CN104479668A - A fluorochrome probe - Google Patents
A fluorochrome probe Download PDFInfo
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- CN104479668A CN104479668A CN201410545164.2A CN201410545164A CN104479668A CN 104479668 A CN104479668 A CN 104479668A CN 201410545164 A CN201410545164 A CN 201410545164A CN 104479668 A CN104479668 A CN 104479668A
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Abstract
A fluorochrome probe is disclosed. The structure of the fluorochrome probe is shown as a formula (I) that is MGVADLIKKFESISKEE-GGGGK-R1-GG-rRrRrRRR, wherein the MGVADLIKKFESISKEE is an amino acid sequence of an actin recognition group, the GGGGK is a first connecting group, the R1 is a fluorochrome group, the GG is a second connecting group, the rRrRrRRR is an amino acid sequence of a cell permeable peptide, the r is D-arginine, and the R is L-arginine. The fluorochrome probe can specifically label living-cell endogenic actin, and obtain super-resolution microimaging with a higher resolution after fluorescence imaging of the living-cell endogenic actin is achieved.
Description
Technical field
The invention belongs to micro-imaging field, more specifically, relate to a kind of fluorescence probe dye.
Background technology
In viable cell the fluorescent mark of Actin muscle have at present two kinds can way of realization, fluorescent protein labeling and fluorochrome label.Wherein, being most widely used of fluorescin.With the Actin muscle of fluorochrome label, need the difficult problem overcoming specific marker and viable cell endogenous mark, little by the indicia means of Successful utilization at present.But the general brightness of fluorescence dye is higher, there are more outstanding bleach-resistant ability and light stability, and have broader emmission spectrum overlay area, the common fluorescent imaging of shades of colour can be met easily.Moreover, the field of resolution limit cannot be broken through in ordinary optical imaging, i.e. super-resolution micro-imaging field, fluorescence dye has more significantly advantage: the high brightness direct reaction of dyestuff, in the positioning precision of higher super-resolution imaging, thus can obtain higher spatial resolution; Stable photophysical property and bleach-resistant ability, more have the potential superiority in temporal resolution.Therefore from microoptic imaging, comprise common fluorescent imaging and super-resolution microscopic imaging fluorescence angle, the viable cell endogenous Actin muscle of fluorochrome label all has a wide range of applications.
The mode that can realize viable cell labelling at present has two classes: Actin muscle is connected with fluorescence dye and purifying by a class in vitro, by modes such as microinjections, this part labelled protein is imported in viable cell, utilize the characteristic of Actin muscle changeable assembling in viable cell, reach viable cell Actin muscle mark object.Although this mode can be incorporated into endogenous protein, it needs to rely on special equipment and technical support, is difficult to extensively be used.In addition, this mode is subject to the application limitations of technology itself, such as, need the clone selecting size larger during microinjection, therefore successfully can only realize in part cell.Another kind of i.e. label protein/peptide technology, the realization of technology needs two parts to import to by different way respectively in viable cell: first one section of label protein/peptide and Actin muscle are formed fusion gene, this part imported in cell by rotaring transfecting mode.Again by a specific small molecules and fluorescence dye covalent attachment, this part and viable cell permeable membrane voluntarily after hatching.Latter part of special small molecules can with front portion labelled protein/peptide specific be combined, reach with this with the object of fluorochrome label Actin muscle.But the range of application of this mode is very restricted.Because in practical application, latter part of permeable membrane ability is subject to the restriction of kind of dyes and clone, only have the mixture after several fluorescence dye of minority and small molecules covalent attachment can permeates cell membranes voluntarily, and for different clone, the difference of mixture permeable membrane ability be larger.In addition, the Actin muscle marked by this way is ectogenic Actin muscle, and namely in cell, the endogenous Actin muscle of itself is not labeled.
At present, there is not a kind of fluorochrome label method of universality, the technical problem of fluorescence dye specific marker and viable cell endogenous mark when marking Actin muscle can be solved, in order to the fluorescence imaging of the endogenous Actin muscle of viable cell, comprise common fluorescence imaging and improve the super-resolution micro-imaging of optical imagery resolving power further.
