Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).
Section 1: Obtaining your fluorescently labeled actin for FSM
Materials required: purified actin, fluorophore (Alexa, X-rhodamine recommended). G-buffer, ultracentrifuge
Section 2: Preparation of Cells and Microinjection of labeled actin
Materials required: microinjection needles, microsyringe, microloaders, prewarmed cell media, injection buffer, bucket of ice, microscope (phase ring, phase contrast objective 40X, transjector, needle puller, micromanipulator)
Section 3: Assembling the Imaging Chamber
Materials Required: coverglass, double sided tape, q-tips, forceps, valap, kim wipe, oxyrase, imaging media, razor blade, p200 pipette, pure ethanol
Section 4: Imaging cells
Materials required: inverted widefield microscope, mecury lamp, appropriate filters, cooled CCD camera with 6.7 micron pixels, High numerical aperture, oil-immersion plan-Apochromatic objectives (60X to 100X, phase or DIC). Vibration table. Imaging software.
Section 5: qFSM analysis
This section is not discussed in detail, but the information on the analysis software can be found here [5]. Software download requests can be made on our website (lccb.scripps.edu).
Topics Covered:
Several key factors are crucial for successfully obtaining speckle images, however good speckles begin and end with the quality of your fluorescently labeled actin. A high labeling ratio of dye to actin monomer, set around 0.4 to 0.7 will ensure that speckles will appear bright and discrete, while the labeled protein itself should be soluble and free of aggregates. Equally important is the microinjection procedure. To ensure normal cell homeostasis (and survival), cells need to be injected with a low flow pressure, introducing a low concentration of labeled protein. This requires some degree of technical expertise/experience with respect to the use of the microinjection system. As a general rule, shorter injection times (< 1s) are preferred over longer injection times. The last crucial component is the microscope. An inverted microscope paired with a mercury-lamp epi-illuminator, found in most core facilities, will serve as an adequate platform for speckle imaging. Assuming the proper fluorescence filters are in place, the combination of a high NA objective and a cooled CCD camera will produce high-resolution images of bright speckles. We recommend using high magnification (100X), high NA (>1.3), plan-apochromat objectives that will provide superior resolution and brightness. Under ideal injection conditions, the magnification can be lowered to 60X, increasing the brightness and SNR per pixel. Low noise, high quantum efficient, cooled CCD cameras, with pixel sizes of ~6.5 microns are ideal detectors, and in some cases can compensate for poor quality fluorescent probes. Additionally, fluorescently labeled protein probes can be co-injected with cDNA constructs (nucleoinjection) to express GFP/RFP conjugated proteins within the same cell. This requires the nucleoinjection of cDNA into the nuclei of cells, followed by a second injection in the surrounding cytoplasm. In summary, FSM provides unprecedented insights into cytoskeleton dynamics. Obtaining good speckles requires the proper probes, equipment and a little bit patience (training).
The authors have nothing to disclose.
The development of qFSM is funded by the NIH grant U01 GM06230.
Injection buffer (IB)
50 mM potassium glutamate
0.5 M MgCl2 (APPENDIX 2A)
Store up to 2 years at −20° C
G-buffer
2 mM Tris⋅Cl, pH 8.0 (APPENDIX 2A)
0.2 mM CaCl2 (APPENDIX 2A)
Add just before use:
0.2 mM ATP (see recipe for 100 mM)
0.5 mM 2-mercaptoethanol