Lee EEL and Bezanilla F (2019), Methodological improvements for fluorescence re...

XB-ART-55592

J Gen Physiol 2019 Feb 04;1512:264-272. doi: 10.1085/jgp.201812189.

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Methodological improvements for fluorescence recordings in Xenopus laevis oocytes.

Lee EEL , Bezanilla F .

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Xenopus laevis oocytes are a widely used model system because of their capacity to translate exogenous mRNA, but their high intrinsic background fluorescence is a disadvantage for fluorescence recordings. Here, we developed two distinct methods for improving fluorescence recordings from oocytes. One was a pharmacological method in which a small-molecule salt-inducible kinase inhibitor was co-injected with the mRNA of interest to stimulate melanin production. We interrogated the oocytes using cut-open voltage clamp with simultaneous fluorescence recording and found that by increasing the amount of light-absorbing melanin in these oocytes, we decreased their intrinsic background fluorescence. The treated oocytes produced fluorescence signals that were approximately four times larger. The second method consisted of direct injection of synthetic melanin. This method also significantly improved (doubled) fluorescence signals and allowed any oocyte to be used for fluorescence recording. These two methods provide significant improvements of the signal quality for fluorescent oocyte recordings and allow all healthy oocytes to be used for high-sensitivity recordings.

???displayArticle.pubmedLink??? 30606741
???displayArticle.pmcLink??? PMC6363413
???displayArticle.link??? J Gen Physiol
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Species referenced: Xenopus laevis
Genes referenced: map9 sik1

