scholarly journals Multiphoton Bleaching of Red Fluorescent Proteins and the Ways to Reduce It

2022 ◽  
Vol 23 (2) ◽  
pp. 770
Author(s):  
Mikhail Drobizhev ◽  
Rosana S. Molina ◽  
Jacob Franklin

Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. Although all these proteins contain the chromophore with the same chemical structure, the mechanisms of their multiphoton bleaching are different. The number of photons required to initiate a photochemical reaction varies, depending on wavelength and power, from 2 (all four proteins) to 3 (jREX-GECO1) to 4 (mCherry, mPlum, tdTomato), and even up to 8 (tdTomato). We found that at sufficiently low excitation power P, the rate K often follows a quadratic power dependence, that turns into higher order dependence (K~Pα with α > 2) when the power surpasses a particular threshold P*. An optimum intensity for TPLM is close to the P*, because it provides the highest signal-to-background ratio and any further reduction of laser intensity would not improve the fluorescence/bleaching rate ratio. Additionally, one should avoid using wavelengths shorter than a particular threshold to avoid fast bleaching due to multiphoton ionization.

2009 ◽  
Vol 96 (3) ◽  
pp. 400a-401a
Author(s):  
Mikhail Drobizhev ◽  
Shane Tillo ◽  
Nikolay Makarov ◽  
Aleksander Rebane ◽  
Thomas E. Hughes

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Caleb R. Stoltzfus ◽  
Lauren M. Barnett ◽  
Mikhail Drobizhev ◽  
Geoffrey Wicks ◽  
Alexander Mikhaylov ◽  
...  

2012 ◽  
Vol 209-211 ◽  
pp. 1596-1599
Author(s):  
Gui Yin Zhang ◽  
Yi Dong Jin ◽  
Hai Ming Zheng

NO is one of the key substances of air pollution. This paper presents the use of the technique of resonant enhanced multi-photon ionization (REMPI) for NO ambient detection. NO is ionized by absorbing four photons and via A2Σ intermediate resonant state when use 452.4nm laser as radiation source. A physical model concerning the ionization process is presented. It is shown that the ion signal depends on laser character and the dynamic parameters of NO. Two-photon absorption and ionization cross section about the resonant state are obtained from the ion decay curve and the model. The detection limit of this work, which can reach 1.4 ppm, is determined by measuring the variation of the ion signal with the concentration of NO.


2010 ◽  
Vol 100 (5) ◽  
pp. 415-423 ◽  
Author(s):  
Oliver C. Caasi ◽  
Nathan R. Walker ◽  
Stephen M. Marek ◽  
James N. Enis ◽  
Thomas K. Mitchell

Spring dead spot, caused by Ophiosphaerella herpotricha, is the most important disease of turf-type bermudagrass (Cynodon spp.) in the transition zone of the United States. Despite the importance of the disease, only limited information is available about the host–pathogen interaction at the cellular level. To evaluate the host plant interaction, an isolate of O. herpotricha expressing green fluorescent proteins (GFP) or red fluorescent proteins (tdTomato) was used to study the infection and colonization of roots and stolons of several bermudagrass cultivars. Roots of cultivars Tifway 419 and Midlawn were colonized similarly, resulting in extensive root necrosis, whereas an accession of Cynodon transvaalensis was less necrotic. The stele of C. transvaalensis roots was colonized but not those of Tifway 419 and Midlawn. For intact stolons, colonization was limited to the epidermis and defined macroscopic necrotic lesions were observed on Tifway 419 and Midlawn while C. transvaalensis stolon tissues remained mostly nonnecrotic. Internal colonization of stolons occurred when hyphae grew into wounds, resulting in necrosis in Tifway 419 and Midlawn, but not in C. transvaalensis. These studies suggest that the interaction of O. herpotricha with bermudagrass varies across host genotypes and the host tissues infected. The limited necrosis in C. transvaalensis tissues, though colonized, suggests an inherent tolerance to O. herpotricha.


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