Fig. 1. Ionization penalty in multiphoton bioimaging^[25]. Simulation parameters are as follows: two-photon absorption cross section of green fluorescent protein σ_2PA=6.5 GM, 0.1 mmol/L solution, quantum efficiency η=1, ionization potential of water U₀=6.5 eV, laser wavelength λ=800 nm, and laser pulse width τ_L=100 fs

Fig. 2. Illustration of mechanisms leading to photodamage in multiphoton imaging^[35]

Fig. 3. Energy deposition and heat accumulation in multiphoton imaging. (a) Left: spatial distribution of irradiance in multiphoton imaging; right: spatial distribution of laser induced low-density plasma at focus^[29]. Due to nonlinearity, plasma region (dotted line) is much smaller than irradiance region (dashed line). (b) Illustration of heat accumulation. Heat accumulates if typical heat diffusion time t_d is longer than repetition period T_PRF, otherwise heat hardly accumulates^[29,63]

Fig. 4. In-vivo 2PEF of murine intestinal mucosa and photodamage^[12]. (a) Anaesthetized mouse on homothermic table; (b) illustration of isolated intestinal loop that was prepared in situ without disturbing blood supply; (c) schematic diagram of intestinal mucosa [yellow dashed line indicates imaging plane at depth of 1.6 μm for (d)-(f)]; (d) first scan of cross-sectional autofluorescence image of intestinal villus in vivo; (e) hyperfluorescence appeared after 7 times repeated scanning; (f) dark spots appeared after 12 times repeated scanning

Fig. 5. 2PEF imaging of living murine cortex and photodamage^[14,66]. (a) Schematic of in-vivo labeling of live and dead cell fluorescent dyes; (b) schematic of two-photon laser scanning of neuron cell nucleus; (c) schematic for evaluating changes in fluorescence intensity of dendrites around target cell for fixed-spot irradiation; (d) two-photon imaging laser scanning damaged neuronal structures surrounding target cell at P_avg=176 mW and for scanning time of 2 s; (e) reduction in fluorescence intensity of YFP-expressing neurites around target cell after fixed-spot imaging laser irradiation with different exposure time at P_avg=300 mW

Fig. 6. Nonlinear energy deposition by single laser pulse and heat accumulation by laser pulse series^[37]. (a) Temporal evolution of electron number density n_e and average kinetic energy ε_avg; (b) temporal evolution of temperature increase of water at focus by single pulse; (c) heat accumulation by pulse series for NA=1.1 at 80 MHz; (d) electron energy spectrum at end of pulse. Simulation parameters are τ_L=140 fs, λ=930 nm, I₀=2.61×10¹² W/cm², f_PRF=80 MHz. (a), (b) and (d) are obtained by Eqs. (4), (5) and (9), and (c) by Eq. (14)

Fig. 7. Two-photon excited fluorescence imaging of retinal cryosections from pigmented Rpe65-/- mice and photodamage. (a) Schematic of retinal substructures^[55]. (b)-(e) 2PEF images of retinal obtained consecutively at 80 MHz^[57]; (f)-(i) 2PEF images of retinal obtained consecutively at 8 MHz^[57]. Regions to the left of blue dashed lines in (f)-(i) indicate portion of the sample that was exposed to 80 MHz light (e). Laser and objective parameters are λ=750 nm, pulse duration τ_L=75 fs and NA=1.0

Fig. 8. Simulation results of plasma-mediated and photothermal effects^[57]. (a) Electron number density and average kinetic energy after single pulse as function of peak irradiance I₀; (b) peak temperature rise due to nonlinear absorption after single pulse and after pulse series as function of I₀; (c) schematic of linear absorption of melanosome particle located at laser spot center; (d) temperature increase of melanosome after single pulse as function of average power for repetition rates of 8 MHz and 80 MHz; (e), (f) heat accumulation by pulse series at 80 MHz and at 8 MHz, respectively; (g), (h) enlarged views of (e) and (f). (a) and (b) are obtained by Eqs. (4), (5) and (9), (d) is simulated by Eq. (8), and (e)-(h) are calculated using Eq. (14)

Fig. 9. Brain heating and thermal damage induced by continuous scanning of 1320 nm three-photon microscopy. (a) Attenuation length of light in mouse brain cortex^[5,77]; (b) pulse energy required at brain surface to generate same signal strength for 2PEF and 3PEF at different imaging depths^[11]; (c) immunostaining results revealing brain heating effects by 1320 nm 3PEF after 20 min continuous scanning with 150 mW average power at 1 mm imaging depth^[11], in which damage is indicated by white arrowheads and scale bar represents 0.5 mm; (d) Monte-Carlo simulation of light intensity of 1320 nm excitation light focused at 1 mm below brain surface^[11]; (e) temperature maps under 1320 nm illumination after 60 s of continuous scanning with 100 mW and 150 mW average power^[11]; (f) maximum temperature versus imaging power for 1 mm and 1.2 mm focal depth^[11]

Fig. 10. Simulation results of plasma-mediated effects. (a) Temporal evolution of electron number density n_e and average kinetic energy ε_avg. Dashed line indicates seed electron density. (b) Electron energy spectrum at the end of pulse. Simulation parameters are same as those in 150 mW experiments in Ref. [11]: τ_L=60 fs, λ=1320 nm, NA=0.75, P_avg=150 mW, average power at brain surface P_surf=128 mW, f_PRF=1 MHz, pulse energy at focus E_z=1.15 nJ, I_z=1.97×10¹² W/cm². Obtained by Eqs. (4) and (5)