Two‐photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP and EYFP

Abstract

SummaryTwo-photon (TP) excitation (820 -1150 nm) and emission (280 -700 nm) spectra for the fluorescent proteins (FPs) ECFP 3 , EGFP 3 and EYFP 3 produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their onephoton (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue-shifted and emission red-shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out-of-maximum excitation, common practice in TP excitation microscopy, can be used for routine application.

Intermolecular charge transfer enhances two-photon absorption in yellow fluorescent protein

“…Indeed, Spiess et al 13 do not report wavelengths below 435 nm (λ laser = 870 nm) and Blab et al 12 not below 375 nm (λ laser = 750 nm) for the TPA spectrum of YFP. Drobizhev et al report the TPA spectrum of Citrine (an optimized YFP variant) down to 600 nm.…”

“…From experimental work it is known that the TPA maximum of the lowest excitation in YFP (480 nm 12 , 485 nm 13 ) is blue-shifted with respect to the one-photon absorption (OPA) maximum by approximately 29 13 to 34 12 nm. Regardless of the type of excitation (OPA or TPA), the excitation maximum is around 530 nm.…”

“…Regardless of the type of excitation (OPA or TPA), the excitation maximum is around 530 nm. 13 The study of absorption properties of fluorescent proteins is usually limited to OPA. TPA of fluorescent proteins has been studied theoretically, but often takes into account only the lowest excitation energy [14][15][16] and -relevant in the case of YFP -only the chromophore.…”

Fluorescence excitation by enhanced plasmon upconversion under continuous wave illumination

“…As a second control experiment, we 1-photon fluorescence is adopted from Ref. [55] where EYFP molecules are contained in a solution. Note that peak of the 2-photon fluorescence with AuNPs (2p on AuNP) curve looks shifted due to the presence of the filter.…”

“…Second, one may question if the EYFP-AuNP coupling modifies the spectrum. Comparing the 1-photon florescence curves for EYFP in solution [55] and EYFP on AuNPs; one can observe that EYFP-AuNP spectrum shifts to higher energies by 4 nm. So, if this 4 nm shift does not originate from the differences in the calibration of two experiments [53] or the solution effect, 2-photon absorption should be less favored compared to the bare EYFPs.…”