CN104745177A - Light activated fluorescent probe having protein label positioning function as well as preparation method and application thereof - Google Patents
Light activated fluorescent probe having protein label positioning function as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a light activated fluorescent probe having a protein label positioning function, and further discloses a preparation method and application thereof. The light activated fluorescent probe having the protein label positioning function has excellent photophysical properties, such as high-efficiency light control property and high fluorescence quantum yield, and characteristics, such as simple small molecule dye synthesis; furthermore, in combination with a chemical label technology, the light activated fluorescent probe is capable of positioning target molecular specificity in living cells precisely; real-time super-resolution fluorescence imaging in the living cells can be realized; and therefore, the light activated fluorescent probe has important application value in the field of super-resolution, especially super-resolution imaging in the living cells.
Description
Technical field
The present invention relates to fluorescent probe technique field, be specifically related to a kind of photoactivation fluorescent probe and its preparation method and application with protein tag location.
Background technology
In biological study field, opticmicroscope is a kind of being widely used and indispensable imaging tool.Compared with other microscopes, opticmicroscope, especially fluorescent microscope has unique advantage: non-invasive (allowing viable cell, tissue and organic observation) and high precision (by fluorescent mark, carrying out accurate certain observation to targeted molecular).We know, conventional opticmicroscope is if confocal fluorescent microscope is due to the diffraction of light wave, and namely by the restriction of diffraction limit, its limiting resolution could not break through 200nm all the time.In order to study the constitutional features of molecule nano yardstick in born of the same parents, improve the research direction that opticmicroscope resolving power becomes scientists.In recent years, scientists is invented and has been developed many super-resolution fluorescence microscopies to overcome the barrier of diffraction limit.Random reconstruction optical microscopy (the STORM) (Rust that such as Harvard University professor Zhuan little Wei proposes, M.J., Bates, M., and Zhuang, X. (2006) .Nat.Methods3,793-795), in single molecular fluorescence technical foundation, successfully breach diffraction limit by means such as photoactivation, optics reconstructions, reach the fluorescence imaging of nanoscale.Also has photoactivation position finding microscope (PALM) (the Betzig E that Eric Betzig proposes, PattersonGH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, DavidsonMW, Lippincott-Schwartz J, Hess HF (2006) .Science 313 (5793): 1642 – 1645), the fluorescence imaging of nanoscale is also successfully reached by photoactivation fluorescin.But no matter super-resolution fluorescence imaging is PALM or STORM technology, its core hinge is all the fluorescent probe with light regulation and control luminescence feature, and light regulation and control luminescence mainly refers to photoactivation and light conversion (reversible and irreversible).At present, be applicable to super-resolution imaging probe mainly: 1) there is the fluorescin that light regulation and control are luminous; 2) there is the small molecule dyes of light adjusting function.The former normally realizes visual by gene fusion technique express fluorescent protein in biological cell, and the latter mainly depends on chemosynthesis.
The fluorescin luminous due to light regulation and control has very high specificity, is widely developed and is applied to super-resolution imaging field in the last few years.Ratio is if any the irreversible conversion photoactivation fluorescin (PAFPs) by " secretly " change " bright ": photoactivation green fluorescent protein (PA-GFP), red fluorescent protein derivative (PAmRFP1 and PAmCHerry1) etc.; The reversible transformation PAFPs:FP595 of fluorescence ON/OFF, Dronpa and mutant Padron etc. thereof; Fluorescence color irreversible conversion PAFPs:Kaede, Monomeric Eos, KiKGR etc.Although fluorescin new these years emerges in an endless stream, but fluorescin still has some obvious defects, the size of such as fluorescin is relatively large, the physiological function being labeled albumen may be disturbed, affect the accuracy of fluorescence localization, in addition, the fluorescence quantum yield of fluorescin is lower simultaneously, light stability is poor, even if the brightest color irreversible conversion PAFPs is if Eos is than the low lightness of some small molecule fluorescent dyestuffs.
