Comparison of higher‐order multiphoton signal generation and collection at the 1700‐nm window based on transmittance measurement of objective lenses

One benefit of excitation at the 1700‐nm window is the more accessible modalities of multiphoton signal generation. It is demonstrated here that the transmittance performance of the objective lens is of vital importance for efficient higher‐order multiphoton signal generation and collection excited at the 1700‐nm window. Two commonly used objective lenses for multiphoton microscopy (MPM) are characterized and compared, one with regular coating and the other with customized coating for high transmittance at the 1700‐nm window. Our results show that, fourth harmonic generation imaging of mouse tail tendon and 5‐photon fluorescence of carbon quantum dots using the regular objective lens shows an order of magnitude signal higher than those using the customized objective lens. Besides, the regular objective lens also enables a 3‐photon fluorescence imaging depth of >1600 μm in mouse brain in vivo. Our results will provide guidelines for objective lens selection for MPM at the 1700‐nm window.     

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

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.      

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

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.      

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.     

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

Multiphoton action cross‐sections are the prerequisite for excitation light selection. At the 1700‐nm window suitable for deep‐tissue imaging, wavelength‐dependent 3‐photon action cross‐sections ησ₃ for RFPs are unknown, preventing wavelength selection. Here we demonstrate: (1) ex vivo measurement of wavelength‐dependent ησ₃ for purified RFPs; (2) a multiphoton imaging guided measurement system for in vivo measurement; and (3) in vivo measurement of wavelength‐dependent ησ₃ in RFP labeled cells. These fundamental results will provide guidelines for excitation wavelength selection for 3‐photon fluorescence imaging of RFPs at the 1700‐nm window, and augment the existing database of multiphoton action cross‐sections for fluorophores.      

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.      

Three‐dimensional cellular imaging in thick biological tissue with confocal detection of one‐photon fluorescence in the near‐infrared II window

Fluorescence imaging in the second near‐infrared optical window (NIR‐II, 900‐1700 nm) has become a technique of choice for noninvasive in vivo imaging in recent years. Greater penetration depths with high spatial resolution and low background can be achieved with this NIR‐II window, owing to low autofluorescence within this optical range and reduced scattering of long wavelength photons. Here, we present a novel design of confocal laser scanning microscope tailored for imaging in the NIR‐II window. We showcase the outstanding penetration depth of our confocal setup with a series of imaging experiments. HeLa cells labeled with PbS quantum dots with a peak emission wavelength of 1276 nm can be visualized through a 3.5‐mm‐thick layer of scattering medium, which is a 0.8% Lipofundin solution. A commercially available organic dye IR‐1061 (emission peak at 1132 nm), in its native form, is used for the first time, as a NIR‐II fluorescence label in cellular imaging. Our confocal setup is capable of capturing optically sectioned images of IR‐1061 labeled chondrocytes in fixed animal cartilage at a depth up to 800 μm, with a superb spatial resolution of around 2 μm.      

Self-phase-modulated femtosecond laser source at 1603 nm and its application to deep-brain 3-photon microscopy in vivo

3-photon microscopy (3PM) excited at the 1700 nm window enables deep-tissue imaging in vivo, especially in brain. PC rod soliton source has previously been exclusively used as the excitation source, which is rather costly and difficult to align. Here we demonstrate a novel nonlinear optical technique to build femtosecond laser source at the 1700 nm window, based on self-phase modulation (SPM) in a short span of large-mode-area fiber. The spectral broadening experienced by the pump pulse leads to the generation of a red-shifted sidelobe at 1603 nm. After spectral filtering, this sidelobe corresponds to 170-fs, 167-nJ pulses at 1603 nm. Using this SPM source, we further demonstrate deep-brain 3 PM to a depth of 1500 μm below the mouse brain surface in vivo. Our SPM femtosecond laser source thus provides a cost effective and easy-to-align alternative excitation source to the PC rod soliton source.     

Two‐photon excitation and direct emission from S2 state of U.S. Food and Drug Administration approved near‐infrared dye: Application of anti‐Kasha's rule for two‐photon fluorescence imaging

In recent years, two‐photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near‐infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two‐photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two‐photon excitation property of ICG to the second excited singlet (S₂) state in aqueous solution. Furthermore, in this work, we demonstrate the two‐photon cellular imaging application of ICG using direct fluorescence emission from S₂ state for the first time. Our results show that two‐photon excitation to S₂ state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well‐known “tissue‐optical window.” This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two‐photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.      