Summary of the invention
For above defect or the Improvement requirement of prior art, the invention provides a kind of fluorescence probe dye, its object is to, by combining to be formed with specific linking group and mode of connection by wearing film peptide, Actin muscle recognition group and fluorescence dye there is the fluorescence probe dye wearing film ability, solve the technical problem that fluorescence dye marks viable cell endogenous Actin muscle in fluorescence imaging field thus.
For achieving the above object, according to one aspect of the present invention, provide a kind of fluorescence probe dye, described fluorescence probe dye has the structure of formula (I):
MGVADLIKKFESISKEE-GGGGK-R
1-GG-rRrRrRRR
Formula (I)
Wherein, MGVADLIKKFESISKEE is the aminoacid sequence of Actin muscle recognition group; GGGGK is the first linking group; R
1for fluorescent dye groups; GG is the second linking group; RRrRrRRR is the aminoacid sequence of cell-penetrating peptide, and r is D type arginine, and R is L-type arginine.
Preferably, described fluorescence probe dye, its fluorescent dye groups R
1for dye stuff of rhodamine kinds, fluoresceins dyestuff or photoswitch fluorescence dye.
Preferably, described fluorescence probe dye, dye stuff of rhodamine kinds described in it is rhodamine B.
Preferably, described fluorescence probe dye, fluoresceins dyestuff described in it is fluorescein isothiocyanate.
Preferably, described fluorescence probe dye, photoswitch rhodamine B described in it, has the structure of formula (II),
In general, the above technical scheme conceived by the present invention compared with prior art, can obtain following beneficial effect:
(1) owing to solving fluorescence dye specificity and the dialytic problem of fluorescent probe, can specific marker to the endogenic Actin muscle of viable cell;
(2) owing to the range of application of fluorescence dye to be expanded to the fluorescence dye of photoswitch, after realizing viable cell endogenous Actin muscle fluorescence imaging, can obtain that there is more high-resolution super-resolution micro-imaging;
(3) experiment confirms, the fluorescent probe of photoswitch rhodamine B mark provided by the invention, has the outstanding super-resolution micro-imaging ability exempting from activation and bleach-resistant.
Accompanying drawing explanation
Fig. 1 is the probe of embodiment 4 marked by fluorescein isothiocyanate enters cell marked situation with different time, wherein Fig. 1 a is the flag state that probe enters 5 minutes, Fig. 1 b is the flag state that probe enters 10 minutes, Fig. 1 c is the flag state that probe enters 15 minutes, and Fig. 1 d is the flag state that probe enters 30 minutes;
Fig. 2 is that the probe of embodiment 5 rhodamine B mark enters the marked situation of cell, and wherein Fig. 2 a is the situation entering cell containing the probe of cell-penetrating peptide, and Fig. 2 b is the situation not entering cell containing the probe of cell-penetrating peptide;
Fig. 3 is the probe of embodiment 6 rhodamine B mark and the common marked situation in viable cell of standard Actin muscle marker, wherein Fig. 3 a is that rhodamine B probe is marked as picture in BSC-1 cell, Fig. 3 b is that standard Actin muscle marker is marked as picture in BSC-1 cell, the imaging importing of Fig. 3 c to be Fig. 3 a be Fig. 3 b, Fig. 3 d is that rhodamine B probe is marked as picture in HeLa cell, Fig. 3 e is that standard Actin muscle marker is marked as picture in HeLa cell, the imaging importing of Fig. 3 f to be Fig. 3 d be Fig. 3 e, Fig. 3 g is that rhodamine B probe is marked as picture in NIH/3T3 cell, Fig. 3 h is that standard Actin muscle marker is marked as picture in NIH/3T3 cell, the imaging importing of Fig. 3 i to be Fig. 3 g be Fig. 3 h,
Fig. 4 is the probe of the rhodamine B mark of embodiment 7 photoswitch, the probe of rhodamine B mark and the sparse luminous detection of the fluorescein-labeled probe of thiocyanic acid when super-resolution micro-imaging, wherein Fig. 4 a is the rhodamine B mark of photoswitch, Fig. 4 b is rhodamine B mark, and Fig. 4 c is that thiocyanic acid is fluorescein-labelled;
Fig. 5 is the probe bleach-resistant ability detection figure of the rhodamine B mark of embodiment 8 photoswitch, wherein Fig. 5 a ~ f is respectively the imaging effect of probe Continuous irradiation time when being 0,8,16,22,28,35 minute of the rhodamine B mark of photoswitch, the imaging effect that Fig. 5 g ~ j is respectively the contrast probe Continuous irradiation time when being 0,2,4,6 minute;
Fig. 6 is the super-resolution micro-imaging figure of the viable cell endogenous Actin muscle of the probe mark of the rhodamine B mark of photoswitch, and wherein Fig. 6 a is common fluorescent imaging results, and Fig. 6 b is super-resolution micro-imaging result.
