A long and expanding list of fluorophores have been used with 3-photon excitation.
Early experiments paired 1700 nm excitation with red fluorophores. With the advent of 1300 nm lasers suitable for 3-photon excitation, the use of GFP-based indicators such as the GCaMP series has become more common. In addition, a some indicators and endogenous biological molecules have 3-photon cross-sections at shorter wavelengths (e.g. monoamines at ~700 nm; Maiti et al., 1997).
3-photon excitation at 1300 nm
Many green-emitting fluorophores are 3-photon excitable at 1300 nm, including fluorescein, green fluorescent protein (GFP) and GFP-based indicators:
fluorescein (Cheng et al., 2014; Wang et al., 2018b; Liu et al., 2020b).
Oregon green 488 BAPTA-1 (Liu et al., 2020b).
wtGFP (Cheng et al., 2014).
eGFP (Chen et al., 2018; Hsu et al., 2018; Rodriguez et al., 2018).
YFP (Chen et al., 2018).
GCaMP5 (Chen et al., 2018).
GCaMP6f and GCaMP6s (Ouzounov et al., 2017; Wang et al., 2018b; Ouzounov et al., 2019; Weisenberger et al., 2019; Yildirim et al., 2019; Takasaki et al., 2019; Takasaki et al., 2020).
What's the optimal wavelength for 3-photon excitation of GCaMP indicators? Several competing factors are relevant: the peak of the 3-photon cross-section is 1300 nm (Ouzounov et al., 2019); the fractional change in fluorescence of GCaMP6s upon binding calcium (ΔF/F) increases with wavelength from 1250 to 1375 nm; and water absorption increases across this wavelength range. Empirical measurements indicate that the most efficient excitation wavelength is 1300 nm (Ouzounov et al., 2019).
Unfortunately 3-photon excitation cross-sections have been measured for few fluorophores. The cross-sections of fluorescein, wtGFP and GCaMP6f are comparable at 1300 nm (Cheng et al., 2014; https://www.janelia.org/lab/harris-lab/research/photophysics/two-photon-fluorescent-probes.)
Discrimination index, a measure of the effectiveness of excitation, peaks at 1300 nm for GCaMP6s (from Ouzounov et al., 2019).
3-photon excitation at 1700 nm
Many red-emitting fluorophores are 3-photon excitable at 1700 nm, including:
Texas red (Horton et al., 2013; Horton et al., 2015; Wang et al., 2019; Liu et al., 2020b).
DsRed tdimer2(12) (Horton et al., 2013).
tdTomato (Rodriguez et al., 2018; Liu et al., 2020b).
mCherry (Tao et al., 2017; Liu et al., 2020b).
mRaspberry (Liu et al., 2020b).
Alexa fluor 633 and 647 (Wang et al., 2019; Liu et al., 2020a).
Sulforhodamine 101 (Cheng et al., 2014; Liu et al., 2020b).
jRCaMP1b (Tao et al., 2017).
jRGECO1a (Li et al., 2019).
Qtracker655, 705 and 800 quantum dots (Liu et al., 2019).
3-photon cross section of calcium-bound GCaMP6f. Adapted from https://www.janelia.org/lab/harris-lab/research/photophysics/two-photon-fluorescent-probes.
2-photon excitation at 1300 nm
Some red fluorophores are 2-photon excitable at 1300 nm, enabling simultaneous 2- and 3-photon excitation of green and red fluorophores. An example is Alexa fluor 680 (Kobat et al., 2011; Wang et al., 2018b).
4-photon excitation at 1700 nm
4-photon excitation of fluorescein and of wtGFP has been demonstrated at 1700 nm (Cheng et al., 2014), raising the possibility of simultaneous 3- and 4-photon excitation of red and green indicators.
Second and third harmonics
Many biological structures generate second and third harmonics (SHG and THG) at 1300 and 1700 nm, providing label-free contrast. Although SHG and THG signals are generated in the forward direction (with respect to the incident illumination), backscatter by biological tissue permits detection in the epi-configuration. SHG and THG signals occur at 1/2 and 1/3 the incident wavelength, respectively, so are often easily separated from fluorescence.
SHG arises from a relatively small subset of biological molecules and has been employed to image collagen, axons and blood vessel walls at 1300 or 1700 nm (Witte et al., 2011; Small et al., 2018.)
THG occurs at interfaces where there is a refractive index mismatch, such as between water and lipid, is associated with many biological molecules, and has been used to image blood vessel walls, erythrocytes, atheroschlerotic plaques, myelinated fibres, muscle fibers, and bone (Farrar et al., 2011; Rehberg et al., 2011; Witte et al., 2011; Horton et al., 2013; Ouzounov et al., 2017; Small et al., 2018; Wang et al., 2018a; Wang et al., 2018b; Ahn et al., 2020; Liu et al., 2020). In Drosophila, THG has been used to image brain tissue and the tissue underlying the cuticle (Tao et al., 2017; Hsu et al., 2018).
THG is dependent on the spatial extent of the interface, with structures of approximately half the axial extent of the PSF driving strong THG (Debarre et al., 2005). Axial PSFs for 3-photon excitation are commonly ~2-3 µm FWHM so many cellular structures, such as dendrites and myelinated axons, are of an appropriate size to generate strong THG signals (Witte et al., 2011).