Efficient non-degenerate two-photon excitation for fluorescence microscopy

Sanaz Sadegh; Sanaz Sadegh; Mu-Han Yang; Mu-Han Yang; Christopher G. L. Ferri; Christopher G. L. Ferri; Martin Thunemann; Payam A. Saisan; Zhe Wei; Erik A. Rodriguez; Stephen R. Adams; Kivilcim Kiliç; David A. Boas; Sava Sakadžić; Anna Devor; Anna Devor; Anna Devor; Anna Devor; Yeshaiahu Fainman; Yeshaiahu Fainman

doi:10.1364/OE.27.028022

Optics Express
Vol. 27,
Issue 20,
pp. 28022-28035
(2019)
•https://doi.org/10.1364/OE.27.028022

Efficient non-degenerate two-photon excitation for fluorescence microscopy

Sanaz Sadegh, Mu-Han Yang, Christopher G. L. Ferri, Martin Thunemann, Payam A. Saisan, Zhe Wei, Erik A. Rodriguez, Stephen R. Adams, Kivilcim Kiliç, David A. Boas, Sava Sakadžić, Anna Devor, and Yeshaiahu Fainman

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Sanaz Sadegh, Mu-Han Yang, Christopher G. L. Ferri, Martin Thunemann, Payam A. Saisan, Zhe Wei, Erik A. Rodriguez, Stephen R. Adams, Kivilcim Kiliç, David A. Boas, Sava Sakadžić, Anna Devor, and Yeshaiahu Fainman, "Efficient non-degenerate two-photon excitation for fluorescence microscopy," Opt. Express 27, 28022-28035 (2019)

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Abstract

Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microscopy. However, a systematic study of the efficiency of ND-TPE to guide the selection of fluorophore excitation wavelengths is missing. We measured the relative non-degenerate two-photon absorption cross-section (ND-TPACS) of several commonly used fluorophores (two fluorescent proteins and three small-molecule dyes) and generated 2-dimensional ND-TPACS spectra. We observed that the shape of a ND-TPACS spectrum follows that of the corresponding degenerate two-photon absorption cross-section (D-TPACS) spectrum, but is higher in magnitude. We found that the observed enhancements are higher than theoretical predictions.

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Fig. 1. ND-TPE spectroscopy. a) Color-coded, normalized ND-TPACS spectra for EGFP, mKO2, Fluorescein, Coumarin 343, and SR101 showing the dependence of the normalized ND-TPACS on NIR and IR wavelengths. The ND-TPACS at each combination of wavelengths was normalized by the peak D-TPACS value of the fluorophore. The isoclines corresponding to the ground to excited state transition energy (

$hc/\lambda _{\mathrm {NIR}}+hc/\lambda _{\mathrm {IR}}=2.66$

eV for EGFP,

$2.6$

eV for mKO2,

$2.7$

eV for Fluorescein, and

$2.76$

eV for SR101) are overlaid as dashed black lines. b) ND-TPACS normalized by the peak D-TPACS as a function of the equivalent degenerate wavelength

$2/\lambda _{\mathrm {D}}=1/\lambda _{\mathrm {NIR}}+1/\lambda _{\mathrm {IR}}$

are shown in red. Along any energy isocline, multiple excitation wavelength combinations within our tuning range sum to the same total energy and, therefore, the same equivalent wavelength. Thus, for each

$\lambda _{\mathrm {D}}$

value we report several values of ND-TPACS (red dots). Independently measured D-TPACS normalized by its peak within the equivalent range of the total photon energy (

$2.3-2.9$

eV) are shown in black. The black dashed vertical line indicates the position of the peak D-TPACS used for the normalization procedure (

$930$

nm for EGFP,

$950$

nm for mKO2,

$920$

nm for Fluorescein,

$860$

nm for Coumarin 343, and

$900$

nm for SR101). These wavelengths correspond to the energy isoclines shown in panel (a).

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Fig. 2. Choice of excitation wavelengths in ND-TPE microscopy. a) Effects of scattering and absorption (adapted from [21]): photon fraction at depth of

$1$

mm, considering both absorption and scattering, for average brain tissue optical properties is shown as blue line. Percent of the photons absorbed by brain tissue in

$1$

mm is shown as red line. b) Simulation results for tissue heating by a scanned focused light: ratio of the maximum tissue temperature change under simultaneous excitation by NIR and IR beam to maximum temperature change under excitation with a single beam with equivalent degenerate wavelength,

$2hc/\lambda _{\mathrm {D}}=hc/\lambda _{\mathrm {NIR}}+hc/\lambda _{\mathrm {IR}}$

and

$\lambda _{\mathrm {D}}=920$

nm, versus

$\lambda _{\mathrm {IR}}$

assuming equal total excitation power

$P_{\mathrm {NIR}}+P_{\mathrm {IR}}=P_{\mathrm {D}}=100$

mW at focal point (

$250$

$\mu$

m below surface).

