows single cells to easily be transfected, without affecting neighbouring cells. The image shows six cells, three of which were transfected with a plasmid encoding for green fluorescent protein (green & blue cells), and three of which that were not. Cells are co-stained with the blue nuclear dye, DAPI, so only the nuclei of untransfected cells are visible. Early transfection work employed a Ti:Sapphire femtosecond pulsed laser using a standard Gaussian beam profile. More recent work has employed the use of a Bessel Beam profile, obviating the need for exact focussing and opening the possibility of a “point-and-shoot” device for the biological community. Current work is ongoing to apply this technology to an axicon tipped fibre, thereby freeing the operator from bulky objectives.

OPTOINJECTION

A femtosecond Infra-Red laser can be used to create a transient pore in the cellular membrane. This allows the micromolecules present in the medium to diffuse into the cytoplasm.

This technique offers a non-contact sterile alternative to micro-injection. It can be easily scaled up to a high throughput cell-selective automated system. Our recently developed system (Antkowiak2010a) provides over 40% injection efficiency in viable cells.

Typical images acquired in the optoinjection efficiency and viability study: (a) PI signal 5 minutes after irradiation, (b) phase contrast image 5 minutes after irradiation, (c) Calcein AM 90 min after irradiation. Examples of necrotic (solid arrows) and viable cells (dashed arrows) are shown.

PHOTOTRANSFECTION

Depending on the requirements of the experiment, phototransfection can be used to introduce the genetic modification by using:

plasmid DNA

Chinese Hamster Ovary (CHO) cells phototransfected with a plasmid expressing green fluorescent protein and co-stained with the blue nuclear dye, DAPI.

Non-differentiated colony of E14g2a cells phototransfected with DsRed2-Mito pDNA :

Phototransfecion of the endoderm associated transcription factor Gata-6 gene into non-differentiated E14g2a cells produces identifiable individual cells with spindle and stellate shaped morphology which is characteristic of extraembryonic endoderm tissue.

mRNA

CHO-K1 cells photo-transfectedwith in vitro transcribed EGFP mRNA. Image (A) is the brightfield picture and (B) fluorescence signal acquired twelve hours following photo-transfection.

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iRNA

Gene knockdown using a violet diode system. (A) A TREXwillin-GFP-HEK cell fluorescing red due to the expression of the Mito-DsRed and (B) under brightfield imaging. (C) Fluorescence image of the same field of view using a FITC HYQ. Red arrow points to a cell which has been co-transfected with Mito-DsRed and willin specific siRNA.

We have succesfully used phototransfection on the following, ever-expanding, list of cell types:

CHO-K1
HEK293
SK-N-SH
non-differentiated E14g2a cells

	24%	Europe
	16%	United Kingdom
	14%	United States
	9%	China
	6%	Germany
	5%	Korea (South)
	4%	India
	3%	France
	2%	Russia
	2%	Japan

06/05/2012 - 5 urgent post-doctoral openings in the group

27/03/2012 - Article in Nano Letters

03/12/2011 - - Top download in OE

Photoporation

OPTOINJECTION

PHOTOTRANSFECTION