Compared with above-mentioned fluorescin, light regulation and control small molecule fluorescent dyestuff presents many advantages: excellent Photophysical Behaviors, the less spatial accuracy not affecting fluorescence localization of size, synthesis is simple, and color category is many etc.In theory, any suitable fluorophore with light conversion characteristic can be used to super-resolution fluorescence imaging field.But current, the small molecule fluorescent dyestuff be applied in the super-resolution fluorescence imaging field Cy dyestuff (Cy2-Cy7) that mainly light regulation and control are luminous, there is photochromic Rhodamine Derivatives dyestuff and Alexa Fluor series dyes.In random super-resolution imaging, the Cy dyestuff of light reversible transformation most widely used.Village little Wei study group utilizes Cy3-Cy5 probe to rebuild super-resolution imaging to being achieved random optical by immunofluorescence label method.But, this probe system still has many deficiencies to be worth us to note: 1) by immunofluorescence label method, and Cy3-Cy5 probe is to being marked on antibody, and antibody does not have cross-film ability, therefore can only be applied to fixed cell or surface of cell membrane, viable cell imaging can not be used for; 2) Cy3-Cy5 probe is changed the mechanism not clear to light, and very harsh to the environmental requirement of surrounding, because the conversion of its light needs just can carry out under the condition of reduction, this just needs to add the reductive agent containing sulfydryl and oxygen scavenger; 3) operation easier is larger, because the distance of Cy3 and Cy5 will meet 1-3nm just can complete pairing.In addition, compare fluorescin, although small molecule fluorescent dyestuff has numerous advantage, also there are some defects: 1) it is for chemosynthesis, and external source joins in cell, and its specific marker degree is lower, often need a large amount of washing and fluorescence background is higher; 2) many small-molecule fluorescent probe (as Sulfonated Cy dyestuff) do not have spontaneous cross-film ability, and this limits it can only be applied to fixed cell or surface of cell membrane.
For the deficiency of above-mentioned existing fluorescent probe, present inventor proposes a kind of photoactivation fluorescent small molecule probe and preparation method thereof with protein tag location.Low in order to overcome above-mentioned small-molecule fluorescent probe specific marker degree, and the defect such as fluorescin own vol is excessive, photophysical property is poor, this fluorescent probe is in conjunction with chemical tags technology, it accurately can be located target molecule in viable cell, realize real-time super-resolution fluorescence imaging in viable cell.Simultaneously this probe has the features such as efficient light control, the photophysical property of the high excellence of fluorescence quantum yield and small molecule dyes synthesis be simple.
Summary of the invention
An object of the present invention is to provide a kind of photoactivation fluorescent probe with protein tag location.
Another object of the present invention is to provide the preparation method of above-mentioned photoactivation fluorescent probe.
Another object of the present invention is to provide the application of above-mentioned photoactivation fluorescent probe in viable cell super-resolution imaging.
There is a photoactivation fluorescent probe for protein tag location, there is the structure shown in following general formula:
Wherein, R
1be selected from following substituting group :-OH ,-NMe
2,-NEt
2,-OCH
2cOOH etc.; R
2be selected from following substituting group: halogenic substituent F, Cl, Br, I etc.; R
3and R
4be selected from the substituting group of following group: halogen atom as F, Cl, Br, I etc., H, alkyl, methylol, sulfonic group, alkylene etc.; R
5be selected from the label-ligand substituting base for target location: Halo-tag, SNAP-tag, TMP-tag, CLIP-tag etc.
A kind of preparation method with the photoactivation fluorescent probe of protein tag location of the present invention comprises the following steps:
(1) under reflux conditions, 5 (6)-Fluoresceincarboxylic acids are reacted about 2h in diacetyl oxide, reaction terminates, and purification thing is dissolved in the trimethyl carbinol after purifying by column chromatography, and adds DMAP and Boc
2o, reaction overnight under 40 degree of conditions, reaction terminates rear pillar Chromatographic purification.Purification thing is sloughed under THF neutral and alkali condition two ethanoyl protections, simple rear drying in oven of purifying dewaters.To be dissolved in by products therefrom in THF again and to add and activate nitroxyl chloride phenyl formate, the rear pillar Chromatographic purification that reacts completely obtains product A;
(2) tonka bean camphor and tin anhydride are reacted 24h under reflux conditions in p-Xylol, after simple purification, purification thing is dissolved in methyl alcohol after reacting completely, reduce with a certain amount of sodium borohydride, after reacting completely, column chromatography is purified, purification thing is dissolved in anhydrous methylene chloride, activate under condition of ice bath Yu to nitroxyl chloride phenyl formate, reaction terminates directly in system, to add N-methyl-N '-tertiary oxygen carbonyl reacting ethylenediamine again and obtain product afterwards, column chromatography obtains product after purifying, purification thing is also obtained by reacting product under further anhydrous methylene chloride and trifluoroacetic acid volume ratio 3:1 condition, and obtain B after simple purification,
(3) above-mentioned product A and product B are reacted in anhydrous methylene chloride, column chromatography obtains product after purifying, again purification thing is sloughed the tertiary butyl under anhydrous methylene chloride and trifluoroacetic acid volume ratio 1:1 condition and obtain product, and obtain product C through column chromatography purification
(4) final product C and part are bonded together, the whole photoactivation fluorescent probe molecule with protein tag location of final synthesis.