In all of the figs, the scale except Fig. 5 is 10 μm, and the scale of Fig. 5 is 2 μm.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.In addition, if below in described each embodiment of the present invention involved technical characteristic do not form conflict each other and just can mutually combine.
Fluorescence probe dye provided by the invention, has the structure of formula (I):
MGVADLIKKFESISKEE-GGGGK-R
1-GG-rRrRrRRR
Formula (I)
Wherein, MGVADLIKKFESISKEE is the aminoacid sequence of Actin muscle recognition group; GGGGK is the first linking group; R
1for fluorescent dye groups; GG is the second linking group, and rRrRrRRR is the aminoacid sequence of cell-penetrating peptide, and r is D type arginine, and R is L-type arginine.
The N end of peptide chain for Actin muscle recognition group MGVADLIKKFESISKEE, this be at present known to the shortest Actin muscle marker, because its length is short and specifically in conjunction with upper Actin muscle, and can not change the kinetics of Actin muscle itself after the coupling.When recognition group is positioned at N end, be combined optimum with Actin muscle, our design continuity recognition group MGVADLIKKFESISKEE is placed in the conclusion of N end.Cell-penetrating peptide rRrRrRRR is positioned at C end, and its effect is that carry can not the other parts (MGVADLIKKFESISKEE-GGGGK-R of permeable membrane
1-GG) enter in cell.This is a kind of cell-penetrating peptide efficiently, and its special character is, can permeates cell membranes being formed in tenuigenin be uniformly distributed at very low concentrations.In our design, consider that Actin muscle major part is positioned in tenuigenin, the direct tenuigenin that cell-penetrating is formed is distributed with the combination being beneficial to recognition group MGVADLIKKFESISKEE and Actin muscle.And be distributed in endosome after current most cell-penetrating peptide permeable membrane, be unfavorable for the identification of recognition group MGVADLIKKFESISKEE to Actin muscle.Is luminescent dye molecule among these two parts, and for the ease of chemosynthesis, fluorescence dye is connected on the side chain of Methionin K by we.
Sterically hindered when considering Peptide systhesis, we add two glycine G as buffer sequence wearing between film peptide rRrRrRRR and Methionin K.In chemosynthesis, G
nconventional in order to reduce sterically hindered sequence.In addition, sterically hindered when considering Peptide systhesis, and recognition sequence MGVADLIKKFESISKEE and Actin muscle in conjunction with time sterically hindered, we add four glycine G as buffer sequence between Methionin K and recognition group MGVADLIKKFESISKEE.
