Nonlinear Fluorescence Microscopy Research Articles

Fast wavefront shaping for two-photon brain imaging with multipatch correction.

Nonlinear fluorescence microscopy promotes in-vivo optical imaging of cellular structure at diffraction-limited resolution deep inside scattering biological tissues. Active compensation of tissue-induced aberrations and light scattering through adaptive wavefront correction further extends the accessible depth by restoring high resolution at large depth. However, those corrections are only valid over a very limited field of view within the angular memory effect. To overcome this limitation, we introduce an acousto-optic light modulation technique for fluorescence imaging with simultaneous wavefront correction at pixel scan speed. Biaxial wavefront corrections are first learned by adaptive optimization at multiple locations in the image field. During image acquisition, the learned corrections are then switched on the fly according to the position of the excitation focus during the raster scan. The proposed microscope is applied to in vivo transcranial neuron imaging and demonstrates multi-patch correction of thinned skull-induced aberrations and scattering at 40-kHz data acquisition speed.

Tight Focus Vector Light Field of Hybrid Double Circularly Polarized Beams

Based on the Richard-Wolf diffraction theory, we demonstrate the tightly focused optical field distribution with two beams circularly polarized light. We deduced the expression of the double-beam superimposed vortex light intensity of left-handed circularly polarized light and right-handed circularly polarized light under the focusing condition of a high numerical aperture objective lens. Based on the different vortex topological charge values, amplitudes and initial phases of the dual beams, we numerically simulated the light intensity distribution of the superimposed hybrid focused light field. It is found that a variety of peculiar vortex spots can be obtained through the tight focus stacking of the double beams. These complex hybrid focused light fields with different polarizations can be used in fields such as optical field micro-manipulation or nonlinear fluorescence microscopy imaging.

Enhanced collection of scattered photons in nonlinear fluorescence microscopy by extended epi-detection with a silicon photomultiplier array

To maximize signal collection in nonlinear optical microscopy, non-descanned epi-detection is generally adopted for in vivo imaging. However, because of severe scattering in biological samples, most of the emitted fluorescence photons go beyond the collection angles of objectives and thus cannot be detected. Here, we propose an extended detection scheme to enhance the collection of scattered photons in nonlinear fluorescence microscopy using a silicon photomultiplier array ahead of the front apertures of objectives. We perform numerical simulations to demonstrate the enhanced fluorescence collection via extended epi-detection in the multi-photon fluorescence imaging of human skin and mouse brain through craniotomy windows and intact skulls. For example, with red fluorescence emission at a depth of 600 µm in human skin, the increased collection can be as much as about 150% with a 10×, 0.6-NA objective. We show that extended epi-detection is a generally applicable, feasible technique for use in nonlinear fluorescence microscopy to enhance signal detection.

Near-field imaging of femtosecond propagating surface plasmon and regulation of excitation efficiency

Near-field imaging and active control of excitation efficiency of femtosecond propagating surface plasmon (fs-PSP) are the prerequisites for its application. Here, we perform near-field imaging of fs-PSP excited at the trench etched on silver nano-film by using photoemission electron microscopy (PEEM). As an excellent near-field microscopy technique of in situ imaging with a high spatial resolution (< 20 nm), it needs neither molecular reporters nor scanning probes as required in nonlinear fluorescence microscopy in nonlinear fluorescence microscopy or scanning near-field optical microscopy, both of which may potentially bias PSP derived from such measurements. The period of the interference patterns induced by the incident femtosecond laser and the laser-induced fs-PSP and the wavelength of fs-PSP in a range of 720–900 nm of the incident laser wavelength are systematically measured. The fringe period of the interference pattern between fs-PSP and the incident laser is a range of 5.9–7.7 µm, and the wavelength of fs-PSP is in a range of 700–879 nm. The experimental results are consistent with the theoretical simulation results. Furthermore, we demonstrate that the excitation efficiency of fs-PSP can be actively controlled by adjusting the polarization direction of the incident laser in the femtosecond pump-probe experiments. Specifically, it is found that when the incident laser is polarized to 0° (p-polarization light), the excitation efficiency of PSP reaches a maximum value, and when the incident light is polarized to 90° (s-polarization light), the excitation efficiency of fs-PSP is the lowest. Unlike the simulation result by the finite difference time domain (FDTD) method, a plateau area of the intensity of the photoemission signal with the polarization direction of the incident laser appears in the femtosecond pump-probe experiment. This phenomenon is attributed to the background noise of the detection laser that masks the change of the fs-PSP excitation efficiency. In a word, this research realizes the experimental measurement of the basic parameters of fs-PSP and the manipulation of fs-PSP excitation efficiency by adjusting the polarization angle of the incident laser. This research lays a foundation for realizing the engineering manipulation of fs-PSP excitation efficiency and optimizing the performance of plasmonic devices.

