Iontophoretic dye injections into Drosophila cells

Computer managed iontophoretic device was constructed and successfully applied in regeneration study. General scheme of the device, the method of dye injection, and associated software is discussed. The Device and Results Iontophoresis can be used as a method to mark a living cell and its progeny cells with a vital charged dye or to deliver active substances to a cell. Of particular interest are the injections of antisence oligonucleotide probes, giving an opportunity to make a single gene silent (Aramaki et al., 2003). In Drosophila research this method was used for marking cells from the wound edges of imaginal discs in regeneration study and to test the cell communications through the newly formed cell contacts (Bryant and Fraser, 1988). The aim of the present communication is to decribe the iontophoretic device according to current technology and to show its use. As it was mentioned in Bryant and Fraser (1988), the positioning of the iontophoretic capillary within a cell is a critically important factor for successful injection. The correct position can be determined by measuring the membrane potential of the cell. Special scheme was constructed to 148 Technique Notes Dros. Inf. Serv. 90 (2007) register a membrane potential of the cell, as well as to inject a fluorescent dye into this cell. The scheme is presented in Figure 1. The scheme contains the microelectrode, preamplifier, ADC/DAC board L-761, voltage controlled depended current source and reed relay. Physically the preamplifier, relay and current source are positioned onto the same board and shielded. While membrane potential polarizes the microelectrode, voltage produced is delivered to the input terminal of the preamplifier. Preamplifier is assembled on operational amplifier chip LPC-662 manufactured by National Semiconductor Corp. It has a high input resistance >1 Tera Ω and ultra low leakage current 2 fA. The preamplifier is intended to transfer signals from the microelectrode to ADC without any distortion as well as to hamper leakage current occurrence. To reduce the leakage current value, a special chip assembling on the printed-circuit board surface was employed. The output signal is digitized by PCI ADC/DAC board L-761 (L-Card Company). This board also supplied with doublechannel DAC. The first channel is employed to control the relay, commutating the microelectrode to the preamplifier input and to the current source output. The second channel controls the current source. The current source is intended to deliver alternate current to microelectrode. Typically current represents a sequence of 200 ms pulses with amplitude of 4 nA and porosity of 2. Such current causes fluorescent dye injection into cell. With use of LabVIEW 6.1 package the program to control L-761 board was developed. This program realizes virtual oscilloscope and voltmeter to register a membrane potential that depends on microelectrode penetration depth into cell. This program has the following features. • Microelectrode signal registration and its shape examination on screen. • Relay commutation control. • Delivering of current signal of any given shape to the microelectrode to inject dye into cell. The iontophoretic capillaries with the tip diameter less than 1 micrometer were filled first by the 200mg/ml rhodamine-conjugated dextran 40S (Sigma) and second by 1.2M LiCl. The fluorescent dye used is positively charged and was injected by pulses of the positive polarity. The capillary was Figure 1. Connection layout of the iontophoretic device. Microelectrode is denoted as a triangle, the cell as a grey oval. Dros. Inf. Serv. 90 (2007) Technique Notes 149 mounted on Narishige micromanipullator MWO-202 attached to Axiovert-200 Carl Zeiss inverted microscope. Transmitted and fluorescent light images (10× and 20× objectives) were taken by Axiocam MBC digital camera attached to the microscope. Injections of imaginal discs into adult female abdomen were done similarly to those already described (Bryant and Fraser, 1988). Figure 2. Membrane potential measurement. Oscillogram showing a polarization of microelectrode during introduction and moving off the capillary into the salivary gland polytene cell (right, one rectangular impulse marked with white arrow) and into the diploid imaginal disc cells (left, two negative impulses marked with white arrows). A window of LabVIEW software is shown. Figure 2 illustrates the oscillogram after introduction and moving off the capillary into the salivary gland polytene cell (right, one rectangular impulse marked with white arrow) and into the imaginal disc cells (left, two negative impulses marked with white arrows). Figure 3 shows the phase contrast image of fragment of salivary gland containing target polytene cell (left, white arrow) and fluorescent image of inotophoretically injected cell (right, position where the capillary was introduced is marked by yellow by the remainder of capillary staying in the cell wall). It can be seen that the rhodamine label does not penetrate into nuclei (denoted by white arrow), but is localized in the cytoplasm. The example of iontophoretic injections into diploid Drosophila cells of the wing imaginal disc are given in Figure 4 (merged fluorescent and phase-contarast images). The most left photo freshly injected cells of imaginal disc. At the next photo the same disc after culturing for 1 day in adult female abdomen is represented. The right photo – the same disc after 2 days of cultivation, note the right rhodamine spot was subdivided into two parts as a result of intercalary proliferation of nearby non-labeled cells or by allocation and proliferation of marked cells. Therefore, the injections made are stable through several cell generations. The application of the system for the study of D. melanogaster imaginal discs regeneration was published earlier (Mattila et al., 2004). References: Aramaki, Y., H. Arima, M. Takahashi, E. Miyazaki, T. Sakamoto, and S. Tsuchiya 2003, Intradermal delivery of antisense oligonucleotides by the pulse depolarization iontophoretic system. Biol. Pharm. Bull. 10: 1461-1466; Bryant, P., and S. Fraser 1988, Wound 150 Technique Notes Dros. Inf. Serv. 90 (2007) healing, cell communication, and DNA synthesis during imaginal disc regeneration in Drosophila. Devel. Biol. 127: 197-208; Mattila, J., L. Omelyanchuk, and S. Nokkala 2004, Dynamics of Figure 3. Injections into polytene cell of the larval salivary gland. Left: phase contrast image of fragment of salivary gland, containing target polytene cell (white arrow). Right: fluorescent image (red) of iontophoretically injected target cell (position where the capillary was introduced is marked by yellow by the remainder of capillary staying in the cell wall). Figure 4. Diploid cells of the wing imaginal disc injections. Merged fluorescent (red) and phase-contrast images are shown. Left: freshly injected cells of imaginal disc. Middle: the same disc after culturing for 1 day in adult female abdomen is represented. Right: the same disc after 2 days of