The above-mentioned application with photoactivation fluorescent probe super-resolution imaging field in viable cell of protein tag location.
Fluorescent probe provided by the invention is a kind of small-molecule fluorescent probe of photoactivation type, owing to make use of mature chemical tags technology, acceptor (Halo-tag, dehalogenase) and part (halogenated alkane) specific combination, fluorescent probe will specific target-marking molecule.The main light emitting host of this fluorescent probe is fluorescein, but based on Intramolecular electron transfer (ICT) mechanism, hydroxy-end capped fluorescein itself is not luminous, probe original state sends the blue-fluorescence of tonka bean camphor, but under 380nm illumination, tonka bean camphor 4 ester bond generation light fractures, to the end-blocking event resolves of fluorescein, now fluorescein is activated and is recovered autofluorescence, will observe bright fluorescein green glow under 490nm irradiates.By STORM technology, this probe can be used for realizing super-resolution imaging.
The present invention compared with prior art has following innovative point:
(1) the invention discloses a kind of synthetic method with the photoactivation fluorescent probe of protein tag location, raw material sources are very extensive, and application cost is low, and preparation method is simple;
(2) the photoactivation fluorescent probe with protein tag location disclosed by the invention is a kind of activated form fluorescent probe, and photoactivation speed is fast, and fluorescence quantum yield is high;
(3) the photoactivation fluorescent probe with protein tag location disclosed by the invention is a kind of small-molecule fluorescent probe, and relative to fluorescin, volume is less, can not affect the physiological function of target molecule;
(4) the photoactivation fluorescent probe with protein tag location disclosed by the invention, can only compared with the probe (as Cy3-Cy5) doing super-resolution fluorescence imaging in fixed cell with majority, this probe utilizes chemical tags technology, by acceptor (tag) and target protein co expression, by the part on probe and its specific binding, realize high precision specific localization, and complete super-resolution imaging in viable cell.
Accompanying drawing explanation
Fig. 1 is the synthetic route chart of the photoactivation fluorescent probe with protein tag location.
Fig. 2 is the uv-visible absorption spectrum figure of the photoactivation fluorescent probe photoactivation process with protein tag location.At 10mw/cm
2under the irradiation of the 365nm light of light intensity, through about 10min, fluorescein reaches top value at the absorption peak of about 490nm, and this illustrates that tonka bean camphor 4 ester bonds rupture, N, and N '-dimethyl quadrol from eliminating, discharges fluorescein fast.
Fig. 3 is the fluorescent emission spectrogram of the photoactivation fluorescent probe photoactivation process with protein tag location.As can be seen from the figure the background fluorescence of probe is very low, at 10mw/cm
2under the irradiation of the 365nm light of light intensity, through about 10min, the fluorescence intensity of fluorescein enhances 220 times, achieves the photoactivation of fluorescence.
Fig. 4 uses the photoactivation fluorescent probe with protein tag location to carry out image checking in viable cell, the cell strain used is 293 cells that have expressed NLS-Halo label in advance in core, 10uM probe of the present invention and cell incubation 30min, wash 3 times afterwards, carry out photoactivation fluorometric investigation with microscope light source.Imaging data cell compartment and background area quantitative analysis are found that probe effectively can enter cell, and specific localization is in nucleus, and probe fluorescence when illumination is about about 10min reaches maximum, illustrate that probe can effectively be chemically activated by light in viable cell, be therefore expected to the super-resolution imaging be applied in viable cell.
