Capillary Microinjection Research Articles

Seeing and Cleaving: Turn-Off Fluorophore Uncaging and Its Application in Hydrogel Photopatterning and Traceable Neurotransmitter Photocages.

The advancements in targeted drug release and experimental neuroscience have amplified the scientific interest in photolabile protecting groups (PPGs) and photouncaging. The growing need for the detection of uncaging events has led to the development of reporters with fluorescence turn-on upon uncaging. In contrast, fluorescent tags with turn-off properties have been drastically underexplored, although there are applications where they would be sought after. In this work, a rhodamine-based fluorescent tag is developed with signal turn-off following photouncaging. One-photon photolysis experiments reveal a ready loss of red fluorescence signal upon UV (365 nm) irradiation, while no significant change is observed in control experiments in the absence of PPG or with irradiation around the absorption maximum of the fluorophore (595 nm). The two-photon photolysis of the turn-off fluorescent tag is explored in hydrogel photolithography experiments. The hydrogel-bound tag enables the power-, dwell time-, and wavelength-dependent construction of intricate patterns and gradients. Finally, a prominent caged neurotransmitter (MNI-Glu) is modified with the fluorescent tag, resulting in the glutamate precursor named as GlutaTrace with fluorescence traceability and turn-off upon photouncaging. GlutaTrace is successfully applied for the visualization of glutamate precursor distribution following capillary microinjection and for the selective excitation of neurons in a mouse brain model.

Single cell optical transfection

The plasma membrane of a eukaryotic cell is impermeable to most hydrophilic substances, yet the insertion of these materials into cells is an extremely important and universal requirement for the cell biologist. To address this need, many transfection techniques have been developed including viral, lipoplex, polyplex, capillary microinjection, gene gun and electroporation. The current discussion explores a procedure called optical injection, where a laser field transiently increases the membrane permeability to allow species to be internalized. If the internalized substance is a nucleic acid, such as DNA, RNA or small interfering RNA (siRNA), then the process is called optical transfection. This contactless, aseptic, single cell transfection method provides a key nanosurgical tool to the microscopist-the intracellular delivery of reagents and single nanoscopic objects. The experimental possibilities enabled by this technology are only beginning to be realized. A review of optical transfection is presented, along with a forecast of future applications of this rapidly developing and exciting technology.

System for Quantitation of Gene Expression in Single Cells by Computerized Microimaging: Application to c- fos Expression after Microinjection of Anti-Casein Kinase II Antibody

A system which allows sensitive and fast automated analysis of weakly labeled fluorescent specimens is described. It is tested in the analysis of c- fos expression stimulated by fetal calf serum and calibrated by quantitation of defined solutions injected into cells with the automated microinjection system. Low light level imaging technology combined with quantitative image processing methods and computer control of the hardware allows fully automated analysis of fluorescent molecules in single living or fixed cells. Reliable methods for subtraction of fluorescent background and automated identification of objects of interest in double-stained cells are described. The accuracy of quantitation is considerably improved by normalizing the fluorescence intensities of respective fluorophores in the same object by the method of ratio imaging. The error rate in determining the relative protein content in single cells is less than 15%. The method is applied to microinjection studies with a monoclonal antibody against casein kinase II subunit β. Microinjection of this antibody into synchronized cells specifically inhibits c- fos expression stimulated by fetal calf serum. In combination with the computer-automated capillary microinjection system, the technique will become a useful tool in experiments requiring quantitative single cell analysis.

Regeneration of Triticum aestivum apical explants after microinjection of germ line progenitor cells with DNA

A highly efficient method of regenerating fertile, phenotypically normal plants from shoot apex cultures of T. aestivum was developed. The hypodermal layer (L2) of the vegetative apex containing germ line precursor cells could be located with bright field microscopy and targeted for microinjection. Fluorescently labelled dextrans were used as markers to develop a microinjection procedure which did not disrupt nuclear or cytoplasmic structure. This procedure was used to inject plasmid DNA into L2 cells. Capillary microinjection did not shear the plasmid DNA and delivery of DNA was confirmed by polymerase chain reaction analysis of DNA isolated from injected apices. The significance of these findings in relation to the development of cereal transformation systems will be discussed.

