In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy

In this study, we use combined two‐photon excitation fluorescence (TPEF), second‐harmonic generation (SHG) and third‐harmonic generation (THG) measurements to image cellular structures of the nematode Caenorhabditis elegans, in vivo. To our knowledge, this is the first time that a THG modality is employed to image live C. elegans specimens. Femtosecond laser pulses (1028 nm) were utilized for excitation. Detailed and specific structural and anatomical features can be visualized, by recording THG signals. Thus, the combination of three image‐contrast modes (TPEF‐SHG‐THG) in a single instrument has the potential to provide unique and complementary information about the structure and function of tissues and individual cells of live biological specimens.     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.     

Second harmonic generation imaging of the deep shade plant Selaginella erythropus using multifunctional two‐photon laser scanning microscopy

Background : Multifunctional two‐photon laser scanning microscopy provides attractive advantages over conventional two‐photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were achieved from the deep shade plant Selaginella erythropus. Results : These measurements show that the S. erythropus leaves produce high SHG signals in both directions and the SHG signals strongly depend on the laser's status of polarization and the orientation of the dipole moment in the molecules that interact with the laser light. The novelty of this work is (1) uncovering the unusual structure of S. erythropus leaves, including diverse chloroplasts, various cell types and micromophology, which are consistent with observations from general electron microscopy; and (2) using the multifunctional two‐photon laser scanning microscopy by combining three platforms of laser scanning microscopy, fluorescence microscopy, harmonic generation microscopy and polarizing microscopy for detecting the SHG signals in the forward and backward directions, as well as two photon excitation fluorescence. Conclusions : With the multifunctional two‐photon laser scanning microscopy, one can use noninvasive SHG imaging to reveal the true architecture of the sample, without photodamage or photobleaching, by utilizing the fact that the SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation characteristic.     

Fluorescence lifetime imaging microscopy using near-infrared contrast agents

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging.     

A white light confocal microscope for spectrally resolved multidimensional imaging

Spectrofluorometric imaging microscopy is demonstrated in a confocal microscope using a supercontinuum laser as an excitation source and a custom‐built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. The supercontinuum laser provides a broad spectrum light source and is coupled with an acousto‐optic tunable filter to provide continuously tunable fluorescence excitation with a 1‐nm bandwidth. Eight different excitation wavelengths can be simultaneously selected. The prism spectrometer provides spectrally resolved detection with sensitivity comparable to a standard confocal system. This new microscope system enables optimal access to a multitude of fluorophores and provides fluorescence excitation and emission spectra for each location in a 3D confocal image. The speed of the spectral scans is suitable for spectrofluorometric imaging of live cells. Effects of chromatic aberration are modest and do not significantly limit the spatial resolution of the confocal measurements.     

Multifunctional imaging of endogenous contrast by simultaneous nonlinear and optical coherence microscopy of thick tissues

A variety of high resolution optical microscopy techniques have been developed in recent years for basic and clinical studies of biological systems. We demonstrate a trimodal microscope combining optical coherence microscopy (OCM) with two forms of nonlinear microscopy, namely two-photon excited fluorescence (2PF) and second harmonic generation (SHG), for imaging turbid media. OCM combines the advantages of confocal detection and coherence gating for structural imaging in highly scattering tissues. Nonlinear microscopy enables the detection of biochemical species, such as elastin, NAD(P)H, and collagen. While 2PF arises from nonlinear excitation of fluorescent species, SHG is a form of nonlinear scattering observed in materials that lack a center of inversion symmetry, such as type I collagen. Characterization of the microscope showed nearly diffraction-limited spatial resolution in all modalities. Images were obtained in fish scales and excised human skin samples. The primary endogenous sources of contrast in the dermis were due to elastin autofluorescence and collagen SHG. Multimodal microscopy allows the simultaneous visualization of structural and functional information of biological systems.     

Time-domain whole-field fluorescence lifetime imaging with optical sectioning

A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode-locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all-solid-state diode-pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.     

A series of flexible design adaptations to the Nikon E‐C1 and E‐C2 confocal microscope systems for UV,multiphoton and FLIM imaging

Multiphoton microscopy is widely employed in the life sciences using extrinsic fluorescence of low‐ and high‐molecular weight labels with excitation and emission spectra in the visible and near infrared regions. For imaging of intrinsic and extrinsic fluorophores with excitation spectra in the ultraviolet region, multiphoton excitation with one‐ or two‐colour lasers avoids the need for ultraviolet‐transmitting excitation optics and has advantages in terms of optical penetration in the sample and reduced phototoxicity. Excitation and detection of ultraviolet emission around 300 nm and below in a typical inverted confocal microscope is more difficult and requires the use of expensive quartz optics including the objective. In this technical note we describe the adaptation of a commercial confocal microscope (Nikon, Japan E‐C1 or E‐C2) for versatile use with Ti‐sapphire and OPO laser sources and the addition of a second detection channel that enables detection of ultraviolet fluorescence and increases detection sensitivity in a typical fluorescence lifetime imaging microscopy experiment. Results from some experiments with this setup illustrate the resulting capabilities.     