Anti‐Stokes fluorescence from endogenously formed protoporphyrin IX – Implications for clinical multiphoton diagnostics

Multiphoton imaging based on two‐photon excitation is making its way into the clinics, particularly for skin cancer diagnostics. It has been suggested that endogenously formed protoporphyrin IX (PpIX) induced by aminolevulinic acid or methylaminolevulinate can be applied to improve tumor contrast, in connection to imaging of tissue autofluorescence. However, previous reports are limited to cell studies and data from tissue are scarce. No report shows conclusive evidence that endogenously formed PpIX increases tumor contrast when performing multiphoton imaging in the clinical situation. We here demonstrate by spectral analysis that two‐photon excitation of endogenously formed PpIX does not provide additional contrast in superficial basal cell carcinomas. In fact, the PpIX signal is overshadowed by the autofluorescent background. The results show that PpIX should be excited at a wavelength giving rise to one‐photon anti‐Stokes fluorescence, to overcome the autofluorescent background. Thus, this study reports on a plausible method, which can be implemented for clinical investigations on endogenously formed PpIX using multiphoton microscopy (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Non‐Invasive Multi‐Dimensional Two‐Photon Microscopy enables optical fingerprinting (TPOF) of immune cells

Mucosal surfaces are constantly exposed to pathogens and show high immunological activity. In a broad variety of ocular surface disorders inflammation is common, but underlying mechanisms are often not fully understood. However, the main clinical problem is that inflammatory processes are difficult to characterize and quantify due to the impossibility of repeated tissue probing of the delicate ocular surface. Therefore non‐invasive optical methods are thought to have the potential for intravital investigation of ocular surface inflammation. This study demonstrates the general potential of two‐photon microscopy to non‐invasively detect and discriminate key players of inflammation in the ocular surface by using intrinsic fluorescence‐based features without the necessity of tissue probing or the use of dyes. The use of wavelength dependent measurements of fluorescence lifetime, in addition to autofluorescence intensity enables a functional differentiation of isolated immune cells in vitro at excitation wavelengths between 710 to 830 nm. Mixed cell cultures and first in vivo results indicate the use of excitation wavelength of 710 to 750 nm for further experiments and future use in patients.

Two photon based autofluorescence features of immune cells enables non‐invasive differentiation.     

Bond‐selective imaging of deep tissue through the optical window between 1600 and 1850 nm

We report the employment of an optical window between 1600 nm and 1850 nm for bond‐selective deep tissue imaging through harmonic vibrational excitation and acoustic detection of resultant pressure waves. In this window where a local minimum of water absorption resides, we found a 5 times enhancement of photoacoustic signal by first overtone excitation of the methylene group CH₂ at 1730 nm, compared to the second overtone excitation at 1210 nm. The enhancement allows 3D mapping of intramuscular fat with improved contrast and of lipid deposition inside an atherosclerotic artery wall in the presence of blood. Moreover, lipid and protein are differentiated based on the first overtone absorption profiles of CH₂ and methyl group CH₃ in this window. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Self‐referenced axial chromatic dispersion measurement in multiphoton microscopy through 2‐color third‐harmonic generation imaging

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2‐color third‐harmonic generation imaging excited by a 2‐color soliton source with tunable wavelength separation. Our technique is self‐referenced, eliminating potential measurement error when 1‐color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2‐color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.      

Visible‐near infrared‐II skull optical clearing window for in vivo cortical vasculature imaging and targeted manipulation

Skull optical clearing window permits us to perform in vivo cortical imaging without craniotomy, but mainly limits to visible (vis)‐near infrared (NIR)‐I light imaging. If the skull optical clearing window is available for NIR‐II, the imaging depth will be further enhanced. Herein, we developed a vis‐NIR‐II skull optical clearing agents with deuterium oxide instead of water, which could make the skull transparent in the range of visible to NIR‐II. Using a NIR‐II excited third harmonic generation microscope, the cortical vasculature of mice could be clearly distinguished even at the depth of 650 μm through the vis‐NIR‐II skull clearing window. The imaging depth after clearing is close to that without skull, and increases by three times through turbid skull. Furthermore, the new skull optical clearing window promises to realize NIR‐II laser‐induced targeted injury of cortical single vessel. This work enhances the ability of NIR‐II excited nonlinear imaging techniques for accessing to cortical neurovasculature in deep tissue.     