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Fig. 3. a) Experimental setup for ND-TPACS measurement. L, lens; PBS, polarizing beam splitter;

$\lambda$

/2, half wave plate; GS, glass slide; M, mirror; DM, dichroic mirror; BD, beam dump; FS, fluorescent sample; OL, objective lens; BPF, band pass filter; PM, power meter; and PMT, photomultiplier tube. b) A typical plot of fluorescence intensity as a function of the temporal offset between NIR and IR pulses. The increase in the signal at zero temporal offset is due to ND-TPE. The red line shows the fitted model (Eq. (3)).

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Fig. 4. Linear dependence of non-degenerate two-photon fluorescence excitation on a) NIR excitation power (

$P_{\mathrm {IR}}= 15$

mW), and b) IR excitation power (

$P_{\mathrm {NIR}}$

= 5 mW). The power dependence was tested at wavelength combinations

$\lambda _{\mathrm {NIR}}=740$

nm and

$\lambda _{\mathrm {IR}}=1230$

nm for all measured fluorophores.

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Fig. 5. Characterization of the experimental error in ND-TPACS measurements via repetitive measurements of ND-TPACS along one isocline. Here we show

$10$

measurements of normalized ND-TPACS of EGFP along the

$hc/\lambda _{\mathrm {NIR}}+hc/\lambda _{\mathrm {IR}}=2.66$

eV isocline vs

$\lambda _{\mathrm {NIR}}$

. The measured relative experimental error is

$\sim 15\%$

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Fig. 6. Normalized one-photon absorption spectra of Coumarin 343 (cyan), EGFP (green), Fluorescein (yellow), mKO2 (red), and SR101 (dark red).

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Fig. 7. Graphical representation of Table 1 for comparison of the experimental and theoretical (two-level approximation, Eq. (1)) values of the ISRE for

$\hbar \omega _{\mathrm {NIR}}+\hbar \omega _{\mathrm {IR}}=2.66$

eV for EGFP,

$2.6$

eV for mKO2,

$2.7$

eV for Fluorescein, and

$2.76$

eV for SR101. Experimental data (solid line) and theoretical results (dashed line) are overlaid.

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Fig. 8. Tissue heating simulations. Simulation code was provided by K. Podgorski [37]. a-d) Simulated spatial temperature profile in brain tissue with cover glass and immersion water (temperature of the immersion water

$1$

mm above cover glass was kept constant at

$25$

$^{\circ }$

C). Profiles are shown for no illumination,

$100$

mW at

$920$

nm,

$50$

mW at

$1230$

nm, and

$50$

mW at

$740$

nm. e-g) Simulated temperature change for

$100$

mW at

$920$

nm,

$50$

mW at

$1230$

nm, and

$50$

mW at

$740$

nm. Contour lines are shown for

$1$

$^{\circ }$

C intervals. All powers are given for a focal plane

$250$

$\mu$

m below the surface.

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Tables (3)

Table 1. Comparison between experimental and theoretical (two-level approximation, Eq. (1)) values of the ISRE₂ for $ℏ ω_{N I R} + ℏ ω_{I R} = 2.66$ eV for EGFP, $2.6$ eV for mKO2, $2.7$ eV for Fluorescein, and $2.76$ eV for SR101. (See Fig. 7 for the graphical representation of the results)

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Table 2. Fluorophore concentrations.

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Table 3. Optical parameters for average brain tissue [21,47] that were used in our heating simulations. $μ_{a}$ and $μ_{s}$ are the absorption and scattering coefficients, respectively, and $T = 100 \times e^{- (μ_{a} + μ_{s}) z}$ is the light transmission percentage at depth $z$ . We set $z = 250$ $μ$ m for our tissue heating simulations.

