Re: 2d To 3d Photo Conversion Software Mac
Herta Adel
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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. Such proteins are finding application in following the dynamics of particular proteins or labelled organelles within the cell. 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. Here we describe a fast, localized and non-invasive method for GFP photoconversion from green to red. We demonstrate its use in transgenic plant, Drosophila and mammalian cells in vivo. 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.
Fusions of Green Fluorescent Protein (GFP) and its derivatives are extremely valuable tools for examining gene expression, protein and RNA localization, protein-protein interactions, protein synthesis and degradation and organelle movement. The development of photoconvertible fluorescent proteins such as mEosFP, tdEosFP, Dendra, Dronpa, Kaede, KikGR, mOrange allows new questions to be asked concerning dynamic processes in living cells1,2,3,4,5,6. However, a number of collections of GFP-tagged organisms had been made before these proteins became available for use. For example, a collection of strains ( ) expressing full-length GFP-tagged proteins is available for yeast7,8. In Drosophila, there is a collection ( ) in which GFP has tagged full-length endogenous proteins expressed from their endogenous loci or used in an enhancer trap9,10. In Arabidopsis, lines are available with GFP tags on transcription factors11, with GFP as promoter-reporters11 and with GFP labels on subcellular structures12. In addition to specific collections, many investigators with interests in particular proteins or subcellular locations have produced cell lines or organisms with GFP tags. For example, various transgenic mouse lines ( ) have been produced with GFP labels on proteins13; retransforming such lines with genes encoding new photoconvertible proteins would require considerable time and expense. Because many proteins have already been tagged with EGFP or GFP modified by an S65T mutation, being able to photoconvert these common forms of GFP would allow the use of existing transgenic lines for studies of dynamic changes.
(a) Alignment of the coding region of the GFP variants. The alignment was created using GeneDoc ( ). (b) Schematic representation of GFP transgenes used in photoconversion experiments. Pr1b-SP, secretory signal peptide from the tobacco pathogenesis-related protein 1; EGFP, enhanced green fluorescent protein; L, linker (GGGS)3; KDEL, endoplasmic reticulum retrieval signal peptide; HttQ103, exon 1 of human HttQ103 gene; Cytosolic mGFP4-T in tobacco cell culture and cytosolic S65T-GFP in Drosophila gut cells. In cytosolic mGFP4-T, V (valine) was replaced by A (alanine) and Q (glutamine) was replaced by R (arginine) relative to EGFP. In cytosolic S65T-GFP, L (leucine) was replaced by F (phenylalanine) relative to EGFP.
We could also observe the photoconversion of EGFP in other eukaryotic model organisms, including Drosophila and rat cells. We obtained a Drosophila line in which S65T-GFP was expressed in gut cells from 3rd instar larvae20 (Fig. 4d) and a rat PC12 cell line expressing EGFP fused to exon 1 of the human Huntington poly Q (HttQ103) gene21 (Fig. 4e). When Drosophila or rat cells were irradiated using conditions similar to those that were effective for plant cells, we observed photoconversion from the green to red state (Fig. 4d,e).
Transient expression methods are also frequently used to study the subcellular localization of GFP-tagged proteins. In plants, several methods exist for transient expression, including particle bombardment, infiltration of Agrobacterium tumefaciens (Agroinfiltration), or protoplast transfection. In addition to photoconversion of stably transformed tobacco cells, we were able to photoconvert EGFP that was transiently expressed in Nicotiana benthamiana epidermal leaf cells by Agroinfiltration (Fig. 5, Supplementary Movies S3-4). We were able to label the endoplasmic reticulum (ER) with GFP targeted to the ER and observe movement through the ER (Fig. 5a). Also, GFP located in the cytosol could be photoconverted and we could follow its movement through the cell (Fig. 5b).
Variant fluorescent proteins that have been designed for photoconversion applications will usually be superior to GFP in terms of rapidity of shift in emission and magnitude and may be brighter at low concentration. Thus, the primary advantage of using GFP in photoconversion is the ability to use existing transgenic lines.