State fluorescent dye groups R
1for dye stuff of rhodamine kinds, fluoresceins dyestuff or photoswitch fluorescence dye.Dye stuff of rhodamine kinds, as rhodamine B, rhodamine 6G, Rhodamine 123, tetramethylrhodamine etc., the rhodamine B selected here is cheap, and cost compare is low.Fluoresceins dyestuff, as fluorescein isothiocyanate, Fluoresceincarboxylic acid etc., the fluorescein isothiocyanate selected here is normal biomarker dyestuff, has higher quantum yield.Described photoswitch fluorescence dye has two states, comprises luminous on state of and non-luminous dark-state, under the second activating light source being different from excitation light source irradiates, possesses the character changed between dark-state on state of.The rhodamine B wherein with the dyestuff photoswitch of one of photoswitch character has the structure of formula (II),
The photoswitch fluorescence dye adopted is due to its special luminosity, when the exciting light of a given intensity, only has sub-fraction simultaneously luminous, after this part molecular imaging, then make other small portion molecular luminescence in all fluorescence molecules.So obtain the fluorescence molecule that a width width is separated into picture in time, by more accurately locating of algorithm determination fluorescence molecule, finally the imaging results of multiple image is superposed, finally obtain the overall super-resolution micro-imaging figure of Actin muscle.Because fluorescence molecule luminous in every single width figure is spatially not intensive, therefore can do accurately location to it with the micro-imaging algorithm of super-resolution, thus improve the optical resolution of Actin muscle imaging.
Fluorescent probe provided by the invention can adopt the synthetic method of solid-phase peptide to obtain.
Be below embodiment:
Embodiment 1
A kind of fluorescence probe dye has following structure:
MGVADLIKKFESISKEE-GGGGK-R
1-GG-rRrRrRRR
Wherein, MGVADLIKKFESISKEE is the aminoacid sequence of Actin muscle recognition group; GGGGK is the first linking group, and fluorescent dye groups is rhodamine B, R
1structure is
gG is the second linking group; RRrRrRRR is the aminoacid sequence of cell-penetrating peptide, and r is D type arginine, and R is L-type arginine.
Described fluorescence probe dye is synthesized by Ao Bo bio tech ltd, Shanghai, and its synthetic method adopts the solid phase method of peptide synthesis.
Embodiment 2
A kind of fluorescence probe dye has following structure:
MGVADLIKKFESISKEE-GGGGK-R
1-GG-rRrRrRRR
Wherein, MGVADLIKKFESISKEE is the aminoacid sequence of Actin muscle recognition group; GGGGK is the first linking group, and fluorescent dye groups is fluorescein isothiocyanate, R
1structure is
gG is the second linking group; RRrRrRRR is the aminoacid sequence of cell-penetrating peptide, and r is D type arginine, and R is L-type arginine.
Described fluorescence probe dye is synthesized by Ao Bo bio tech ltd, Shanghai, and its synthetic method adopts the solid phase method of peptide synthesis.
Embodiment 3
A kind of fluorescence probe dye has following structure:
MGVADLIKKFESISKEE-GGGGK-R
1-GG-rRrRrRRR
Wherein, MGVADLIKKFESISKEE is the aminoacid sequence of Actin muscle recognition group; GGGGK is the first linking group, and fluorescent dye groups is photoswitch rhodamine B, R
1structure is
gG is the second linking group; RRrRrRRR is the aminoacid sequence of cell-penetrating peptide, and r is D type arginine, and R is L-type arginine.
Described fluorescence probe dye is synthesized by Ao Bo bio tech ltd, Shanghai, and its synthetic method adopts the solid phase method of peptide synthesis.
Embodiment 4
The fluorescent probe of marked by fluorescein isothiocyanate wears the experiment of film time gradient:
Cell prepares: the BSC-1 cell (1.5x10 that growth conditions is good
4cells/well) solution 200 μ l is inoculated in aseptic round ware (glass bottom Φ 15mm, NEST Biotechnology Co., Ltd., China).37 DEG C, 5%CO
2, containing overnight incubation in the MEM solution of 10% foetal calf serum, again adherent to cell.
The probe pure water of the marked by fluorescein isothiocyanate provided in embodiment 2 is made into the solution of high density (1mM), and is diluted to final concentration 15 μMs with PBS.Before experiment, take out cell and suck substratum, wash remaining serum off by PBS solution.Respectively getting the above-mentioned probe solution of 200 μ l joins in four round wares, respectively at 37 DEG C, 5%CO
2in environment, lucifuge hatches 5 minutes, 10 minutes, 15 minutes and 30 minutes.Remove probe solution, add the blue solution of platform phenol of the 1mg/ml of 200 μ l, leave standstill and after one minute, remove platform phenol indigo plant, carefully clean cell three times by PBS solution, add the MEM solution containing 10% serum, upper sem observation.