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	Figure 1. Oocytes are variable in endogenous background fluorescence at multiple wavelengths. (A) X. laevis oocytes from different batches of oocytes. Scale bar, 1 mm. (B) Endogenous background fluorescence of the animal pole excited with a 420-nm LED reflected onto the oocytes by a 455-nm long-pass dichroic through a ×40 water-immersion objective, where the emission was collected through the dichroic and a 475-nm long-pass filter. Batches were placed in order of most endogenous background to least. Batch 1, n = 22; batch 2, n = 1; batch 3, n = 14; batch 4, n = 20; batch 5, n = 10; batch 6, n = 8; batch 7, n = 8; and batch 8, n = 7. A one-way ANOVA together with a post hoc Tukey multiple comparisons test. **, P < 0.0001. For further information, see Table S1. (C) Endogenous background fluorescence of the animal pole excited with a 470-nm LED that was passed through a filter cube housing a 480/40-nm excitation filter, a 505-nm long-pass dichroic, and a 535/50-nm emission filter. Batches are placed in order of most endogenous background to least. Batch 1, n = 6; batch 2, n = 8; batch 3, n = 6; batch 4, n = 5; batch 5, n = 7; batch 6, n = 4; batch 7, n = 4; and batch 8, n = 7. A one-way ANOVA together with a post hoc Tukey multiple comparisons test. **, P < 0.0001. For further information, see Table S2. The batch numbers in B and C do not correspond and are different batches of oocytes. The measurements are in comparable optical conditions.
	Figure 2. Addition of the SIK inhibitor decreases background fluorescence. (A) Chemical structure of the small-molecule SIK inhibitor HG 9-91-01. (B) Comparison of endogenous fluorescence between several batches of uninjected and 0.5% DMSO-injected oocytes. A one-way ANOVA with a post hoc Holm–Sidak multiple comparisons test found no significant difference compared with uninjected oocytes (uninjected: n = 11, 19, and 11; DMSO: n = 10, 18, and 11). For further information, see Table S3. (C) Effect of HG 9-91-01 endogenous fluorescence under different conditions: uninjected (circles, n = 14), 0.1% DMSO injected (diamonds, n = 9), 10 µM HG 9-91-01 bath (squares, n = 22), 10 µM HG 9-91-01 injected (triangles, n = 21), and 100 µM HG 9-91-01 injected (upside-down triangles, n = 21) measured on d 4. **, P < 0.0001 compared to uninjected oocytes, one-way ANOVA with a post hoc Holm–Sidak multiple comparisons test. For further information, see Table S4. (D) Endogenous fluorescence under different conditions measured over time, as described in B. Panel on the right is a zoom in to demonstrate the effect of the different conditions described in B. , P < 0.001, 10 µM HG injected and 100 µM HG injected on day 4 (two-way ANOVA with a post hoc Dunnett multiple comparisons test). For further information, see Table S5. (E) Results from a darker batch of oocytes where HG 9-91-01 was tested (uninjected: n = 11; 100 µM HG 9-91-01 injected: n = 11). , P = 0.0031, unpaired two-tailed t test. (F) Results from a lighter batch of oocytes where HG 9-91-01 was tested (uninjected: n = 22; 100 µM HG 9-91-01 injected: n = 14). **, P < 0.0001, unpaired two-tailed t test. All error bars are ±SEM centered on the mean..
	Figure 3. Addition of the SIK inhibitor improves ATTO 425 signal. (A) A comparison of uninjected (n = 19) and 100 µM HG 9-91-01–injected (n = 18) oocytes on day 4 before labeling with ATTO 425. (B) **, P < 0.0001 after labeling with ATTO 425, unpaired two-tailed t tests. (C) Pulse protocol for ATTO 425 fluorescence, where the oocytes were held at −90 mV and prepulsed to −120 mV for 60 ms, followed by an 80-ms pulse to 80 mV, with a post pulse of 120 mV for 60 ms before returning to the holding potential. (D) Representative trace of fluorescence data of ATTO 425–labeled Shaker W434F M356C (top) and ATTO 425 labeled Shaker W434F M356C + 100 µM HG 9-91-01 (bottom) oocytes with the pulse protocol from A. (E) Endogenous oocyte fluorescence on day 3 was checked for several conditions (uninjected, n = 16; construct, n = 8; HG 9-91-01, n = 21; construct + HG 9-91-01, n = 10). *, P < 0.0001, ordinary one-way ANOVA with a post hoc Bonferroni multiple comparisons test. (F) Comparisons of levels of expressions in Shaker W434F M356C (n = 4) and Shaker W434F M356C + HG 9-91-01 (n = 6) injected oocytes. Unpaired two-tailed t test showed no statistical significance (P = 0.5414). (G) Comparison of background fluorescence of ATTO 425–labeled oocytes Shaker W434F M356C (n = 4) and Shaker W434F M356C + HG 9-91-01 (n = 8). , P = 0.0324, unpaired two-tailed t test. (H) Comparisons of fluorescence signal in oocytes injected with Shaker W434F M356C (n = 4) and Shaker W434F M356C + HG 9-91-01 (n = 8). , P = 0.0085, unpaired two-tailed t test. (I) Comparison of ΔF/F % in oocytes injected with Shaker W434F M356C (n = 4) and Shaker W434F M356C + HG 9-91-01 (n = 8). , P = 0.0011, unpaired two-tailed t test. For further information, see Tables S6–S12. All error bars are ±SEM centered on the mean.
	Figure 4. Synthetic melanin characterization. (A) Absorption spectrum of 0.3 mg/ml melanin from 400 to 1,000 nm. (B) Absorbance of different concentrations of melanin at 600 nm (n = 3). (C) Dynamic light scattering measurement of synthetic melanin size at 30 mg/ml unfiltered (average radius size, 220 nm).
	Figure 5. Synthetic melanin injections significantly improve fluorescence recording conditions. (A) Representative X. laevis oocytes injected with 50.1 nl of 30 mg/ml melanin. Scale bars, 1 mm (top) and 0.3 mm (bottom). (B) Improvement of endogenous background fluorescence of four separate batches of oocytes at 535 nm. Batch 1, n = 4; batch 2, n = 6; batch , n = 10; and batch 4, n = 4. , P < 0.05; , P < 0.01; *, P < 0.0001, two-way ANOVA with a post hoc Tukey multiple comparison test. (C) The improvement in endogenous background fluorescence in “dark” oocytes, <20 AU at 535 nm (vegetal, n = 4; animal, n = 4; and melanin injected, n = 4). *, P < 0.0001, ordinary one-way ANOVA with a post hoc Tukey multiple comparison test; no statistically significant difference was found between the animal pole and melanin. (D) The improvement in endogenous background fluorescence in “light” oocytes, >20 AU at 535 nm (vegetal, n = 4; animal, n = 4; and melanin injected, n = 4). , P < 0.05; ****, P < 0.0001, ordinary one-way ANOVA with a post hoc Tukey multiple comparison test. For further statistical information, see Tables S13–S15.
	Figure 6. Synthetic melanin injections improve the signal of ASAP-Y. (A) Pulse protocol for ASAP-Y fluorescence, where the oocytes were held at −60 mV with a 50-ms prepulse to −160 mV, a 30-ms pulse to 160 mV, and a postpulse back to −160 mV for 50 ms before returning to holding. (B) A representative trace of fluorescence data of ASAP-Y (top) and ASAP-Y and melanin (bottom) with the pulse protocol from A. (C) Comparisons of levels of expressions in oocytes injected with ASAP-Y (n = 9) and ASAP-Y and melanin (n = 5). P = 0.7365, unpaired two-tailed t test showed no statistical significance. (D) Comparison of background fluorescence at a holding potential of −60 mV in ASAP-Y (vegetal, n = 5; animal, n = 4) and ASAP-Y and melanin (n = 5). An ordinary one-way ANOVA with a post hoc Tukey multiple comparison test found no statistically significance differences among the three conditions. (E) Comparisons of fluorescence signal in oocytes injected with ASAP-Y (vegetal, n = 5; animal, n = 8) and ASAP-Y and melanin (n = 5). An ordinary one-way ANOVA with a post hoc Tukey multiple comparison test found no statistically significance differences among the three conditions. (F) Comparisons of ASAP-Y–injected oocytes ΔF/F % (vegetal, n = 5; animal, n = 4; and melanin injected, n = 5). ****, P < 0.0001, ordinary one-way ANOVA with a post hoc Tukey multiple comparison test. For further statistical information, see Tables S16–S19. All error bars are ±SEM centered on the mean.