Another benefit of GFP-based photoconversion is the absence of any random or auto-photoconversion in our tested samples. For some of the non-GFP-based photoconverting proteins, auto-photoconversion has been reported. For example, in mEosFP-cytosolic plants grown in bright fluorescent white light, up to a quarter of hypocotyl epidermal cells contained red nuclei and in cells transiently expressing cytosolic mEosFP, auto-conversion was observed without any intentional photoconversion8. Another concern involved with the use of mEosFP is the partial photoconversion under short exposure times. Photoconversion of mEosFP happens in a concentric manner, therefore producing variability in shades, which is problematic since both partial photoconversion and co-localization of the green and red will produce yellow hues29.
How to cite this article: Sattarzadeh, A. et al. Green to red photoconversion of GFP for protein tracking in vivo. Sci. Rep. 5, 11771; doi: 10.1038/srep11771 (2015).
Metadata management is also at the heart of Avalanche. All Exif and IPTC metadata will correctly flow from your source catalog to your new photo editing software you want to move in. Flags, color labels, GPS information are properly handled too.
In one evening the migration of 25 200 photos (121GB) to a Lightroom catalog was done, and all the photos were rightthere at my fingertips, organized in the same original folders. Genius!Not only that, the software looks slick and professional, and is easy to use.Amazing product, highly recommended!
Avalanche is designed to convert catalogs of photographs from one application to another. For example, if you wish to move your pictures that are currently cataloged in Apple Aperture to another app, such as Adobe Lightroom, Avalanche will do exactly that.
Currently, Avalanche migrates the photographs from your source catalog into a new, initially empty, catalog it creates. When exporting to Lightroom, you can then use the Lightroom catalog merging feature to merge that newly created catalog into your master catalog if you have one.
Avalanche is available for Lightroom, Luminar or Capture One. Avalanche Unlimited compiles the 3 versions into one. All the versions of Avalanche support the same input sources: Aperture, Lightroom, Luminar 4/AI, exports from Google photo, Aperture or even iView MediaPro.
I can do this conversion for you for your previous photo sessions, too! I recently did black and white conversions from a client session that was 4 years ago! Check out this screenshot. Brings new life to that old edit for sure!!
Finally, there were some portraits I took this year (some in Iceland) where I had specifically planned to edit them only using this black and white processing. For these images, the subjects dressed in light clothing and were photographed against dark backgrounds to make the images even more eye catching. Enjoy!
I am a photographer in Northern Virginia specializing in portraits of newborn babies, children and families. My photography has been featured in Northern Virginia Magazine, Front Porch Living magazine, on BabyCenter.com and NewbornPhotography.com, among others. My work is currently on display at Premier Birth Center in Chantilly, VA, South Riding Pediatrics in South Riding, VA, NOVA Birth Partners in Stone Ridge, VA, and the Inova Children's Hospital NICU in Fairfax, VA.
Photo-conversion of hWJCs from green fluorescence to red fluorescence was very robust. hWJCs were gently exposed to UV light at 100 mW every 10 s at a frequency of 1 Hz for 300 s. Green fluorescence intensity and red fluorescence intensity were measured in real-time as hWJCs were photo-converted. There was a rapid decrease in green fluorescence intensity as red fluorescence intensity increased. The critical moment where red fluorescence eclipsed green fluorescence occurred at 125 60 s. Please refer to Supplementary Figure 1 to view the full time-lapse video of a single recording of the photo-conversion that is representative of all attempts. The results displayed were fully consistent with nine attempts to photo-convert cells from three different umbilical cords. AU = Arbitrary Unit; Scale Bar = 100 μm
Individual hWJCs were targeted for photo-conversion. The left column displays the fluorescence of cells before photo-conversion. The right column displays cells after photo-conversion. The amount of Dendra2 produced in each cell varied, thus some targeted cells displayed a strong expression of red fluorescence while other cells display a weak expression of red fluorescence after photo-conversion. The images displayed are a random collection of images taken from three different (n = 3) different populations of hWJCs transfected with Dendra2. Scale Bar = 100 μm.
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