Embodiment
For a more detailed description to the present invention by embodiment below.Below in conjunction with accompanying drawing and embodiment, the invention will be further described, do not have any restriction to scope of the present invention.
Embodiment 1:
Have the synthetic method of the photoactivation fluorescent probe of protein tag location, concrete molecule is made up of following structural formula I:
Concrete synthesis step is as follows:
(1) add 35g Resorcinol and 240ml methylsulphonic acid in 500ml round-bottomed flask, under nitrogen protection, add 30g trimellitic anhydride, after mixing, at 145 DEG C, stir 24h; pour frozen water into after cooling, filter, washing; drying, ethyl alcohol recrystallization obtains 30g, yield 60%.Products therefrom 5 (6)-Fluoresceincarboxylic acid, gets 5g 5 (6)-Fluoresceincarboxylic acid (12.8mmol) and is placed in 50mL round-bottomed flask after drying, then add 24mL Ac
2o (0.26mol), reflux 3h, is cooled to room temperature after complete reaction, is then poured into by reaction solution in about 200mL frozen water, extraction into ethyl acetate (150mL × 3), organic phase washing (150mL × 3), anhydrous Na
2sO
4drying, filters, and vacuum rotary steam, except desolventizing, obtains faint yellow solid A 4.0g (productive rate 80%) after crude product purified by silica gel chromatographic column (DCM:MeOH=100:0 ~ 100:3) purifying.
1HNMR(CDCl
3，400MHz)：δ＝8.40(m，J＝8.0Hz，1H)，8.15(d，J＝8.0Hz，1.2Hz，0.5H)，7.90(s，0.5H)，7.30(s，1H)，7.10(s，2H)，6.80(m，J＝8.0Hz，4H)，2.31(s，6H)；
13CNMR(CDCl
3，100MHz)：171.6，169.0，168.4，157.4，152.2，151.5，136.9，135.6，131.9，128.8，127.6，126.0，125.5，124.5，118.0，115.5，110.7，60.5，21.3；MS(ESI)：461.1[M+H]
+。
(2) 0.5g A (1.1mmol) in 250ml round-bottomed flask, is added 70ml in 50mL round-bottomed flask
tbuOH dissolves, and then adds 1.53ml Boc
2o (7.0mmol) and 0.25gDMAP (2.0mmol), stirs 10h under 40 DEG C of conditions, and vacuum rotary steam is except desolventizing, obtain viscous liquid, be extracted with ethyl acetate again (100mL × 3), organic phase saturated common salt washing (100mL × 2), anhydrous Na
2sO
4drying, filters, and vacuum rotary steam is except desolventizing, and crude product purified by silica gel chromatographic column (PE:EA=4:1) purifying obtains white crystal B 0.35g (productive rate 70%).
1HNMR(CDCl
3，400MHz)：δ＝8.62(s，1H)，8.30(dd，J
1＝6.4Hz，J
2＝7.2Hz，1H)，7.25(d，J＝4.8Hz，1H)，7.10(s，2H)，6.80(m，J＝8.0Hz，4H)，2.31(s，6H)，1.65(s，9H)；
13CNMR(CDCl
3，100MHz)：169.6，163.9，156.1，155.0，152.7，151.2，136.5，134.5，129.3，126.6，125.0，124.2，117.8，115.6，110.5，83.0，82.5，28.5，21.3；MS(ESI)：517.14[M+H]
+。
(3) 0.2g compd B (0.39mmol) is placed in 25mL round-bottomed flask, add 5ml methyl alcohol/5ml tetrahydrofuran (THF) and 0.08g NaOH (2mmol) dissolving, normal-temperature reaction 10min, vacuum rotary steam is except desolventizing, suitable quantity of water is dissolved, under agitation, dilute hydrochloric acid adjust pH to 2 ~ 3, separate out precipitation, filter, filter cake is washed, and dry at vacuum constant temperature loft drier, and crude product purified by silica gel chromatographic column (DCM:MeOH=100:3) purifying obtains yellow solid C 0.13g (productive rate 70%).
