Refractive index and pulse broadening characterization using oil immersion and its influence on three‐photon microscopy excited at the 1700‐nm window

Abstract

Three‐photon microscopy excited at the 1700‐nm window enables deep‐tissue penetration. However, the refractive indices of commonly used immersion oils, and the resultant pulse broadening are not known, preventing imaging optimization. Here, we demonstrate detailed characterization of the refractive index, pulse broadening and distortion for excitation pulses at this window for commonly used immersion oils. On the physical side, we uncover that absorption, rather than material dispersion, is the main cause of pulse broadening and distortion. On the application side, comparative three‐photon imaging results indicate that 1600‐nm excitation yields 5 times higher three‐photon signal than 1690‐nm excitation.

Deep‐brain three‐photon microscopy excited at 1600 nm with silicone oil immersion

“…From the immersion medium perspective, the excitation wavelength is better chosen to suffer from least absorption. Based on our previous results , the measured absorption spectrum for both silicone oil and D 2 O are shown in Figure A. Comparing both absorption spectra, we can immediately see that: (a) 1600‐nm excitation is least absorbed by silicone oil within the 1700‐nm window; (b) around 1600 nm, the absorption due to silicone oil is even smaller than that due to D 2 O.…”

“…Recently, we have performed detailed investigation of the wavelength‐dependent refractive index, absorption and pulse broadening properties of several immersion oils suitable for imaging . Our results point to silicone oil as a promising immersion medium for MPM excited at the 1700‐nm window: (a) the measured refractive index (1.396 at 1600 nm ) is as close to the brain as D 2 O, indicating that both will reduce index mismatch induced spherical aberration equally well.…”

“…Figure B illustrates the wavelength‐dependent refractive index for a commonly used immersion oil (IMMOIL‐F30CC; Olympus, Tokyo, Japan), silicone oil (SIL300CS‐30CC; Olympus) and D 2 O (Cambridge Isotope Laboratories Inc, Tewksbury, Massachusetts). These refractive indices are calculated results from Sellmeier equations, which fit to the measured results with high precision . Although the brain refractive index is not measured within this window, we also schematically show it as the rectangle in the figure (data from ).…”

“…A 1‐MHz, 500‐fs, 1550‐nm fiber laser (FLCPA‐02CSZU; Calmar, Palo Alto, California) was used to pump a 44‐cm photonic‐crystal rod (SC‐1500/100‐Si‐ROD; NKT Photonics, Birkerød, Denmark) with a core diameter of 100 μm. At the output, tunable femtosecond soliton pulses can be generated through power tuning . A 1635‐nm long‐pass filter (1635lpf; Omega Optical, San Diego, California) was used to remove the residual pump, using the angle tuning technique for continuous cut‐on wavelength tuning.…”

“…For deep‐brain 3PM excited at the 1700‐nm window, deuterium oxide (D 2 O) immersion is exclusively used . The selection of D 2 O is based on the following reasons: (a) the refractive index of D 2 O (1.317 at 1600 nm ) is better matched to that of the brain (1.35~1.37 measured at various wavelengths excluding the 1700‐nm window ), in comparison with commonly used immersion oils (1.499 at 1600 nm ). (b) Compared with water (H 2 O) immersion, the absorption coefficients of D 2 O at the 1700‐nm window is an order‐of‐magnitude smaller, which in turn leads to an order‐of‐magnitude higher three‐photon signals .…”