Two-photon fluorescence imaging

CHll BIOLOGICAL APPUCAnONS OF SINGLE- AND TWO-PHOTON FLUORESCENCE 

From Equation (11.3) it can be seen that the two photon absorption probabiUty is dependent on the incident laser intensity. Thus two-photon absorption will take place most readily at the laser focus. The transverse (spatial) dependence of a the output intensity of a laser operating in a fundamental (lowest loss) mode at time t has the form 

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Hanson, K. M., and Clegg, R. M. 2005. Two-photon fluorescence imaging and reactive oxygen species detection within the epidermis. Methods Mol. Biol. 289 413-22. 

Squirrell, J. M., Wokosin, D. L., White, J. G., and Bavister, B. D. 1999. Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. Nat. Biotech. 17 763-67. 

Fig. 30 Two-photon fluorescence image of storage medium before (a) and after (b) two-photon recording, c DIG readout of the storage data after two-photon recording, and d input intensity dependent up-converted fluorescence of fluorene 17...

Fig. 33 Two-photon fluorescent images of photosensitive films developed (via 350-nm broadband exposure, 4.4mW/cm ) using an Air Force resolution target mask, a Image recorded by channel 1, b image recorded by channel 2, and c fluorescence intensity by scanning an xy line across one set of three-membered elements (line across set 5)...

Figure 3.81. Two-photon fluorescent images recorded by channel 1 (a) or channel 2 (b) that is formed via 350-nm broadband exposure of photosensitive polymer films containing 43e2 and triarylsulfonium hexafluoroantimonate as photoinduced acid generator using a light intensity of 4.4mW/cm2. (From Ref. [118] with permission of the American Chemical Society.)...

K.M. Hanson, M.J. Behne, N.P. Barry, T.M. Mauro, E. Gratton, Two-photon fluorescence imaging of the skin stratum comeum pH gradient, Biophys. 

Gradinaru, C. C., Martinsson, R, Aartsma, T. J., and Schmidt, T. 2004. Simultaneous atomic-force and two-photon fluorescence imaging of biological specimens in vivo. Ultramicroscopy 99,235-245. 

Figure 11.13 Schematic representation of a two-photon fluorescence microscope system two-photon excitation of fluorophores within the sample is achieved using the output of a Ti sapphire laser. This is scanned in the X-Y plane to produce an image of a section of the sample. The insert shows a two-photon fluorescence image of pig kidney cells labelled with two-photon fluorophores (image provided courtesy of Dr Mireille Blanchard-Desce (CNRS UMR 6510 Rennes))...

K. T. Yong et al.. Quantum rod bioconjugates as targeted probes for confocal and two-photon fluorescence imaging of cancer cells, Nano Letters, 7(3), 761-765 (2007). 

For demonstrative purposes, multilayer assanblies were constructed by placing an uncoated glass cover slip between two cover slips coated with patterned photosensitive films, with the coated sides against the middle cover slip late 2). Three-dimensional two-photon fluorescence imaging was poformed on the multilayer structures. 

Plate 9.1. Two-photon fluorescence images of photosensitive films developed (via 350 nm broadband exposure, 4.4 mW/cm ) through Air Force targets. 

Fig. 37 Reconstructed 3D two-photon fluorescence images of a a gearwheel and b an icosahedron...

Zhou et al. [177] use a random copolymer of tetrahydropyranyl methacrylate (THPMA) and methyl methacrylate (MMA) polymer doped with BSB-S2 as the PAG for microfabrication. At the laser focal spot, the THPMA groups were converted to carboxylic acid groups due to photogenerated acid-induced ester cleavage reactions, and were therefore rendered soluble in aqueous base developer. Figure 45 shows the 3D microstructure produced by this method. By two-photon fluorescence imaging, it was found that the buried channels are open and a continuous connection was made between the two cavities. 

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