1HNMR(DMSO，400MHz)：δ＝11.20(s，1H)，8.36(s，1H)，8.26(s，1H)，7.40(s，1H)，6.70(s，2H)，6.60(d，J＝8.0Hz，2H)，6.55(m，3H)，1.52(s，9H)；
13CNMR(DMSO，100MHz)：169.5，165.0，160.2，157.0，153.6，137.4，130.1，127.0，126.3，125.4，114.2，110.1，103.6，84.2，83.0，49.8；MS(ESI)：433.1[M+H]
+。
(4) 0.2g Compound C (0.46mmol) is placed in 50mL round-bottomed flask, add 10ml dry tetrahydrofuran dissolve and add 0.1ml dry triethylamine, again 0.1g is joined in 25ml constant pressure funnel to nitroxyl chloride phenyl formate (0.49mmol), and dissolve by 10ml dry tetrahydrofuran, constant pressure funnel is placed on round-bottomed flask, drip with 1/2s speed, 30min is reacted under condition of ice bath, vacuum rotary steam is except desolventizing, and crude product purified by silica gel chromatographic column (PE:EA:DCM=3:1:4) purifying obtains white crystal D 0.15g (productive rate 54%).0.12g7-N, N-diethyl-4-(N, N '-dimethyl quadrol acid methyl esters) tonka bean camphor (0.33mmol) is in 10ml DCM and 4ml TFA mixing solutions, reaction 0.5h removal of solvent under reduced pressure and excessive acid, then be directly dissolved in 10ml DCM with above-mentioned 0.15g D and 0.1ml triethylamine, room temperature reaction 1h, removal of solvent under reduced pressure, crude product purified by silica gel chromatographic column (DCM:MeOH=100:0.8) is purified, and obtains 0.12g E (productive rate 60%).
1HNMR(CDCl
3，400MHz)：δ＝8.55(s，1H)，8.15(m，1H)，7.27(m，2H)，7.15(m，1H)，6.75(m，2H)，6.68(m，1H)，6.50(m，4H)，6.10(m，1H)，5.27(m，2H)，3.60(m，4H)，3.36(t，J＝8.0Hz，4H)，3.10(m，6H)，1.60(m，9H)，1.15(t，J＝5.2Hz，6H)；
13CNMR(CDCl
3，100MHz)：164.0，159.2，156.0，156.1，151.9，136.0，134.1，129.0，130.1，124.3，113.1，109.0，106.2，102.8，97.6，82.4，62.6，47.6，46.8，44.7，35.5，35.0，34.8，34.5，33.1，31.9，31.5，30.2，29.6，29.0，28.0，27.0，25.2，22.7，20.7，20.5，19.6，18.8，14.2，12.4，11.4，9.9；MS(ESI)：820.3[M+H]
+。
(5) 0.1g E (0.12mmol) is in 10mlDCM and 10mlTFA mixing solutions, room temperature reaction 2h, removal of solvent under reduced pressure crude product purified by silica gel chromatographic column (DCM:MeOH=100:2) is purified, and obtains 80mgF (productive rate 86%); Same method, 50mg Halo part (0.15mmol) is 0.5h under 10ml DCM and 2ml TFA room temperature, removal of solvent under reduced pressure, obtain product G directly and 80mg F dissolve with DMF in 25ml round-bottomed flask, add phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl 100mg (0.19mmol) and 0.1ml TEA again, room temperature reaction 1h, removal of solvent under reduced pressure crude product purified by silica gel chromatographic column (DCM:MeOH=100:1) is purified, and obtains 70mg (productive rate 70%).
1HNMR(CDCl
3，400MHz)：δ＝8.40(s，1H)，8.15(m，1H)，7.65(m，1H)，7.30(m，1H)，7.08(m，1H)，6.68(m，3H)，6.55(m，3H)，6.45(m，1H)，5.27(m，2H)，3.70(m，4H)，3.60(m，6H)，3.48(m，4H)，3.35(m，6H)，3.15(m，3H)，3.05(m，3H)，3.00(m，2H)，1.85(m，3H)，1.75(m，3H)，1.55(m，2H)，1.38(m，3H)，1.15(t，J＝4Hz，6H)；
13CNMR(CDCl
3，100MHz)：170.0，160.2，151.0，130.1，124.9，116.0，105.1，103.0，97.1，83.3，72.1，71.0，69.2，58.8，47.6，46.4，45.0，40.6，32.5，29.7，26.5，25.0，18.8，12.5；MS(ESI)：991.3[M+Na]
+。
Embodiment 2
There is the uv-absorbing of photoactivation fluorescent probe in methyl alcohol and the photoluminescent property of protein tag location.In methyl alcohol, (concentration is 10 to fluorescent probe of the present invention
-5mol/L) ultraviolet visible absorption spectra figure-Fig. 2 (0 minute curve) and fluorescent emission spectrogram-Fig. 3 (0 minute curve).