References [+] :

Cha, Structural implications of fluorescence quenching in the Shaker K+ channel. 1998, Pubmed

	Figure 1. Oocytes are variable in endogenous background fluorescence at multiple wavelengths. (A) X. laevis oocytes from different batches of oocytes. Scale bar, 1 mm. (B) Endogenous background fluorescence of the animal pole excited with a 420-nm LED reflected onto the oocytes by a 455-nm long-pass dichroic through a ×40 water-immersion objective, where the emission was collected through the dichroic and a 475-nm long-pass filter. Batches were placed in order of most endogenous background to least. Batch 1, n = 22; batch 2, n = 1; batch 3, n = 14; batch 4, n = 20; batch 5, n = 10; batch 6, n = 8; batch 7, n = 8; and batch 8, n = 7. A one-way ANOVA together with a post hoc Tukey multiple comparisons test. **, P < 0.0001. For further information, see Table S1. (C) Endogenous background fluorescence of the animal pole excited with a 470-nm LED that was passed through a filter cube housing a 480/40-nm excitation filter, a 505-nm long-pass dichroic, and a 535/50-nm emission filter. Batches are placed in order of most endogenous background to least. Batch 1, n = 6; batch 2, n = 8; batch 3, n = 6; batch 4, n = 5; batch 5, n = 7; batch 6, n = 4; batch 7, n = 4; and batch 8, n = 7. A one-way ANOVA together with a post hoc Tukey multiple comparisons test. **, P < 0.0001. For further information, see Table S2. The batch numbers in B and C do not correspond and are different batches of oocytes. The measurements are in comparable optical conditions.
	Figure 2. Addition of the SIK inhibitor decreases background fluorescence. (A) Chemical structure of the small-molecule SIK inhibitor HG 9-91-01. (B) Comparison of endogenous fluorescence between several batches of uninjected and 0.5% DMSO-injected oocytes. A one-way ANOVA with a post hoc Holm–Sidak multiple comparisons test found no significant difference compared with uninjected oocytes (uninjected: n = 11, 19, and 11; DMSO: n = 10, 18, and 11). For further information, see Table S3. (C) Effect of HG 9-91-01 endogenous fluorescence under different conditions: uninjected (circles, n = 14), 0.1% DMSO injected (diamonds, n = 9), 10 µM HG 9-91-01 bath (squares, n = 22), 10 µM HG 9-91-01 injected (triangles, n = 21), and 100 µM HG 9-91-01 injected (upside-down triangles, n = 21) measured on d 4. **, P < 0.0001 compared to uninjected oocytes, one-way ANOVA with a post hoc Holm–Sidak multiple comparisons test. For further information, see Table S4. (D) Endogenous fluorescence under different conditions measured over time, as described in B. Panel on the right is a zoom in to demonstrate the effect of the different conditions described in B. , P < 0.001, 10 µM HG injected and 100 µM HG injected on day 4 (two-way ANOVA with a post hoc Dunnett multiple comparisons test). For further information, see Table S5. (E) Results from a darker batch of oocytes where HG 9-91-01 was tested (uninjected: n = 11; 100 µM HG 9-91-01 injected: n = 11). , P = 0.0031, unpaired two-tailed t test. (F) Results from a lighter batch of oocytes where HG 9-91-01 was tested (uninjected: n = 22; 100 µM HG 9-91-01 injected: n = 14). **, P < 0.0001, unpaired two-tailed t test. All error bars are ±SEM centered on the mean..
	Figure 3. Addition of the SIK inhibitor improves ATTO 425 signal. (A) A comparison of uninjected (n = 19) and 100 µM HG 9-91-01–injected (n = 18) oocytes on day 4 before labeling with ATTO 425. (B) **, P < 0.0001 after labeling with ATTO 425, unpaired two-tailed t tests. (C) Pulse protocol for ATTO 425 fluorescence, where the oocytes were held at −90 mV and prepulsed to −120 mV for 60 ms, followed by an 80-ms pulse to 80 mV, with a post pulse of 120 mV for 60 ms before returning to the holding potential. (D) Representative trace of fluorescence data of ATTO 425–labeled Shaker W434F M356C (top) and ATTO 425 labeled Shaker W434F M356C + 100 µM HG 9-91-01 (bottom) oocytes with the pulse protocol from