Intravital imaging of fluorescent markers and FRET probes by DNA tattooing
- Adriaan D Bins†1,
- Jacco van Rheenen†1, 2Email author,
- Kees Jalink3,
- Jonathan R Halstead4,
- Nullin Divecha4,
- David M Spencer5,
- John BAG Haanen1 and
- Ton NM Schumacher1Email author
© Bins et al; licensee BioMed Central Ltd. 2007
Received: 13 September 2006
Accepted: 03 January 2007
Published: 03 January 2007
Advances in fluorescence microscopy and mouse transgenesis have made it possible to image molecular events in living animals. However, the generation of transgenic mice is a lengthy process and intravital imaging requires specialized knowledge and equipment. Here, we report a rapid and undemanding intravital imaging method using generally available equipment.
By DNA tattooing we transfect keratinocytes of living mice with DNA encoding fluorescent biosensors. Subsequently, the behavior of individual cells expressing these biosensors can be visualized within hours and using conventional microscopy equipment. Using this "instant transgenic" model in combination with a corrected coordinate system, we followed the in vivo behavior of individual cells expressing either FRET- or location-based biosensors for several days. The utility of this approach was demonstrated by assessment of in vivo caspase-3 activation upon induction of apoptosis.
This "instant skin transgenic" model can be used to follow the in vivo behavior of individual cells expressing either FRET- or location-based probes for several days after tattooing and provides a rapid and inexpensive method for intravital imaging in murine skin.
The introduction of fluorescent proteins (FP) and the ability to visualize molecular events using fluorescence microscopy have been of substantial value for understanding intracellular signaling events. FP-based biosensors, and especially those based on Fluorescent Resonance Energy Transfer (FRET), can report on cellular processes such as apoptosis or cell division, the levels of second messengers such as Ca2+, PtdIns(4,5)P2 and cAMP, or the activation status of proteins such as RhoA . To date, the vast majority of analyses utilizing FP-based biosensors have been carried out on cells in tissue culture. However, for cellular processes that are controlled by cellular interactions or by the microenvironment, it would clearly be preferable to perform such analyses in vivo. Indeed, the small number of studies that have analyzed the behavior of single cells in vivo, have been informative with regard to the intracellular interactions of immune cells and early events in tumors formation [2–5]. Intravital images of whole mice or tumors can readily be acquired with macroscopical resolution using simple instrumentations (e.g. LED flashlight, filters and a digital camera ). However, for imaging with subcellular resolution, sophisticated equipment and expertise (e.g. two-photon microscopes  or whole-mouse imaging systems ) are required, which are only available in few specialized laboratories. Furthermore, the generation of biosensor-transgenic mice is a lengthy process and thereby hampers the use of this technique in settings where a number of different biosensors are tested or combinations of biosensors are required. A few studies have previously described fast and simple techniques to target selective genes to specific sites. For example, Li and Hoffman have targeted reporter genes to hair follicles in mice using liposomes . However, it will be important to extend such approaches to other cell types. Here we present a rapid and inexpensive DNA tattoo method to target FP-genes to keratinocytes in the skin of mice. The behavior of these keratinocytes expressing fluorescent or FRET markers were followed for multiple days with subcellular resolution using a standard confocal microscope. The utility of this tattoo-approach was demonstrated by in vivo imaging of caspase-3 activation upon apoptosis induction.
Results and Discussion
Prior experiments have demonstrated the feasibility of introducing transgenes in murine skin cells by use of a rapidly oscillating tattoo machine . In an effort to determine the number of cells that express the introduced transgene upon this type of skin application (see Methods), we tattooed a small area (10 × 15 mm) of the abdominal skin of mice with DNA encoding a GFP reporter protein driven by the CMV immediate early (CMV-IE) promoter. 3 hours thereafter, mice were anesthetized and analyzed for DNA tattoo-induced GFP expression by intravital imaging using a conventional confocal microscope. Expression of the GFP reporter gene was observed in approximately 160 cells per 10 mm2 tattooed skin, showing that the transfection efficiency of DNA tattooing was sufficient for imaging purposes. The majority of fluorescent cells obtained after a GFP-encoding DNA tattoo were keratinocytes (see Additional file 1).
Mouse transgenesis in combination with intravital imaging is time-consuming and can only be performed in specialized laboratories. Here we have presented a tattoo-based intravital imaging assay using techniques that are generally available. Clearly, this new approach that is based on the intradermal delivery of fluorescent reporters by DNA tattoo has its limitations. First, transgene expression obtained by the 'instant-transgenic' method as used here is confined to the epidermis and second, the expression of fluorescent reporters is restricted to a few days. In future studies, the duration of expression may be prolonged by enabling integration  or episomal maintenance of the plasmid used for transfection . Furthermore, in vivo transfection using naked DNA can be achieved by various means in other tissues, including hepatocytes , myocytes  and lymphocytes . More importantly, for many applications the abovementioned drawbacks are balanced by the substantial advantages provided by intravital DNA tattoo imaging. Firstly, mice expressing multiple cellular reporters can be generated in a time span of hours instead of months to years. Secondly, because it is possible to restrict expression of transgenes and reporters to a subset of cells amidst a population of unmodified cells, it is feasible to follow the behavior of gene-modified cells in isolation. In addition, this allows the retracing of individual cells in order to study these cells over a period of days. Thirdly, several tattoos with different combinations of transgenes and reporters can be compared within the same animal, thereby reducing experimental variation. Finally, imaging can be performed on widely available confocal or epifluorescent microscopes. These features suggest that intravital DNA tattoo imaging can form a highly rapid and versatile system to analyze cellular processes such as cell cycle progression, cellular differentiation or apoptosis in an in vivo setting.