Image-forming condition: observed by laser scanning co-focusing microscope FV1000 (Olympus, Japan).Fluorescein isothiocyanate excites in 488nm, receives at 510-530nm.
After fluorescein isothiocyanate probe mark cell, laser confocal imaging the results are shown in accompanying drawing 1.Fig. 1 shows, and probe enters cell and occurs in a uniformly distributed manner (5 minutes), and afterwards along with incubation time increases, the structure of similar actin cytoskeleton is accumulated toward central zone, until filamentary texture is paved with whole cell from cell edges.This process middle probe occurs in mode equally distributed in tenuigenin, proves the permeable membrane character not affecting itself after cell-penetrating peptide rRrRrRRR carries goods (MGVADLIKKFESISKEE-GGGGK-R1-GG).The advantage that fluorescent probe provided by the invention is located in tenuigenin is embodied.
Embodiment 5
The fluorescent probe of rhodamine B mark wears film experiment:
Cell prepares: the BSC-1 cell (1.5x10 that growth conditions is good
4cells/well) solution 200 μ l is inoculated in aseptic round ware (glass bottom Φ 15mm, NEST Biotechnology Co., Ltd., China).37 DEG C, 5%CO
2, containing overnight incubation in the MEM solution of 10% foetal calf serum, again adherent to cell.
The probe pure water that the rhodamine B provided in embodiment 1 marks is made into the solution of high density (1mM), and is diluted to final concentration 15 μMs with PBS.Before experiment, take out cell and suck substratum, wash remaining serum off by PBS solution.Getting the above-mentioned probe solution of 200 μ l joins in round ware, respectively at 37 DEG C, 5%CO
2in environment, lucifuge hatches 30 minutes.Remove probe solution, add the blue solution of platform phenol of the 1mg/ml of 200 μ l, leave standstill and after one minute, remove platform phenol indigo plant, carefully clean cell three times by PBS solution, add the MEM solution containing 10% serum, upper sem observation.
Image-forming condition: observed by laser scanning co-focusing microscope FV1000 (Olympus, Japan).Rhodamine B excites in 543nm, receives at 580-630nm.
After rhodamine B probe mark cell, laser confocal imaging the results are shown in accompanying drawing 2.Fig. 2 shows, and after fluorescence dye is replaced with rhodamine B, (a), mark result is still consistent with the mark result of fluorescein isothiocyanate probe for Fig. 2.Contrast probe not containing cell-penetrating peptide rRrRrRRR then cannot enter cell (Fig. 2 b).Prove that probe that viable cell endogenous Actin muscle provided by the invention marks is by the restriction of fluorescence dye kind, the validity permeable membrane of probe is because cell-penetrating peptide validity is carried.
Embodiment 6
The experiment of the common location of the probe mark that embodiment 1 provides and standard Actin muscle marker GFP-actin:
The BSC-1 cell (preparation method is as embodiment 4 and embodiment 5) that growth conditions is good is inoculated in 24 orifice plates, 37 DEG C, 5%CO
2, containing overnight incubation in the MEM solution of 10% foetal calf serum, again adherent to cell, the rate of being paved with is 80-90%.According to transfection reagent Lipofectamine
tMthe standard step of 2000 (Invitrogen, Grand Island, NY, USA), proceeds to plasmid GFP-actin in cell.Remove supernatant after 24 hours, the trysinization of cell containing 0.25%EDTA, with 1.5x10
4cells/well is inoculated in aseptic round ware (glass bottom Φ 15mm, NEST Biotechnology Co., Ltd., China) and continues to cultivate 12-24 hour.The probe pure water that the rhodamine B provided in embodiment 1 marks is made into the solution of high density (1mM), and is diluted to final concentration 15 μMs with PBS.Before experiment, take out again adherent cell and suck substratum, wash remaining serum off by PBS solution.Getting 200 μ l probe solutions joins in round ware, in 37 DEG C, 5%CO
2in environment, lucifuge hatches half an hour.Remove probe solution, add the blue solution of platform phenol of the 1mg/ml of 200 μ l, leave standstill and after one minute, remove platform phenol indigo plant, carefully clean cell three times by PBS solution, add the MEM solution containing 10% serum, upper sem observation.HeLa cell and NIH/3T3 cell obtain corresponding common positioning result respectively with same experimentation.