Enhanced stability of the focus obtained by wavefront optimization in dynamical scattering media

Focusing scattered light using wavefront shaping provides interesting perspectives to image deep in opaque samples, as e.g. in nonlinear fluorescence microscopy. Applying these technics to in vivo imaging remains challenging due to the short decorrelation time of the speckle in depth, as focusing and imaging has to be achieved within the order of the decorrelation time. In this paper, we experimentally study the focus lifetime after focusing through dynamical scattering media, when iterative wavefront optimization and speckle decorrelation occur over the same timescale. We show experimental situations with heterogeneous stability of the scattering sequences, where the focus presents significantly higher stability than the surrounding speckle.

High-resolution nonlinear fluorescence microscopy using repetitive stimulated transition based on the saturation of stimulated emission implemented with two-color continuous-wave lasers.

High-resolution nonlinear fluorescence (NF) microscopy that utilizes repetitive stimulated transition due to the saturation of stimulated emission caused by two-color continuous-wave lasers was developed. The resulting NF signal, detected via the lock-in technique, is produced by the multiplicative combination of incident beams, which results in an improvement of the optical resolution. The proposed method is demonstrated to have a three-dimensional optical resolution superior to that of conventional NF microscopy. The results of biological imaging reveal the feasibility and superiority of the proposed method.

Time-domain fluorescence lifetime imaging by nonlinear fluorescence microscopy constructed of a pump-probe setup with two-wavelength laser pulses.

We propose a time-domain approach for fluorescence lifetime measurements using nonlinear fluorescence microscopy constructed of a pump-probe setup with two-wavelength laser pulses. Nonlinear fluorescence signals generated by fluorescence reduction due to stimulated emission were detectable through a lock-in technique. Changing the time delay between the two-wavelength pulses enables acquisition of a time-resolved nonlinear fluorescence signal, which directly reflects the fluorescence lifetime of the sample and is thus applicable to fluorescence lifetime imaging. We also quantitatively demonstrate that nonlinear fluorescence microscopy possesses better optical resolution than conventional laser-scanning fluorescence microscopy. Experimental trials indicate that straightforward fluorescence lifetime imaging with high optical resolution is readily available.

Two-Photon Microscopy (TPM) and Fluorescence Lifetime Imaging Microscopy (FLIM) of Retinal Pigment Epithelium (RPE) of Mice In Vivo.

Retinal pigment epithelium (RPE), a monolayer of epithelial cells located between the neural retina and the choroid, plays a significant role in the maintenance of retinal function. Its in vivo imaging is still technically challenging in human eye. With the mouse eye, there is a possibility to look into the RPE through the sclera using two-photon microscopy (TPM). TPM is a two photon-excited nonlinear fluorescence microscopy that enables the observation of deep tissues up to several hundred micrometers. Since the simultaneous absorption of two photons occurs only at the focal plane, spatial resolution of the TPM is quite high, such that pinhole as used in a confocal microscope is not necessary. TPM enables observation of autofluorescence at the cellular level, and thus may provide new insights into the fluorescent molecules in/around RPE cells.The combination of TPM with fluorescence lifetime imaging microscopy (FLIM) may expand the breadth of information about cells and tissues. Fluorescence lifetime is a fluorophore-specific property, which is independent of fluorescence intensity and changes with the alteration of molecular environment. FLIM may have therefore the potentials to distinguish different fluorophores and to indicate the change in the environment of a fluorophore. Some energy metabolisms-related intracellular fluorophores, such as NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), show characteristic fluorescence lifetimes that shift under different molecular environments, and thus their fluorescence lifetime have been used to indicate cell energy metabolic states. These nonlabeling imaging methods offer us the opportunity to engage in the study of the RPE in vivo as well as in vitro both in morphological as well as metabolic aspects.