Deep-skin multiphoton microscopy in vivo excited at 1600 nm: A comparative investigation with silicone oil and deuterium dioxide immersion

Multiphoton microscopy (MPM) excited at the 1700-nm window has enabled deep-tissue penetration in biological tissue, especially brain. MPM of skin may also benefit from this deep-penetration capability. Skin is a layered structure with varying refractive index (from 1.34 to 1.5). Consequently, proper immersion medium should be selected when imaging with high numerical aperture objective lens. To provide guidelines for immersion medium selection for skin MPM, here we demonstrate comparative experimental investigation of deep-skin MPM excited at 1600 nm in vivo, using both silicone oil and deuterium dioxide (D₂O) immersion. We specifically characterize imaging depths, signal levels and spatial resolution. Our results show that both immersion media give similar performance in imaging depth and spatial resolution, while signal levels are slightly better with silicone oil immersion. We also demonstrate that local injection of fluorescent beads into the skin is a viable technique for spatial resolution characterization in vivo.      

Two‐photon excited fluorescence lifetime measurements through a double‐clad photonic crystal fiber for tissue micro‐endoscopy

This paper presents an endoscopic configuration for measurements of tissue autofluorescence using two–photon excitation and time‐correlated single photon counting detection through a double‐clad photonic crystal fiber (DC‐PCF) without pre‐chirping of laser pulses. The instrument performance was evaluated by measurements of fluorescent standard dyes, biological fluorophores (collagen and elastin), and tissue specimens (muscle, cartilage, tendon). Current results demonstrate the ability of this system to accurately retrieve the fluorescence decay profile and lifetime of these samples. This simple setup, which offers larger penetration depth than one‐photon‐based techniques, may be combined with morphology‐yielding techniques such as photoacoustic and ultrasound imaging. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Transmission in near‐infrared optical windows for deep brain imaging

Near‐infrared (NIR) radiation has been employed using one‐ and two‐photon excitation of fluorescence imaging at wavelengths 650–950 nm (optical window I) for deep brain imaging; however, longer wavelengths in NIR have been overlooked due to a lack of suitable NIR‐low band gap semiconductor imaging detectors and/or femtosecond laser sources. This research introduces three new optical windows in NIR and demonstrates their potential for deep brain tissue imaging. The transmittances are measured in rat brain tissue in the second (II, 1,100–1,350 nm), third (III, 1,600–1,870 nm), and fourth (IV, centered at 2,200 nm) NIR optical tissue windows. The relationship between transmission and tissue thickness is measured and compared with the theory. Due to a reduction in scattering and minimal absorption, window III is shown to be the best for deep brain imaging, and windows II and IV show similar but better potential for deep imaging than window I.

     

Two‐ and three‐photon absorption cross‐section characterization for high‐brightness,cell‐specific multiphoton fluorescence brain imaging

We demonstrate an accurate quantitative characterization of absolute two‐ and three‐photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high‐brightness, cell‐specific two‐ and three‐photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two‐photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep‐tissue experiments.     

In vivo autofluorescence in the biological windows: the role of pigmentation

Small animal deep‐tissue fluorescence imaging in the second Biological Window (II‐BW, 1000–1350 nm) is limited by the presence of undesirable infrared‐excited, infrared‐emitted (900–1700 nm) autofluorescence whose origin, spectral properties and dependence on strains is still unknown. In this work, the infrared autofluorescence and laser‐induced whole body heating of five different mouse strains with distinct coat colors (black, grey, agouti, white and nude) has been systematically investigated. While neither the spectral properties nor the magnitude of organ autofluorescence vary significantly between mouse strains, the coat color has been found to strongly determine both the autofluorescence intensity as well as the laser‐induced whole body heating. Results included in this work reveal mouse strain as a critical parameter that has to be seriously considered in the design and performance of small animal imaging experiments based on infrared‐emitting fluorescent markers.

     

Two‐photon microscopy of healthy,infarcted and stem‐cell treated regenerating heart

Two‐photon excitation autofluorescence (produced in myocytes) and second‐harmonic generation (produced mainly by collagen) allow label‐free visualization of these two important components of myocardium. Because of their different emission wavelengths, these two signals can be separated spectrally. Here, we examine two‐photon microscopy images of healthy, infarcted and stem‐cell treated rat hearts. We find that in infarcted heart, regions distant from the site of infarct are similar to healthy tissue in composition (mostly myocytes, very little collagen) and organization (densely packed myocytes), but infarct regions are characterized by sparse myocytes and high collagen content indicative of scar tissue formation. Stem cell treated hearts, in contrast, show regions of intertwined myocytes and collagen throughout the infarct, suggesting reduced tissue damage. Finally, these results offer interesting insights into our ongoing polarized light studies of cardiac tissue anisotropy, and reveal that both tissue composition and tissue micro‐organization are reflected in polarization‐measured linear retardance values. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)