Single cell assay with an automated capillary microinjection system

An automated capillary microinjection system with computer controlled positioning of the cells and of the capillary, and its applications and advantages arc described. About 1500 injections are possible in one hour, with high reproducibility. In cytoplasmic and nuclear injections more than 90% and 85% of the cells are successfully injected. Using FITC-Dextran at a concentration of 0.5% as a fluorescently labelled coinjection marker, 99% of the cells can be retrieved in cultrue medium even 48 hours after unjection. The coordinates of the cells are stored in the computer and accuracy in statistical evaluation of experiments is improved in coparison to the manual techniques. Methods for preparatin and handling of glass capillaries were developed resulting in reproducible form and significantly reduced clogging rate. The improved characteristics offered by this system are demonstrated in studies leading to the confirmation of existence of mRNA(s) inhibiting cell proliferation. Functional screening by cell injections of cDNA libraries and of size fractionated mRNA molecules can be performed efficiently with the automated microinjection system.

Single cell assay with an automated capillary microinjection system

An automated capillary microinjection system with computer-controlled positioning of the cells and of the capillary, and its applications and advantages, is described. The system is easy in handling and manipulation. About 1500 injections are possible in 1 h, with high reproducibility. In cytoplasmic and nuclear injections more than 90 and 85 % of the cells are successfully injected. Using FITC-dextran at a concentration of 0.5 % as a fluorescently labeled coinjection marker, 99 % of the cells can be retrieved in culture medium even 48 h after injection. The coordinates of the cells are stored in the computer and accuracy in statistical evaluation of experiments is improved in comparison to the manual techniques. Methods for preparation and handling of glass capillaries were developed resulting in reproducible form and significantly reduced clogging rate. The improved characteristics offered by this system are demonstrated in studies leading to the confirmation of existence of an mRNA inhibiting DNA synthesis in cells. Functional screening by cell injections of cDNA libraries and of size-fractionated mRNA molecules can be performed efficiently with the automated microinjection system.

Automatic microinjection system facilitates detection of growth inhibitory mRNA.

Naturally quiescent human lymphocytes, consisting predominantly of T cells, contain mRNA(s) that can inhibit DNA synthesis when injected into either human diploid fibroblasts (IMR-90) or transformed recipient cells (HeLa). By using an automated capillary microinjection system and a fluorescent coinjection marker (fluorescein isothiocyanate-dextran), individually injected cells can be retrieved and analyzed for DNA synthesis. mRNA isolated from resting T cells is able to block the cells from entering the S phase. The block is reversible and leads to a delay in DNA synthesis. The inhibitory effect is not observed if the injected mRNA is isolated from growth-activated T cells. The disappearance of the inhibition coincides with the approach of the G1/S boundary in both the donor T cells and the recipient human fibroblasts. The mRNA of resting T cells was size-fractionated and the peak inhibitory activity was recovered in a fraction approximately equal to 1.5 kilobases long.

Performance of an automated system for capillary microinjection into living cells

An automated capillary microinjection system for nuclear and cytoplasmic injections into cells is described. The system has been tested with samples of DNA, RNA and proteins. Movements of the capillary with precise cell positioning and time of injections are controlled by a computer. The first automated microinjection system allows injection of more than 1500 cells per hour with a minimum of practical training, volumes injected are more reproducible and cells are less damaged when compared with the standard manual injection technique. Retrieval of the injected cells is accurate to within 1% without complicated and laborius produced orientation marks on the cell support. The number of successfully injected cells is easily determined with great accuracy and the error of the statistical evaluation of the results is reduced to a minimum. Standardized procedures for pulling, handlin and storage of the injection capillaries were developed.

Microinjection of catalytic subunit of cyclic AMP-dependent protein kinases triggers acute morphological changes in thyroid epithelial cells

In dog thyroid epithelial cells in primary culture, thyrotropin acting through cyclic AMP induced rapid morphological changes associated with complete disruption of actin containing stress fibers. This modification preceded cell retraction and rounding up. These morphological effects were also induced by glass capillary microinjection of purified catalytic subunit of cAMP-dependent protein kinase. This provides the first direct evidence in intact cells that catalytic subunit, which is released upon activation of cAMP-dependent protein kinases, is responsible for cAMP-dependent morphological transformation.