Image-forming condition: cell sample is observed by laser scanning co-focusing microscope FV1000 (Olympus, Japan).Two channels imaging simultaneously, rhodamine B excites in 543nm, receives at 580-630nm; GFP excites in 488nm, receives at 508-532nm.
Laser confocal imaging the results are shown in accompanying drawing 3.Green channel (Fig. 3 b, Fig. 3 e, Fig. 3 h) shows, after transfection, and Actin muscle mark standard substance GFP-actin successful expression in BSC-1, HeLa and NIH/3T3 cell; Red channel (Fig. 3 a, Fig. 3 d, Fig. 3 g) is the mark result of rhodamine B label probe, and the shape of similar actin cytoskeleton and the result of Fig. 1 and Fig. 2 a are consistent.Through the common location (Fig. 3 c, Fig. 3 f, Fig. 3 i) of two channels imaging, yl moiety is with a signal that two, position passage detects jointly.The high coverage rate of yl moiety shows that the structure of above-mentioned (Fig. 1 and Fig. 2) similar actin cytoskeleton is Actin muscle really, sufficient proof probe provided by the invention can to mark the endogenous Actin muscle of viable cell effectively, and effective to multiple clone.
Embodiment 7
The sparse luminous situation observation of the probe super-resolution micro-imaging that embodiment 3 provides:
BSC-1 cell (preparation method is as embodiment 4 and the embodiment 5) (1.5x10 that growth conditions is good
4cells/well) solution 200 μ l is inoculated in aseptic round ware (glass bottom Φ 15mm, NESTBiotechnology Co., Ltd., China).37 DEG C, 5%CO
2, containing overnight incubation in the MEM solution of 10% foetal calf serum, again adherent to cell.
The probe of the marked by fluorescein isothiocyanate that the probe of rhodamine B mark embodiment 1 provided, embodiment 2 provide, and the probe of the rhodamine B mark of the photoswitch that provides of embodiment 3 is made into the solution of high density (1mM) respectively with pure water, and be diluted to final concentration 15 μMs with PBS.Before experiment, take out cell and suck substratum, wash remaining serum off by PBS solution.Getting the different probe solution of 200 μ l respectively joins in round ware, 37 DEG C, 5%CO
2lucifuge hatches half an hour.Remove probe solution, add the blue solution of platform phenol of the 1mg/ml of 200 μ l, leave standstill and after one minute, remove platform phenol indigo plant, carefully clean cell three times by PBS solution, add the MEM solution containing 10% serum, upper sem observation.
Image-forming condition: cell sample is observed by total internal reflection imaging system.This system is by inverted light microscope (IX 71, Olympus), numerical aperture is 100 times of total internal reflection object lens (UAPON100XOTIRF of 1.49, Olympus), the diode pumping solid laser (CNILaser of iXon 897EMCCD detector (Andor Tech.), 561nm, China), the diode pumping solid laser (CNILaser of 473nm, China) and 405nm laser diode (CNILaser, China) composition.
Its sparse luminescence the results are shown in accompanying drawing 4.The probe of the rhodamine B mark of the photoswitch that embodiment 3 provides only just can form sparse luminescence under the irradiation of exciting light (561nm) single light source in viable cell, and (Fig. 4 a).This needs secondary light source different as the super-resolution imaging of exciting light from great majority.Decrease secondary light source, the light injury that the UV-light avoiding short wavelength is formed viable cell.The probe (Fig. 4 b) of the rhodamine B mark that embodiment 1 provides can form sparse luminescence phenomenon to a certain degree in viable cell, but the signal to noise ratio of image is too poor, is unfavorable for obtaining high-quality image; The probe that the fluorescein isothiocyanate (Fig. 4 c) that embodiment 2 provides marks can not sparse luminescence in cell.Owing to forming the basis that sparse luminescence is super-resolution micro-imaging, the therefore probe of common dye mark, namely the probe of rhodamine B mark and the probe of marked by fluorescein isothiocyanate are not suitable for for doing super-resolution micro-imaging.The experiment of this group proves, the probe of the rhodamine B mark of photoswitch has the outstanding super-resolution micro-imaging ability exempting to activate.
