Abstract
The quantum yield of a fluorophore is reduced when two or more identical fluorophores are in close proximity to each other. The study of protein folding or particle aggregation is can be done based on this above-mentioned phenomenon-called self-quenching. However, it is challenging to characterize the self-quenching of a fluorophore at high concentrations because of the inner filter effect, which involves depletion of excitation light and re-absorption of emission light. Herein, a novel method to directly evaluate the self-quenching behavior of fluorophores was developed. The evanescent field from an objective-type total internal reflection fluorescence (TIRF) microscope was used to reduce the path length of the excitation and emission light to ~100 nm, thereby supressing the inner filter effect. Fluorescence intensities of sulforhodamine B, fluorescein isothiocyanate (FITC), and calcein solutions with concentrations ranging from 1 μM to 50 mM were directly measured to evaluate the concentration required for 1000-fold degree of self-quenching and to examine the different mechanisms through which the fluorophores undergo self-quenching.
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📋 Methods
Microplate reader measurement
The fluorescence intensity of solutions with different concentrations of the fluorophores was measured using a microplate reader from PerkinElmer (EnSpire), USA. Fluorophores were dissolved in a buffer solution, which contained 50 mM HEPES (H3375), 100 mM NaCl (S7653), and 5% glycerol (G5516), at pH 7.4. All chemicals used for preparing the buffer solution were purchased from Sigma-Aldrich. A total of 100 μL of each solution was loaded into a 96-well plate (655076 from Greiner) and the fluorescence signals were measured at the following wavelengths with an integration time of 0.1 seconds: excitation 565 nm, emission 586 nm for sulforhodamine B (S1307 from ThermoFisher), excitation 498 nm, emission 517 nm for FITC (F6377 from Sigma-Aldrich), and excitation 493 nm, emission 515 nm for calcein (C0003 from TCI, Japan). All fluorescence signals were corrected with a background signal using the buffer-only solution. The signal was fitted using Origin 8.0 (OriginLab) TIRF microscopy The flow chamber was passivated with polyethylene glycol to minimize the possible interactions between the fluorophores and the glass surface by following a previously described method [ 26 ]. 30 μL of each fluorophore solution, prepared with the same buffer as the microplate reader experiment, was injected into a custom-built flow chamber [ 26 ]. Fluorescence measurements for FITC were performed by using an objective-type TIR microscope based on Ti-E (Nikon, Japan) with CFI APO TIRF 60XH objective lens while Sulforhodamine b and Calcein were measured using a home-built objective-type TIR microscope based on Olympus IX71 (Japan). The detailed configuration of this microscope can be found elsewhere [ 26 ]. In brief, a laser beam was briefly allowed to enter through the backport of the microscope and beam splitters were used to separate the emission wavelength from the laser (Semrock, Di03-R473-t1-25 × 36 for 473 nm and Di02-R532-25 × 36 for 532 nm laser).
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Microplate reader measurement
The fluorescence intensity of solutions with different concentrations of the fluorophores was measured using a microplate reader from PerkinElmer (EnSpire), USA. Fluorophores were dissolved in a buffer solution, which contained 50 mM HEPES (H3375), 100 mM NaCl (S7653), and 5% glycerol (G5516), at pH 7.4. All chemicals used for preparing the buffer solution were purchased from Sigma-Aldrich. A total of 100 μL of each solution was loaded into a 96-well plate (655076 from Greiner) and the fluorescence signals were measured at the following wavelengths with an integration time of 0.1 seconds: excitation 565 nm, emission 586 nm for sulforhodamine B (S1307 from ThermoFisher), excitation 498 nm, emission 517 nm for FITC (F6377 from Sigma-Aldrich), and excitation 493 nm, emission 515 nm for calcein (C0003 from TCI, Japan). All fluorescence signals were corrected with a background signal using the buffer-only solution. The signal was fitted using Origin 8.0 (OriginLab) TIRF microscopy The flow chamber was passivated with polyethylene glycol to minimize the possible interactions between the fluorophores and the glass surface by following a previously described method [ 26 ]. 30 μL of each fluorophore solution, prepared with the same buffer as the microplate reader experiment, was injected into a custom-built flow chamber [ 26 ]. Fluorescence measurements for FITC were performed by using an objective-type TIR microscope based on Ti-E (Nikon, Japan) with CFI APO TIRF 60XH objective lens while Sulforhodamine b and Calcein were measured using a home-built objective-type TIR microscope based on Olympus IX71 (Japan). The detailed configuration of this microscope can be found elsewhere [ 26 ]. In brief, a laser beam was briefly allowed to enter through the backport of the microscope and beam splitters were used to separate the emission wavelength from the laser (Semrock, Di03-R473-t1-25 × 36 for 473 nm and Di02-R532-25 × 36 for 532 nm laser).
Fluorescence from the blue
(FITC and calcein, 473 nm, Thorlabs, USA) or green (sulforhodamine B, 532 nm, Thorlabs USA) laser excitation was recorded with an electron-multiplying charge-coupled device (EM-CCD, iXon 897+, Andor, Northern Ireland) after using long-pass filters to suppress the excitation laser (Semrock, LP02-473RS-25 for FITC and calcein, LP03-532RS-25 for sulforhodamine B). To minimize the noise, the fluorescence signal from a large number of pixels (262,144 for FTIC and 160,000 for Sulforhodamine b and Calcein) were taken and averaged for each image. At least 3 images were taken for each condition.
Supporting information S1 File (DOCX) Click here for additional data file.
📊 Figures
Fig 1
Comparison between the conventional fluorometric system and the nano-cuvette system.
(a) Fluorescence measurement of solutions with low and high fluorophore concentrations in a microplate reader. Compared with solutions of low concentration in which all the fluorophores are excited (l...
Fig 2
Normalized fluorescence signal per fluorophore at different concentrations recorded with conventional fluorometer and TIR microscope.
Fluorescence signals from the conventional fluorometer are significantly underestimated by approximately two to three orders of magnitude (black dots). In TIR, 1000-fold self-quenching is observed at ...
Fig 3
Degree of quenching against concentration.
All of the curves were fitted with the modified Stern-Volmer equation for self-quenching: I 0 I u2245 c + a [ F l u o r o p h o r e ] 0 + b [ F l u o r o p h o r e ] 0 u03bd . The value of u03bd was 2...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
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