A. (E) Endogenous oocyte fluorescence on day 3 was checked for several conditions (uninjected, n = 16; construct, n = 8; HG 9-91-01, n = 21; construct + HG 9-91-01, n = 10). *, P < 0.0001, ordinary one-way ANOVA with a post hoc Bonferroni multiple comparisons test. (F) Comparisons of levels of expressions in Shaker W434F M356C (n = 4) and Shaker W434F M356C + HG 9-91-01 (n = 6) injected oocytes. Unpaired two-tailed t test showed no statistical significance (P = 0.5414). (G) Comparison of background fluorescence of ATTO 425–labeled oocytes Shaker W434F M356C (n = 4) and Shaker W434F M356C + HG 9-91-01 (n = 8). , P = 0.0324, unpaired two-tailed t test. (H) Comparisons of fluorescence signal in oocytes injected with Shaker W434F M356C (n = 4) and Shaker W434F M356C + HG 9-91-01 (n = 8). , P = 0.0085, unpaired two-tailed t test. (I) Comparison of ΔF/F % in oocytes injected with Shaker W434F M356C (n = 4) and Shaker W434F M356C + HG 9-91-01 (n = 8). , P = 0.0011, unpaired two-tailed t test. For further information, see Tables S6–S12. All error bars are ±SEM centered on the mean.
	Figure 4. Synthetic melanin characterization. (A) Absorption spectrum of 0.3 mg/ml melanin from 400 to 1,000 nm. (B) Absorbance of different concentrations of melanin at 600 nm (n = 3). (C) Dynamic light scattering measurement of synthetic melanin size at 30 mg/ml unfiltered (average radius size, 220 nm).
	Figure 5. Synthetic melanin injections significantly improve fluorescence recording conditions. (A) Representative X. laevis oocytes injected with 50.1 nl of 30 mg/ml melanin. Scale bars, 1 mm (top) and 0.3 mm (bottom). (B) Improvement of endogenous background fluorescence of four separate batches of oocytes at 535 nm. Batch 1, n = 4; batch 2, n = 6; batch , n = 10; and batch 4, n = 4. , P < 0.05; , P < 0.01; *, P < 0.0001, two-way ANOVA with a post hoc Tukey multiple comparison test. (C) The improvement in endogenous background fluorescence in “dark” oocytes, <20 AU at 535 nm (vegetal, n = 4; animal, n = 4; and melanin injected, n = 4). *, P < 0.0001, ordinary one-way ANOVA with a post hoc Tukey multiple comparison test; no statistically significant difference was found between the animal pole and melanin. (D) The improvement in endogenous background fluorescence in “light” oocytes, >20 AU at 535 nm (vegetal, n = 4; animal, n = 4; and melanin injected, n = 4). , P < 0.05; ****, P < 0.0001, ordinary one-way ANOVA with a post hoc Tukey multiple comparison test. For further statistical information, see Tables S13–S15.
	Figure 6. Synthetic melanin injections improve the signal of ASAP-Y. (A) Pulse protocol for ASAP-Y fluorescence, where the oocytes were held at −60 mV with a 50-ms prepulse to −160 mV, a 30-ms pulse to 160 mV, and a postpulse back to −160 mV for 50 ms before returning to holding. (B) A representative trace of fluorescence data of ASAP-Y (top) and ASAP-Y and melanin (bottom) with the pulse protocol from A. (C) Comparisons of levels of expressions in oocytes injected with ASAP-Y (n = 9) and ASAP-Y and melanin (n = 5). P = 0.7365, unpaired two-tailed t test showed no statistical significance. (D) Comparison of background fluorescence at a holding potential of −60 mV in ASAP-Y (vegetal, n = 5; animal, n = 4) and ASAP-Y and melanin (n = 5). An ordinary one-way ANOVA with a post hoc Tukey multiple comparison test found no statistically significance differences among the three conditions. (E) Comparisons of fluorescence signal in oocytes injected with ASAP-Y (vegetal, n = 5; animal, n = 8) and ASAP-Y and melanin (n = 5). An ordinary one-way ANOVA with a post hoc Tukey multiple comparison test found no statistically significance differences among the three conditions. (F) Comparisons of ASAP-Y–injected oocytes ΔF/F % (vegetal, n = 5; animal, n = 4; and melanin injected, n = 5). ****, P < 0.0001, ordinary one-way ANOVA with a post hoc Tukey multiple comparison test. For further statistical information, see Tables S16–S19. All error bars are ±SEM centered on the mean.