Ex and in vivo characterization of the wavelength‐dependent 3‐photon action cross‐sections of red fluorescent proteins covering the 1700‐nm window

Cheng

et al. 2019

Self Cite

Three‐photon microscopy excited at the 1700‐nm window (roughly covering 1600‐1840 nm) is especially suitable for deep‐brain imaging in living animals. To match the brain refractive index, D2O has been exclusively used as the immersion medium. However, the hygroscopic property of D2O leads to a decrease of transmittance of the excitation light and as a result a decrease in three‐photon signals over time. Solutions such as replacing D2O from time to time, wrapping both the objective lens and the immersion D2O, and sealing D2O with paraffin liquid have all been demonstrated, which add to the system complexity. Based on our recent characterization of immersion oils, we propose using silicone oil as a potential alternative to D2O for deep‐brain imaging. Excited at 1600 nm, our comparative deep‐brain imaging using both D2O and silicone oil immersion show that silicone oil immersion yields 17% higher three‐photon signal in third‐harmonic generation imaging within the white matter. Besides, silicone oil immersion also enables three‐photon fluorescence imaging of vasculature up to 1460 μm (mechanical depth) into the mouse brain in vivo acquired at 2 seconds/frame. Together with the nonhygroscopic physical property, silicone oil is promising for long‐span three‐photon brain imaging excited at the 1700‐nm window.

Section: Discussionmentioning

confidence: 99%

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

Tong

Cheng

et al. 2019

Self Cite

“…In this research, we demonstrate that 3PM excited at the 1700‐nm window, besides suitable for through‐the‐skull imaging, is also a promising optical imaging technique especially suitable for visualizing osteocytes deep in the skull in vivo. 3PM excited at the 1700‐nm window (covering 1600–1840 nm ) has been demonstrated to be the enabling technique for deepest multiphoton brain imaging . However, bone is a totally different biological tissue from brain (more scattering and less water content).…”

Section: Introductionmentioning

confidence: 99%

Visualizing the “sandwich” structure of osteocytes in their native environment deep in bone in vivo

Wang

et al. 2018

Self Cite

Osteocytes are the most abundant cells in bone and always the focus of bone research. They are embedded in the highly scattering mineralized bone matrix. Consequently, visualizing osteocytes deep in bone with subcellular resolution poses a major challenge for in vivo bone research. Here we overcome this challenge by demonstrating 3‐photon imaging of osteocytes through the intact mouse skull in vivo. Through broadband transmittance characterization, we establish that the excitation at the 1700‐nm window enables the highest optical transmittance through the skull. Using label‐free third‐harmonic generation (THG) imaging excited at this window, we visualize osteocytes through the whole 140‐μm mouse skull and 155 μm into the brain in vivo. By developing selective labeling technique for the interstitial space, we visualize the “sandwich” structure of osteocytes in their native environment. Our work provides novel imaging methodology for bone research in vivo.

“…Equivalently, air‐core fiber solitons are not as efficient as PC rod solitons in exciting three‐photon signals. This can be quantitatively explained by the three‐photon signal S 3 dependence on excitation power < P > and pulse width τ , given by :

S_{3} \propto < P >^{3} / τ^{2} .

…”

Section: Experimental and Simulation Resultsmentioning

confidence: 99%

Air‐core fiber or photonic‐crystal rod, which is more suitable for energetic femtosecond pulse generation and three‐photon microscopy at the 1700‐nm window?

Gan

Zhuang

et al. 2019

Self Cite

Energetic femtosecond pulses at the 1700‐nm window are a prerequisite for deep‐tissue three‐photon microscopy (3PM). Soliton self‐frequency shift (SSFS) in photonic‐crystal (PC) rod has been the only technique to generate such pulses suitable for 3PM. Here we demonstrate through SSFS in an air‐core fiber, we can generate most energetic femtosecond soliton pulses at the 1700‐nm window, 5.2 times higher than that from PC rod. However, the air‐core soliton pulse width is 5.9 times longer than that of PC rod soliton. Based on comparative 3PM excited with both air‐core and PC rod solitons, we propose the more suitable source for 3PM. We further elucidate the challenge of generating shorter soliton pulses from air‐core fibers through numerical simulation.