Near Infrared Fluorescent Protein for Multicolor Invivo Imaging

Presented by, Siddhant Mohapatra

So, what is the paper about ?

Near Infrared Fluorescent Proteins are in boom

Exhibit higher brightness in cells and tissues

Long term uses are suggested 2 color as well as multicolor imaging iRFP670, 682,702,720 from bacterial

phytochromes

Why Near Infrared ?

Infrared region (700nm -1mm)

Through optical imaging in Mammalian tissues

require iRF probes Why not white light ? Water, melanin and hemoglobin make it more transparent iRF introduces less scattering and less auto fluorescence

Bacterial Phytochromes
They are promising for engineering NIF FPs

Found in fungi, bacteria, cyanobacteria

Use tetrapyrroles as photoreceptors They have the most red shifted absorption

spectra They use biliverdin IV@ which is found in mammalian cells Adopt 2 different photochemical states- Far, near and red absorbing Deletion of various domains, followed by mutations and sometimes injection of BV

Continued
Rhodopseudomonas palustris is used as a

template to engineer Absorption maximum 700nm The PAS and GAF domains were used to incorporate BV Followed by mutation and various insertions in Hela cells Various proteins were expressed and their brightness was judged based on the proper folding of the proteins

Variants
Irf702 -673(E) and 702(EM)

3 Variants were chosen based on random

mutations and additions Irf 670/720/682 Absorption spectra showed they incorporated BV chromophore These variants covered around 50nm of NIR region

Data 1-Properties

Observations
The spectrum tells us about the absorption and

emission maximum Tells us at wavelength do they incorporate BV which is lower than the maxima peaks It will also give us an idea about the range of NIR High signal was obtained for IRFP 713 which needed no addition of BV The stability of all IRFPs was lower than IRFP713 A phantom model was introduced to judge the auto fluorescence and light scattering properties

 Through imaging techniques the ratio of

fluorescence by auto fluorescence was calculated The ratios were for each of the IRFs were higher than the FP

Fig1

Fluorescence Toxicity
In long term studies for the study of cell growth

for generations toxicity factor has to be taken into account Then various IRFPs and GFPs were introduced into cells The fluorescence was calculated in 2 stages By comparing the fluorescence produced, signal retention was calculated It showed that IRFPs retained more than that of GFPs

Whole Body Imaging

Mouse xenograft breast cancer model was taken

MTLn3 Cells were expressed in the mammary

glands of the mice and then grown into tumors It enabled detection of tumors at early stages Growth of the tumors was constantly monitored upto 4 weeks For the IRFPs the growth of signal increased in each stage in comparison to FPs The IRFs were then introduced into living mice (670 and 720)

IRFPs- Depth factor

The IRFPs were grown as conjoined tumors

The tissues were distinguished using filter

channels Then 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 liver Using imaging techniques the location was distinguished efficiently

Different Organelles
HeLa cells expressing 2 different IRFPs in

nucleus and mitochondria was seen Detection of 2 types of cells or tissues with spectral imaging techniques 4 types of cells with different IRFPs were taken and detected via confocal microscopy and flow cytometry In living mice, all the tumors can be unmixed and can even be detected at a later stage

Fig 2

Biotechnology Perspective
The effective brightness depends on molecular

brightness, stability, affinity for BV and protein expression level suggests the effective use of IRFPs It promises us whole cell labeling in applications using standard red lasers Dealing with toxicity is still an issue to consider

References
Near Infrared Fluorescent Protein for Multicolor

Invivo Imaging-Shcherbakova D.M, Verkhusha V.V