Super-resolving nonlinear fluorescence microscopy with pump–probe setup using repetitive stimulated transition

Super-resolving nonlinear fluorescence microscopy with a pump–probe setup that utilizes repetitive stimulated absorption and stimulated emission caused by two-color beams was developed. The resulting nonlinear fluorescence that undergoes such a repetitive stimulated transition is detectable as a signal via the lock-in technique. The signal is produced by a multiplicative combination of incident beams, which leads to an improvement of the optical resolution. A phenomenological interpretation of the nonlinear fluorescence process that offers estimation of signal properties is provided with rate equations. The proposed method is demonstrated to provide the scalability of the optical resolution.

Fabrication of a micro-optical lens using femtosecond laser 3D micromachining for two-photon imaging of bio-tissues

We report, for the first time to our knowledge, on the application of a micro-optical lens fabricated by three-dimensional (3D) femtosecond laser direct writing for two-photon fluorescence imaging of biological tissues. We show that the two-photon fluorescence images of a plant leaf tissue acquired with the micro-optical lens are comparable to that of a 5× objective lens. Our result represents an important step towards the application of micro-optical components fabricated by femtosecond laser micromachining in miniaturized nonlinear fluorescence microscopy applications, such as two-photon endoscopy.

Multiphoton excitation fluorescence microscopy in planar membrane systems

The feasibility of applying multiphoton excitation fluorescence microscopy-related techniques in planar membrane systems, such as lipid monolayers at the air-water interface (named Langmuir films), is presented and discussed in this paper. The non-linear fluorescence microscopy approach, allows obtaining spatially and temporally resolved information by exploiting the fluorescent properties of particular fluorescence probes. For instance, the use of environmental sensitive probes, such as LAURDAN, allows performing measurements using the LAURDAN generalized polarization function that in turn is sensitive to the local lipid packing in the membrane. The fact that LAURDAN exhibit homogeneous distribution in monolayers, particularly in systems displaying domain coexistence, overcomes a general problem observed when "classical" fluorescence probes are used to label Langmuir films, i.e. the inability to obtain simultaneous information from the two coexisting membrane regions. Also, the well described photoselection effect caused by excitation light on LAURDAN allows: (i) to qualitative infer tilting information of the monolayer when liquid condensed phases are present and (ii) to provide high contrast to visualize 3D membranous structures at the film's collapse pressure. In the last case, computation of the LAURDAN GP function provides information about lipid packing in these 3D structures. Additionally, LAURDAN GP values upon compression in monolayers were compared with those obtained in compositionally similar planar bilayer systems. At similar GP values we found, for both DOPC and DPPC, a correspondence between the molecular areas reported in monolayers and bilayers. This correspondence occurs when the lateral pressure of the monolayer is 26+/-2 mN/m and 28+/-3 mN/m for DOPC and DPPC, respectively.

Enhancing Signal to Noise Ratio in linear and non-linear excitation microscopy

The resolution capability of an optical system can be completely characterized by the vectorial diffraction theory[1], which defines the intensity distribution of a point like source imaged by a lens assuming ideal imaging conditions. Unfortunately, these conditions can not be completely reached as noise affects a recorded microscope image. A detailed characterization of the imaging process in linear and non-linear fluorescence microscopy allows to evaluate the noise deterioration effect on the resolution capability.

Cover Picture: Two‐Photon Microscopy and Spectroscopy of Lanthanide Bioprobes (ChemPhysChem 14/2007)

The cover picture shows one- and two-photon excited fluorescence microscopy imaging pictures of crystals of lysozyme-containing terbium and europium tris-dipicolinate complexes. Whereas one-photon irradiation results in green (Tb) or red (Eu) luminescence of the whole crystal aggregate, two-photon excitation in the case of terbium gives a three-dimensionally resolved spot corresponding to the confocal excitation volume. In their article on page 2125, D'Aléo, Pompidor et al. describe the photophysical properties and particularly the linear and nonlinear fluorescence microscopy of these complexes in solution and in the crystalline state.