The abdomen of the mouse was shaved and subsequently treated with depilation cream (Veet, Reckitt&Colmann, France). The nude abdominal surface was thoroughly rinsed to remove remaining cream, and a droplet of 20 μg DNA in 10 μl water was applied to the skin. Using a sterile disposable 11-needle bar (fine magnum 15, "challenge in colors" China) mounted on a rotary tattoo/permanent make-up device (Cold skin, B&A trading, Lijnden, the Netherlands) the DNA was tattooed in the skin over a surface of approximately 30 mm2. During the 15 second tattoo, the needles oscillated at 100 Hz to a depth of 0.5 mm. Following application of the DNA, reference points were tattooed with black permanent make-up ink using a single point needle cartridge (Nouveau contour BV, Weert, Netherlands) mounted on a permanent make-up device (fine magnum 15, Aella, Medium-Tech, Berlin, Germany). All animal experiments were approved by the relevant institutional ethical committee (DEC) and performed in accordance with the local guidelines.
Anesthetized mice (GFP-MHC class II mice  were a kind gift of R. Offringa) were transferred to a 37°C heated cage, mounted to an inverted TC-SP-AOBS confocal microscope (Leica, Mannheim, Germany). A 5× dry objective (0.15 HC PL Fluotar) and a 20× dry objective (0.7 HC) were used to collect overview images, and a 63× water objective (1.2 PL APO) to collect high resolution images.
In vivo FRET imaging
in vivo FRET was measured by three independent methods; measurement of sensitized YFP emission, measurement of emission spectra, and measurement of the gain in CFP fluorescence upon YFP photo bleaching. For sensitized emission (YFP fluorescence upon CFP excitation), three images were collected: a CFP image excited at 405 nm and detected between 430 and 480 nm, a sensitized emission image excited at 405 nm and detected between 528 nm and 603 nm, and a YFP image excited at 514 nm and detected between 528 nm and 603 nm. Sensitized emission (FRET) was calculated from these images by correcting the sensitized emission image for leak-through of CFP fluorescence and fluorescence due to direct YFP excitation as described in . Correction factors for leak-through of CFP and indirect excitation of YFP were determined on-line from cells expressing CFP or YFP only at different locations on the same animal. For every collected FRET image, images of cells expressing CFP or YFP only were collected subsequently to re-determine correction factors for laser fluctuations. Emission spectra were measured by collecting series of xyλ-images at 405 nm excitation and at 10 nm bandwidth emission starting at 430 nm and ending at 650 nm. No significant YFP emission was observed in emission spectra of cells expressing only YFP. Moreover, FRET observed by spectrum imaging was confirmed by the gain of CFP fluorescence after photo-destruction of YFP. The CFP gain was not a result of photo conversion of YFP during photo bleaching as described in , because YFP bleaching of skin cells expressing YFP only did not result in a significant increase in fluorescence at CFP wavelengths.
Coordinate system to image the same cells for several days
Cells of interest were retraced using a coordinate system. The coordinate system is based on two black ink reference points, which are tattooed on the mouse abdomen: an origin and a reference. The distance L(ref) and the angle a(ref) between the origin and the reference are determined by a custom-made Visual Basic (v6.0) program by calling commands from the Leica macro tool package. For every cell coordinate, the distance L(cell) and the angle a(cell) between the cell and the origin are determined and saved. During the next imaging session, the re-anaesthetized animal is re-positioned on the confocal microscope. While L(ref) and L(cell) will be similar (but not identical, see below) at subsequent time-points, the reference a(ref) and all cell angles (a(cell)) will vary because of small rotations in the position of the mouse. To correct for this, a(ref) is re-determined, and all cell angles (a(cell)) are corrected for changes in a(ref). The positions of the cells are then recalculated from the corrected a(cell) values and L(cell). Stretching of the skin does result in small deviations in L in subsequent imaging sessions and therefore can result in misalignment of the position of cells up to 0.5 mm. To avoid this issue we use an overview image at low (5×) magnification to determine the exact coordinate of each cell (Figure 4). Once a single misalignment has been resolved, all positions (within a radius of 5 mm) are corrected for this misalignment.
We would like to thank Drs J. Segall, W. Wang, D. Kedrin, S. Goswami and J. Wyckoff for helpful discussions, Dr. L. Oomen for assistance with confocal microscopy and Dr. G. van der Krogt for technical assistance. We would like to thank ARIAD Pharmaceuticals (Cambridge, MA) for the generous supply of AP20187, and R. Offringa for providing the MHCII-eGFP mice. JvR was supported by a fellowship from the Dutch Cancer Society.
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