Fig. 2 is the uv-visible absorption spectrum figure of probe light activation process of the present invention.At 10mw/cm
2under the irradiation of the 365nm light of light intensity, through about 10 minutes, fluorescein reaches top value at the absorption peak of about 490nm, and this illustrates that tonka bean camphor 4 ester bonds rupture, N, and N '-dimethyl quadrol from eliminating, discharges fluorescein fast.
Fig. 3 is the fluorescent emission spectrogram of probe light activation process of the present invention.As can be seen from the figure the background fluorescence of probe is very low, at 10mw/cm
2under the irradiation of the 365nm light of light intensity, through about 10 minutes, the fluorescence intensity of fluorescein enhances 220 times, achieves the photoactivation of fluorescence.
Embodiment 3
The photoactivation fluorescent probe with protein tag location carries out image checking in viable cell.Utilize gene fusion technique by after target protein and Halo-tag (dehalogenase) amalgamation and expression, in physiological conditions just can by direct for Halo-tag part (this part is on probe of the present invention) specific marker on the Halo-tag of fusion rotein.
Fig. 4 uses the photoactivation fluorescent probe with protein tag location to carry out image checking in viable cell, the cell strain used is 293 cells that have expressed NLS-Halo label in advance in core, 10uM probe of the present invention and cell incubation 30min, wash 3 times afterwards, carry out photoactivation fluorometric investigation with microscope light source.Imaging data cell compartment and background area quantitative analysis are found that probe effectively can enter cell, and specific localization is in nucleus, and probe fluorescence when illumination is about about 10min reaches maximum, illustrate that probe can effectively be chemically activated by light in viable cell, be therefore expected to the super-resolution imaging be applied in viable cell.
Claims (3)
1. have a photoactivation fluorescent probe for protein tag location, it is characterized in that, described fluorescent probe is made up of following structural formula I:
2. there is described in claim 1 preparation method of the photoactivation fluorescent probe of protein tag location, it is characterized in that, comprise the following steps:
Under step one, reflux conditions, 5 (6)-Fluoresceincarboxylic acids are reacted about 2h in diacetyl oxide, reaction terminates, and purification thing is dissolved in the trimethyl carbinol after purifying by column chromatography, and adds DMAP and Boc
2o, reaction overnight under 40 degree of conditions, reaction terminates rear pillar Chromatographic purification; Purification thing is sloughed under THF neutral and alkali condition two ethanoyl protections, simple rear drying in oven of purifying dewaters; To be dissolved in by products therefrom in THF again and to add and activate nitroxyl chloride phenyl formate, the rear pillar Chromatographic purification that reacts completely obtains product A;
Step 2, tonka bean camphor and tin anhydride are reacted 24h under reflux conditions in p-Xylol, after simple purification, purification thing is dissolved in methyl alcohol after reacting completely, reduce with a certain amount of sodium borohydride, after reacting completely, column chromatography is purified, purification thing is dissolved in anhydrous methylene chloride, activate under condition of ice bath Yu to nitroxyl chloride phenyl formate, reaction terminates directly in system, to add N-methyl-N '-tertiary oxygen carbonyl reacting ethylenediamine again and obtain product afterwards, column chromatography obtains product after purifying, purification thing is also obtained by reacting product under further anhydrous methylene chloride and trifluoroacetic acid volume ratio 3:1 condition, and obtain B after simple purification,
Step 3, above-mentioned product A and product B to be reacted in anhydrous methylene chloride, column chromatography obtains product after purifying, again purification thing is sloughed the tertiary butyl under anhydrous methylene chloride and trifluoroacetic acid volume ratio 1:1 condition and obtain product, and obtain product C through column chromatography purification;
Step 4, final product C and part are bonded together, the whole photoactivation fluorescent probe molecule with protein tag location of final synthesis.
3. there is described in claim 1 application of the super-resolution imaging of photoactivation fluorescent probe molecule in viable cell of protein tag location.
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