In Vivo Deep-Brain Structural and Hemodynamic Multiphoton Microscopy Enabled by Quantum Dots.

Visualizing deep-brain vasculature and hemodynamics is key to understanding brain physiology and pathology. Among the various adopted imaging modalities, multiphoton microscopy (MPM) is well-known for its deep-brain structural and hemodynamic imaging capability. However, the largest imaging depth in MPM is limited by signal depletion in the deep brain. Here we demonstrate that quantum dots are an enabling material for significantly deeper structural and hemodynamic MPM in mouse brain in vivo. We characterized both three-photon excitation and emission parameters for quantum dots: the measured three-photon cross sections of quantum dots are 4-5 orders of magnitude larger than those of conventional fluorescent dyes excited at the 1700 nm window, while the three-photon emission spectrum measured in the circulating blood in vivo shows a slight red shift and broadening compared with ex vivo measurement. On the basis of these measured results, we further demonstrate both structural and hemodynamic three-photon microscopy in the mouse brain in vivo labeled by quantum dots, at record depths among all MPM modalities at all demonstrated excitation wavelengths.

3-photon fluorescence imaging of sulforhodamine B-labeled elastic fibers in the mouse skin in vivo.

Elastic fibers are key constituents of the skin. The commonly adopted optical technique for visualizing elastic fibers in the animal skin in vivo is 2-photon microscopy (2 PM) of autofluorescence, which typically suffers from low signal level. Here we demonstrate a new optical methodology to image elastic fibers in animal models in vivo: 3-photon microscopy (3 PM) excited at the 1700-nm window combining with preferential labeling of elastic fibers using sulforhodamine B (SRB). First, we demonstrate that intravenous injection of SRB can circumvent the skin barrier (encountered in topical application) and preferentially label elastic fibers, as verified by simultaneous 2 PM of both autofluorescence and SRB fluorescence from skin structures. Then through 3-photon excitation property characterization, we show that 3-photon fluorescence can be excited from SRB at the 1700-nm window, and 1600-nm excitation is most efficient according to our 3-photon action cross section measurement. Based on these results and using our developed 1600-nm femtosecond laser source, we finally demonstrate 3 PM of SRB-labeled elastic fibers through the whole dermis in the mouse skin in vivo, with only 3.7-mW optical power deposited on the skin surface. We expect our methodology will provide novel optical solution to elastic fiber research.

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?

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.

Visualizing the "sandwich" structure of osteocytes in their native environment deep in bone in vivo.

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.