Anti-Stokes fluorescence microscopy using direct and indirect dark state formation

Abstract

Measurements on biological samples are often hampered by auto-fluorescence from inherent compounds in tissue or cells, limiting the achievable contrast. Both the signal of interest and the auto-fluorescence are usually detected on the Stokes side of the excitation laser. In this communication, we present two new microscopy modalities, based on the emission of a red-emitting DNA-stabilized silver nanocluster (DNA-AgNC). Its bright fluorescence can be generated on the anti-Stokes side of the readout laser, allowing easy spectral separation of the signal of interest from the Stokes side auto-fluorescence.

Probing heterogeneity of NIR induced secondary fluorescence from DNA-stabilized silver nanoclusters at the single molecule level

“…The following OADF emission will occur on the anti-Stokes side of the secondary NIR excitation pulse. 11,17 By choosing an appropriate delay time for the secondary NIR pulse, time-gating can easily eliminate all auto-fluorescence coming from a primary excitation pulse. 17 This allows for simple suppression of potential NIR auto-fluorescence by a short-pass filter.…”

“…11,17 By choosing an appropriate delay time for the secondary NIR pulse, time-gating can easily eliminate all auto-fluorescence coming from a primary excitation pulse. 17 This allows for simple suppression of potential NIR auto-fluorescence by a short-pass filter. 6 The intensity of the OADF signal depends essentially on the quantum yield of dark state formation (Q D1 ) and the efficiency to generate the emissive state upon secondary NIR excitation (Q D1-S1 ).…”

“…18 We have already demonstrated that these DNA-AgNCs can undergo OADF and upconversion fluorescence (UCF) when immobilized in a poly(vinyl alcohol) (PVA) matrix. 17 The intensity of the NIR pulse used to optically pump the DNA-AgNCs from the D 1 to the S 1 state can also induce a two consecutive photon absorption process. This UCF process leads from S 0 via D 1 to S 1 , and results in emission in the same time range as the OADF signal.…”

“…This UCF process leads from S 0 via D 1 to S 1 , and results in emission in the same time range as the OADF signal. 17 However, at the experimental NIR excitation intensities used in this study, we tried to minimize the UCF contribution. Based on our previous results, we estimate the average contribution of UCF to the SF signal to be maximum 20 percent.…”

Simultaneous Increase in Brightness and Singlet Oxygen Generation of an Organic Photosensitizer by Nanocrystallization

“…Excitation and emission spectra are independent of excitation wavelength for both nanoaggregates and nanocrystals, revealing that there is only one type of emitter in the samples (Figure S5, Supporting Information). To further explore the homogeneity of the nanoaggregates and nanocrystals, single particle fluorescence spectra of individual particles were measured using a confocal fluorescence microscope . The results show that the individual particles in nanoaggregates and nanocrystals have PL spectra similar to that of the bulk, confirming the homogeneity of samples at single particle level (see Figures S6 and S7 in the Supporting Information for details).…”