Embodiment 8
The photoswitch rhodamine B probe bleach-resistant experiment that embodiment 3 provides:
The photoswitch rhodamine B probe provided by embodiment 3 carries out marking and imaging, and with the actin probe tdEosFP-Lifeact of the bleach-resistant reported in document in contrast, compare the bleach-resistant ability of the two.Probe mark and image-forming step are as embodiment 7.
The sparse scintillation that the probe of the photoswitch rhodamine B mark that embodiment 3 provides is formed significantly is not bleached sign within half an hour.This phenomenon is conducive to long-time imaging, is seldom reported in viable cell super-resolution micro-imaging.We with the actin probe tdEosFP-Lifeact of the bleach-resistant reported in document in contrast, the results are shown in accompanying drawing 5.Under laser apparatus maximum luminous power width is penetrated, (power density on sample is 0.33kWcm to the probe of the rhodamine B mark of photoswitch
-2) after Continuous irradiation half an hour, probe is not still by obviously bleaching (Fig. 5 a); And the cell of tdEosFP-Lifeact transfection, under exciting light (405nm) associated image, with maximum luminous power half, (power density on sample is for 0.165kWcm
-2) when being radiated on cell sample, still in 4 minutes by full bleaching (Fig. 5 b).The experiment of this group proves, the probe of the rhodamine B mark of photoswitch has the super-resolution micro-imaging ability of outstanding bleach-resistant.
According to the sparse luminance of sample, select high-density location reconstruction algorithm PALMER process image.Obviously, (Fig. 6 a) obtains higher spatial resolution compared with common imaging (Fig. 6 b) to super-resolution micro-imaging.Prove that the probe of the rhodamine B mark of photoswitch is as the probe of an outstanding super-resolution micro-imaging, can obtain the viable cell endogenous Actin muscle image that structure is meticulousr.
Those skilled in the art will readily understand; the foregoing is only preferred embodiment of the present invention; not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. a fluorescence probe dye, is characterized in that, described fluorescence probe dye has the structure of formula (I):
MGVADLIKKFESISKEE-GGGGK-R
1-GG-rRrRrRRR
Formula (I),
Wherein, MGVADLIKKFESISKEE is the aminoacid sequence of Actin muscle recognition group; GGGGK is the first linking group; R
1for fluorescent dye groups; GG is the second linking group; RRrRrRRR is the aminoacid sequence of cell-penetrating peptide, and r is D type arginine, and R is L-type arginine.
2. fluorescence probe dye as claimed in claim 1, is characterized in that, described fluorescent dye groups R
1for dye stuff of rhodamine kinds, fluoresceins dyestuff or photoswitch fluorescence dye.
3. fluorescence probe dye as claimed in claim 2, it is characterized in that, described dye stuff of rhodamine kinds is rhodamine B.
4. fluorescence probe dye as claimed in claim 2, it is characterized in that, described fluoresceins dyestuff is fluorescein isothiocyanate.
5. fluorescence probe dye as claimed in claim 2, it is characterized in that, described photoswitch rhodamine B, has the structure of formula (II),
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|---|---|---|---|---|
| CN107417793A (en) * | 2017-04-28 | 2017-12-01 | 华中科技大学 | A kind of small peptide, fluorescence probe and preparation method thereof |
| WO2018196831A1 (en) * | 2017-04-28 | 2018-11-01 | 奇点荧光南京生物科技有限公司 | Short peptide, fluorescence probe and preparation method thereof |
| CN107417793B (en) * | 2017-04-28 | 2019-11-22 | 奇点荧光南京生物科技有限公司 | A kind of small peptide, fluorescence probe and preparation method thereof |
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