Intracellular fate of ferritin in HeLa cells following microinjection

It is known that following iron overload newly synthesized ferritin molecules accumulate in lysosomes. However, the way in which these molecules enter the lysosomes has not been clarified. In order to assess if these molecules can be taken up by lysosomes from the cell sap, i.e., by way of autophagy, ferritin was introduced into HeLa cells through microinjection with a glass capillary. The fate of the ferritin was studied after varying intervals with the electron microscope. Shortly after microinjection ferritin molecules could be observed in the cell sap. After both 1 and 2 h, they were found in clusters and still mainly in the cell sap. After 4 h, ferritin molecules were present not only in the cell sap and in autophagic vacuoles but also in occasional secondary lysosomes. After 12 h, they were seen mainly in lysosomes, undergoing degradation. In no instance were ferritin molecules translocated into other organelles such as mitochondria, Golgi apparatus, or endoplasmic reticulum. The present study demonstrates that ferritin can be introduced into cells by glass capillary microinjection without cell damage. From its initial location in the cell sap ferritin is taken up into the lysosomal vacuome. Autophagy is considered to be the principal mechanism for the transfer of the ferritin molecules into lysosomes.

Capillary Microinjection into Protoplasts and Intranuclear Localization of Injected Materials

Capillary Microinjection into Protoplasts and Intranuclear Localization of Injected Materials

Expression of cloned genes microinjected into cultured mouse and human cells

The direct approach of capillary microinjection was used to study exogenous gene expression in mouse and human cells. The efficiencies of biochemical and morphological transformation in mouse cells and the differential ras gene expression in mouse and human cells were determined. Biochemical transformation efficiency of mouse cells after microinjection was ≈2.5% with the thymidine-kinase plasmid and ≈30% with the neomycin-resistant gene, pSV2 neo, which contains the SV-40 enhancer sequence. Mouse NIH3T3 cells microinjected with genes of the ras family—including viral isolates, the human bladder oncogene EJ, and N- ras—produced morphologically transformed foci with an efficiency of 10–20%, whereas the normal human cellular homologue of the ras gene in Harvey sarcoma virus showed an efficiency of 0.5–1%. Expression of v- ras genes was compared in mouse and human cells by both immunofluorescence detection of the ras protein, p21, 24 hr after microinjection and by the focus assay. The data suggest that microinjection is a useful approach for the investigation of early exogenous gene expression and control in eukaryotic cells and has the potential to allow insight into oncogenic events.

Protoplast derived tobacco cells can survive capillary microinjection of the fluorescent dye Lucifer Yellow

The first steps to successful microinjection in plant cells of different types of macromolecules (RNA, DNA and protein) are reported. Evidence is also given that plant cells can survive the mechanical injury of the injection procedure. By attaching the cells to coverslips with polylysine, the progeny of an individual cell can be followed microscopically. Using the fluorescent dye Lucifer Yellow, the injection procedure can be monitored and the injected volume calculated.

Improved system for capillary microinjection into living cells

A technique for microinjection into cells is described, with several simplifications as to preparation and handling of micropipettes, sample filling and injecting system. The simplified procedure enables us to pull and fill the capillaries in an easy, reproducible and fast way, requiring only about 1 min. The injecting system uses three positive pressure levels (high, injecting, holding) controlled from one button. It provides for quick and reproducible changes among the three pressures. Furthermore, it limits the chances for clogging the microcapillary by elimination of the temporary negative pressure (suction at its tip), which would normally occur when the pressure is reduced from the injecting to a zero (atmospheric) pressure value. The estimated sample ejection rate which is achieved with this system could correspond to 10 −11–10 −13 ml/sec, according to [6]. Compared with other known techniques using pressurized gas in bottles, this ejected volume is three orders of magnitude smaller and makes the system of interest for work with cells smaller than 50 μm. Whereas the sample volume injected into cells is reproducible, further improvement in the control of the amount actually injected is desirable. The manipulation of the sample ejection system is very easy and no time is needed to learn it. The user can inject the sample volume either in the cytoplasm or the nucleus, as desired, by manipulating a single button. Operational aspects and problems with capillary clogging are discussed. The reliability of the whole system and technique is shown in the results obtained on microinjection of living cells. Cells could be injected at an initial speed of about 600–800 cells per hour, the actual number of cells injected in 1 h being about 400. After injection of SV40DNA, 70–80% of the injected cells have synthesized the T-antigen. The above improvements make the injection technique easily accessible to molecular biologists who need to screen for expression of the recombinants made in vitro, or to study the distribution of fluorescently labelled structural proteins. The pressure, the valves and the penetration of the cell (piezo driver) may be regulated electronically. The system would be of interest for any automated injection process. When the common pressure regulator (from 150 to 1 bar) is available, the cost of the additional equipment shown in fig. 2 b is as low as 500 DM.