Two-photon Fluorescent Probes For Application In Biological Imaging

Two-photon fluorescence microscopy (TPM) has been proved to be a powerful technique for the study of dynamic processes, live-cell imaging and especially living phenomena in living tissues. It offers several advantages for imaging of cells and tissues: deeper penetration depth (>500μm), lower tissue autofluorescence and self absorption, and reduced photodamage and photoleaching, in addition to the intrinsically locallized excitation. This allows imaging deep inside the intact tissue for a long period of time without tissue preparation artifacts. However, most of the one-photon fluorescent probes used for TPM have low two-photon excited fluorescent action cross section(φ×δ) that limit their use in TPM. Therefore, there is a pressing need to develop efficient two-photon fluorescent probes with largerφ×δfor in vivo imaging. In this thesis, series of crabazole derivatives type of two-photon fluorescent nucleic acid and acidic vesicles probe were synthesized, they have optimized two-photon properties and could image living cells and/or living tissues by TPM.1. Two-photon fluorescence imaging of nucleic acid in living plant turbid tissue with dicationic carbazole derivatives:At every excitation wavelength, the values ofφ×δof dicationic carbazole derivatives(9E-BHVC,9E-BMVC and BMVC) in various solvents are larger than that of DAPI. It is worth to note that theφ×δof 9E-BHVC,9E-BMVC and BMVC are in the range of 45-96 GM in the presence of DNA, whileφ×δof DAPI is 2.18 GM. With addition of DNA, electronic absorption spectra of the three molecules show significant bathochromic shifts (20-23 nm) and large absorbance changes in opposite directions; introduced circle dichroism in the range of 350-550 nm are found; one-photon fluorescent intensities are strongly enhanced, with exaltation of 485 (9E-BHVC), 83(9E-BMVC) and 374(BMVC); the binding constants reach 106-107 order. With the two-photon excitation, carbazole derivtives also display sensitive response to DNA, and their two-photon fluorescence exaltation are 36-54 fold. Their imaging DNA ability in living cells and living tissues at the excitation wavelength of 800 nm and 740 nm were envalued. At 800 nm, carbazole derivatives can stain the DNA in protoplasts and the positions and regions stained are consistent with those of DAPI. However, when staining living tissue of Arabidopsis thaliana, the abilities of 9E-BHVC and 9E-BMVC are superior to BMVC, and 9E-BHVC are best. Therefore, the imaging performance of 9E-BHVC in imaging highly scattering specimens were studies in detail. A series of doublestaining microscopic photos of DAPI and 9E-BHVC under the same imaging conditions and at different depths were obtained. In the same imaging depth, the fluorescent intnesity of 9E-BHVC are stronger than that of DAPI. Similarity, at the same power, the fluorescent intnesity of 9E-BHVC are also more intensitive than that of DAPI. Experimental results show:compared to DAPI,9E-BHVC can be used to carry out deeper observation using the same incident power, or can be used to obtain usable fluorescent images by using a lower incident power. At 740 nm,9E-BHVC also exhibits better imaging ability in living tissues than that of DAPI.Thus, we can say that 9E-BHVC is an efficient two-photon fluorescence nucleic acid probe. It possesses potential to become a commercial two-photon fluorescent DNA probe:(i) high (i)highφ×δwhen binding to DNA (90 GM) and lowφ×δin buffer solution in the absence of DNA (2.4 GM), (ii) large intrinsic binding constant to DNA (1.02×l07 M-1), (iii) good DNA "light-switch" properties, (iv) exclusively and fully labeling nuclei in living plant tissue same as DAPI, especially deeper imaging than DAPI. (v) high photostability and low biological toxicity. These excellent properties make 9E-BHVC important value to biologist for significant biological discovery.2. Analysis of interaction between dicationic carbazole derivatives and DNA by spectra:Using electronic absorption spectra, circle dichroism spectra and one-and two-photon emission spectra, the binding modes between BHVC and DNA have been studied. All spectral show such ratio of [phosphate of DNA]/[BHVC] in the range of 2～4.3. When less than the ratio, the absorbance of BHVC decrease, negative introduced circle dichroism in 320-550 nm are observed, positive circle dichroism signal of DNA at 275 nm change to be negative, peak position of one-and two-photon spectra red shift, fluorescence intensities of BHVC in the presence of denatured DNA are higher than that in normal DNA. When larger than the ratio, the absorbance of BHVC increase, negative/positive mode introduced circle dichroism in 320-550 nm are observed, intensity of positive circle dichroism signal of DNA at 275 nm decrease, peak position of one-and two-photon spectra blue shift, fluorescence intensities of BHVC in the presence of denatured DNA are lower than that in normal DNA. In addition, whether in low ratio of [phosphate]/[BHVC] or high ratio of [phosphate]/[BHVC] system, the one-photon fluorescence intensity of BHVC decrease with addition of KI. Therefore, BHVC bind to DNA with carbazole motif intercalate into base pair of DNA and pyridium motif exposed outside at low ratio, while BHVC bind to DNA with aggregation on the surface of DNA at high ratio.3. Basic research on properties of carbazole derivatives sensitive to acid enviroment:TheΦ×δof the title compound (9E-BVC) in acidic (0.1 M trifluoroacetic acid, TFA) ethanolic solution reach 48.1 GM, while at the same excitation wavelength, theΦ×δin ethanol only 2.9 GM. On the other hand, the two-photon fluorescent peak position in acidic (0.1 M trifluoroacetic acid, TFA) ethanolic solution is 595 nm, while that in ethanol only 470 nm. Moreover,9E-BVC can stain acidic vesicles in living cell by TPM.The imaging ablitiy of DNA probe in living turbid tissues are envalued in detail and the binding modes are disussed; Besides, a basic research on imaging ability of two-photon fluorescence acidic vesicles probe which possess both greatly enhanced emission intensity and large stokes shift, has been carried out. These provide guidance for the design and synthesis of two-photon fluorescence probe and will further promote the application of TPM in biological field.