#PHOTOCONVERT LINAGE TRACKING SOFTWARE#

Bars 10 μm.ĮGFP photoconversion occurs in N. Images acquired by 30 iterations with the duration of 190 ms each. White arrowheads highlight cytosolic inclusions. (e) Rat PC12 cells expressing EGFP fusion to exon 1 of human HttQ103 gene. (d) Drosophila gut cells from 3rd instar larvae expressing S65T-GFP. (c) Changes in fluorescence intensity in the irradiated area in (a) over time indicate increasing red and decreasing green fluorescence. Cytoplasmic strands exist as extensions of the cytosol between organelles. The endoplasmic reticulum network (gray) surrounds the vacuole and the nucleus (white) and extends through the rest of the cytosol (white). A large vacuole (blue) occupies the majority of the space inside the cell. The cell wall (brown) surrounds the plasma membrane (red). (b) Schematic representation of a tobacco suspension culture cell. Photoconversion was performed at 50% laser power and 30 iterations of 111 milliseconds (ms). Photoconverted GFP spreads through the cytosol via cytoplasmic strands. (a) Tobacco suspension cell culture in which GFP is targeted to cytosol. Single snapshots from the pre-photoconversion and post-photoconversion are shown. Photoconversion of GFP from green to red state in vivo. Returned values of α were ~0.14 s−1 for all data sets except the decay of the pH 8.0 green channel (which was 0.24 s−1). Black lines are fits to exponential increase for the red channel (1-exp(-αt)) and decrease (exp(-αt)) for the green. Error bars represent the SEM of three trials at pH 5.0 and two at pH 8.0. Plotted data points are the average pixel values across the entire field of view which estimates the total photo-product produced. The 405 nm power at the sample was at 2.5 mW, corresponding to 6.8 MW/cm2 at the focus of the 1.2 NA objective lens. EGFP (30 mM) was subjected to a sequence of 10 “bleach” iterations over the ROI (1.8 seconds) followed by image acquisition using 488 and 561 nm illumination. (d) Time course of the increase in red fluorescence with successive 405 nm irradiations within the region of interest (ROI). (c) Absorption spectra of EGFP (~2 μM) at pH 5.0 and pH 8.0. (b) Emission spectra of photoconverted red state EGFP acquired using the Zeiss 710 spectral detector (5 nm bandwidth intervals). 10 iterations of 257 ms each were performed.

#PHOTOCONVERT LINAGE TRACKING SOFTWARE#

(a) Typical pre- and post-photoconversion images of a thin (~10 μm) layer of 30 μM EGFP using 405 nm excitation delivered through a 1.2 NA objective to the region within the circle using the Zen software bleaching mode (Green channel: 505–525 nm Red channel: 580–670 nm scale bar: 50 μm). Photoconversion of purified EGFP in vitro. While genes encoding fluorescent proteins specifically designed for photoconversion will usually be advantageous when creating new transgenic lines, our method for photoconversion of GFP allows the use of existing GFP-tagged transgenic lines for studies of dynamic processes in living cells. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. However, before genes encoding these fluorescent proteins were available, many proteins have already been labelled with GFP in transgenic cells a number of model organisms feature collections of GFP-tagged lines and organisms. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. A variety of fluorescent proteins have been identified that undergo shifts in spectral emission properties over time or once they are irradiated by ultraviolet or blue light.

Photoconvert linage tracking