Near Infrared Fluorescent Protein for Multicolor Invivo Imaging

Presented by,Siddhant MohapatraNear Infrared Fluorescent Protein for Multicolor Invivo ImagingSo, what is the paper about ?Near Infrared Fluorescent Proteins are in boomExhibit higher brightness in cells and tissuesLong term uses are suggested2 color as well as multicolor imagingiRFP670, 682,702,720 from bacterial phytochromesWhy Near Infrared ?Infrared region (700nm -1mm)Through optical imaging in Mammalian tissues require iRF probesWhy not white light ? Water, melanin and hemoglobin make it more transparentiRF introduces less scattering and less auto fluorescence

Bacterial PhytochromesThey are promising for engineering NIF FPsFound in fungi, bacteria, cyanobacteriaUse tetrapyrroles as photoreceptorsThey have the most red shifted absorption spectraThey use biliverdin IV@ which is found in mammalian cellsAdopt 2 different photochemical states- Far, near and red absorbingDeletion of various domains, followed by mutations and sometimes injection of BVContinued Rhodopseudomonas palustris is used as a template to engineerAbsorption maximum 700nmThe PAS and GAF domains were used to incorporate BVFollowed by mutation and various insertions in Hela cellsVarious proteins were expressed and their brightness was judged based on the proper folding of the proteins

VariantsIrf702 -673(E) and 702(EM)3 Variants were chosen based on random mutations and additionsIrf 670/720/682Absorption spectra showed they incorporated BV chromophoreThese variants covered around 50nm of NIR region

Data 1-Properties

ObservationsThe spectrum tells us about the absorption and emission maximum Tells us at wavelength do they incorporate BV which is lower than the maxima peaksIt will also give us an idea about the range of NIRHigh signal was obtained for IRFP 713 which needed no addition of BVThe stability of all IRFPs was lower than IRFP713A phantom model was introduced to judge the auto fluorescence and light scattering propertiesThrough imaging techniques the ratio of fluorescence by auto fluorescence was calculated The ratios were for each of the IRFs were higher than the FPFig1

Fluorescence ToxicityIn long term studies for the study of cell growth for generations toxicity factor has to be taken into accountThen various IRFPs and GFPs were introduced into cells The fluorescence was calculated in 2 stagesBy comparing the fluorescence produced, signal retention was calculatedIt showed that IRFPs retained more than that of GFPsWhole Body ImagingMouse xenograft breast cancer model was takenMTLn3 Cells were expressed in the mammary glands of the mice and then grown into tumorsIt enabled detection of tumors at early stagesGrowth of the tumors was constantly monitored upto 4 weeksFor the IRFPs the growth of signal increased in each stage in comparison to FPsThe IRFs were then introduced into living mice (670 and 720)IRFPs- Depth factorThe IRFPs were grown as conjoined tumorsThe tissues were distinguished using filter channelsThen further to investigate if IRFPs were able to be distinguished at different depths, a mammary gland expressing a IRFP was infected with another IRFP at the liverUsing imaging techniques the location was distinguished efficiently

Different Organelles HeLa cells expressing 2 different IRFPs in nucleus and mitochondria was seenDetection of 2 types of cells or tissues with spectral imaging techniques4 types of cells with different IRFPs were taken and detected via confocal microscopy and flow cytometryIn living mice, all the tumors can be unmixed and can even be detected at a later stage

Fig 2

Biotechnology PerspectiveThe effective brightness depends on molecular brightness, stability, affinity for BV and protein expression level suggests the effective use of IRFPsIt promises us whole cell labeling in applications using standard red lasersDealing with toxicity is still an issue to considerReferencesNear Infrared Fluorescent Protein for Multicolor Invivo Imaging-Shcherbakova D.M, Verkhusha V.V Thank you