View Table | View all tables in this article

Equations (11)

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I S R E_{2} = \frac{σ_{N D} (ω_{N I R}, ω_{I R})}{σ_{D} (ω_{D})} = \frac{(ℏ ω_{N I R} + ℏ ω_{I R})^{2}}{4 ℏ ω_{N I R} ℏ ω_{I R}}

⟨ F ⟩_{D} = \frac{\sqrt{π} C P^{2} λ ϕ η σ_{D}^{(2)} (λ)}{4 f c^{2} h^{2} Γ} .

\begin{aligned} ⟨ F (τ) ⟩ & = \frac{\sqrt{π} C P_{N I R}^{2} λ_{N I R} ϕ η σ_{D}^{(2)} (λ_{N I R})}{4 f c^{2} h^{2} Γ_{N I R}} + \frac{\sqrt{π} C P_{I R}^{2} λ_{I R} ϕ η σ_{D}^{(2)} (λ_{I R})}{4 f c^{2} h^{2} Γ_{I R}} \\ + \frac{2 \sqrt{π} C P_{N I R} P_{I R} λ_{N I R} λ_{I R} ϕ η σ_{N D}^{(2)} (λ_{N I R}, λ_{I R})}{f c^{2} h^{2} Γ_{x} \sqrt{λ_{N I R}^{2} + λ_{I R}^{2}}} \exp (\frac{- τ^{2}}{2 Γ_{x}^{2}}) . \end{aligned}

| S^{(f)} |^{2} = | \sum_{i} [\frac{(e_{1} \cdot μ_{i 0}) (μ_{f i} \cdot e_{2})}{E_{i} - ℏ ω_{1} + i ξ_{i}} + \frac{(e_{1} \cdot μ_{i 0}) (μ_{f i} \cdot e_{2})}{E_{i} - ℏ ω_{2} + i ξ_{i}}] |^{2},

σ_{N D} (ω_{1}, ω_{2}) \sim ω_{1} ω_{2} | S^{(f)} |^{2} ρ_{f} (ω_{N I R} + ω_{I R} - ω_{f}),

| S^{(f)} |^{2} = | \sum_{i} [\frac{μ_{i 0} μ_{f i}}{E_{i} - ℏ ω_{1} + i ξ_{i}} + \frac{μ_{i 0} μ_{f i}}{E_{i} - ℏ ω_{2} + i ξ_{i}}] |^{2} .

| S^{(f)} |^{2} = | [\frac{μ_{00} μ_{f 0}}{E_{0} - ℏ ω_{1} + i ξ_{0}} + \frac{μ_{00} μ_{f 0}}{E_{0} - ℏ ω_{2} + i ξ_{0}}] + [\frac{μ_{f 0} μ_{f f}}{E_{f} - ℏ ω_{1} + i ξ_{f}} + \frac{μ_{f 0} μ_{f f}}{E_{f} - ℏ ω_{2} + i ξ_{f}}] |^{2} .

| S^{(f)} |^{2} \sim [μ_{f 0} (μ_{f f} - μ_{00})]^{2} (\frac{1}{ℏ ω_{1}} + \frac{1}{ℏ ω_{2}})^{2} .

σ_{N D} (ω_{1}, ω_{2}) \sim \frac{(ℏ ω_{1} + ℏ ω_{2})^{2}}{ℏ ω_{1} ℏ ω_{2}},

I S R E_{2} = \frac{σ_{N D} (ω_{N I R}, ω_{I R})}{σ_{D} (ω_{D})} = \frac{(ℏ ω_{D})^{2}}{ℏ ω_{N I R} ℏ ω_{I R}} = \frac{(ℏ ω_{N I R} + ℏ ω_{I R})^{2}}{4 ℏ ω_{N I R} ℏ ω_{I R}},

I S R E_{3} = \frac{ℏ ω_{N I R} ℏ ω_{I R} | μ_{f 0} (μ_{00} - μ_{f f}) (\frac{1}{ℏ ω_{N I R}} + \frac{1}{ℏ ω_{I R}}) + μ_{i 0} μ_{f i} (\frac{1}{E_{i} - ℏ ω_{N I R}} + \frac{1}{E_{i} - ℏ ω_{I R}}) |^{2}}{(ℏ ω_{D})^{2} | μ_{f 0} (μ_{00} - μ_{f f}) (\frac{2}{ℏ ω_{D}}) + μ_{i 0} μ_{f i} (\frac{2}{E_{i} - ℏ ω_{D}}) |^{2}} .

EGFP				mKO2
$ℏ ω_{N I R}$	$ℏ ω_{I R}$	${I S R E}_{2}$	${I S R E}_{e x p e r i m e n t}$	$ℏ ω_{N I R}$	$ℏ ω_{I R}$	${I S R E}_{2}$	${I S R E}_{e x p e r i m e n t}$
$1.33$	$1.33$	$1.000$	$-$	$1.30$	$1.30$	$1.000$	$-$
$1.51$	$1.15$	$1.019$	$1.1 \pm 0.2$	$1.49$	$1.11$	$1.022$	$1.0 \pm 0.2$
$1.63$	$1.03$	$1.054$	$1.4 \pm 0.2$	$1.55$	$1.05$	$1.038$	$1.2 \pm 0.2$
$1.67$	$0.99$	$1.070$	$1.5 \pm 0.2$	$1.63$	$0.97$	$1.069$	$1.6 \pm 0.2$

Fluorescein				SR101
$ℏ ω_{N I R}$	$ℏ ω_{I R}$	${I S R E}_{2}$	${I S R E}_{e x p e r i m e n t}$	$ℏ ω_{N I R}$	$ℏ ω_{I R}$	${I S R E}_{2}$	${I S R E}_{e x p e r i m e n t}$
$1.35$	$1.35$	$1.000$	$-$	$1.38$	$1.38$	$1.000$	$-$
$1.53$	$1.17$	$1.018$	$1 \pm 0.2$	$1.55$	$1.21$	$1.015$	$1.2 \pm 0.2$
$1.61$	$1.09$	$1.039$	$1.1 \pm 0.2$	$1.61$	$1.15$	$1.029$	$1.4 \pm 0.2$
$1.68$	$1.02$	$1.064$	$1.3 \pm 0.2$	$1.66$	$1.10$	$1.043$	$1.4 \pm 0.2$

Fluorophores	Solvent	Extinction Coefficient M $^{- 1}$ mm $^{- 1}$	Concentration $μ$ M
Fluorescein	PBS	$7500$ [38]	$90$
EGFP	PBS	$5600$ [38]	$45$
mKO2	PBS	$6380$ [39]	$20$
SR101	PBS	$13900$ [40]	$5$
Coumarin 343	DMSO	$4430$ [27]	$135$

$λ_{N I R}$ (nm)	$μ_{s}$ (1/mm)	$μ_{a}$ (1/mm)	$T$ ( $%$ )	$λ_{N I R}$ (nm)	$μ_{s}$ (1/mm)	$μ_{a}$ (1/mm)	$T$ ( $%$ )
$700$	$6.9$	$5 \times 10^{- 4}$	$18$	$1340$	$2.8$	$0.2$	$47$
$740$	$6.4$	$2.1 \times 10^{- 3}$	$20$	$1230$	$3.2$	$8.9 \times 10^{- 2}$	$44$
$780$	$6.0$	$2 \times 10^{- 3}$	$22$	$1120$	$3.6$	$1.9 \times 10^{- 2}$	$40$
$820$	$5.6$	$1.7 \times 10^{- 3}$	$25$	$1050$	$4.0$	$1.2 \times 10^{- 2}$	$37$
$860$	$5.2$	$3.3 \times 10^{- 3}$	$27$	$990$	$4.3$	$3.4 \times 10^{- 2}$	$34$
$900$	$4.9$	$4.8 \times 10^{- 3}$	$29$	$940$	$4.6$	$1.3 \times 10^{- 2}$	$31$
$920$	$4.8$	$6.5 \times 10^{- 3}$	$30$	$920$	$4.8$	$6.5 \times 10^{- 3}$	$30$

EGFP				mKO2
$ℏ ω_{N I R}$	$ℏ ω_{I R}$	${I S R E}_{2}$	${I S R E}_{e x p e r i m e n t}$	$ℏ ω_{N I R}$	$ℏ ω_{I R}$	${I S R E}_{2}$	${I S R E}_{e x p e r i m e n t}$
$1.33$	$1.33$	$1.000$	$-$	$1.30$	$1.30$	$1.000$	$-$
$1.51$	$1.15$	$1.019$	$1.1 \pm 0.2$	$1.49$	$1.11$	$1.022$	$1.0 \pm 0.2$
$1.63$	$1.03$	$1.054$	$1.4 \pm 0.2$	$1.55$	$1.05$	$1.038$	$1.2 \pm 0.2$
$1.67$	$0.99$	$1.070$	$1.5 \pm 0.2$	$1.63$	$0.97$	$1.069$	$1.6 \pm 0.2$

Abstract

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Tables (3)

Equations (11)

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