The use of optical parametric oscillator for harmonic generation and two-photon UV fluorescence microscopy

Ultrafast lasers have found increasing use in scanning optical microscopy due to their very high peak power in generating multiphoton excitations. A mode-locked Ti:sapphire laser is often employed for such purposes. Together with a synchronously pumped optical parametric oscillator (OPO), the spectral range available can be extended to 1,050-1,300 nm. This broader range available greatly facilitates the excitation of second harmonic generation (SHG) and third harmonic generation (THG) due to better satisfaction of phase matching condition that is achieved with a longer excitation wavelength. Dental sections are then investigated with the contrasts from harmonic generation. In addition, through intra-cavity doubling wavelengths from 525-650 nm are made available for effective two-photon (2-p) excitation with the equivalent photon energy in the UVB range (290-320 nm) and beyond. This new capacity allows UV (auto-) fluorescence excitation and imaging, for example, from some amino acids, such as tyrosine, tryptophan, and glycine.     

A practical implementation of multifrequency widefield frequency‐domain fluorescence lifetime imaging microscopy

Widefield frequency‐domain fluorescence lifetime imaging microscopy (FD‐FLIM) is a fast and accurate method to measure the fluorescence lifetime, especially in kinetic studies in biomedical researches. However, the small range of modulation frequencies available in commercial instruments makes this technique limited in its applications. Herein, we describe a practical implementation of multifrequency widefield FD‐FLIM using a pulsed supercontinuum laser and a direct digital synthesizer. In this instrument we use a pulse to modulate the image intensifier rather than the more conventional sine‐wave modulation. This allows parallel multifrequency FLIM measurement using the Fast Fourier Transform and the cross‐correlation technique, which permits precise and simultaneous isolation of individual frequencies. In addition, the pulse modulation at the cathode of image intensifier restores the loss of optical resolution caused by the defocusing effect when the cathode is sinusoidally modulated. Furthermore, in our implementation of this technique, data can be graphically analyzed by the phasor method while data are acquired, which allows easy fit‐free lifetime analysis of FLIM images. Here, our measurements of standard fluorescent samples and a Föster resonance energy transfer pair demonstrate that the widefield multifrequency FLIM system is a valuable and simple tool in fluorescence imaging studies. Microsc. Res. Tech. 76:282–289, 2013. © 2013 Wiley Periodicals, Inc.     

Label‐free detection of fibrillar collagen deposition associated with vascular elements in glioblastoma multiforme by using multiphoton microscopy

Glioblastoma multiforme (GBM‐WHO grade IV) is the most common and the most aggressive form of brain tumors in adults with the median survival of 10–12 months. The diagnostic detection of extracellular matrix (ECM) component in the tumour microenvironment is of prognostic value. In this paper, the fibrillar collagen deposition associated with vascular elements in GBM were investigated in the fresh specimens and unstained histological slices by using multiphoton microscopy (MPM) based on two‐photon excited fluorescence (TPEF) and second harmonic generation (SHG). Our study revealed the existence of fibrillar collagen deposition in the adventitia of remodelled large blood vessels and in glomeruloid vascular structures in GBM. The degree of fibrillar collagen deposition can be quantitatively evaluated by measuring the adventitial thickness of blood vessels or calculating the ratio of SHG pixel to the whole pixel of glomeruloid vascular structure in MPM images. These results indicated that MPM can not only be employed to perform a retrospective study in unstained histological slices but also has the potential to apply for in vivo brain imaging to understand correlations between malignancy of gliomas and fibrillar collagen deposition.     

Label‐free imaging characteristics of colonic mucinous adenocarcinoma using multiphoton microscopy

Colorectal carcinoma (CRC) has high mortality and increased incidence rates. An early detection of CRC is very important. Multiphoton microscopy (MPM) with high resolution and high sensitivity is used to effectively distinguish the microstructure changes of normal and mucinous adenocarcinoma slices of ex vivo human colonic tissues. In mucinous adenocarcinoma mucosa, the glands are distorted and elongated, the gland cavity is indistinct, and the mesh collagen fibers are diminished. In the submucosa, the collagens are seriously disordered, elongated, pushed aside, and sparsely visible, the content of elastic fibers is also broken and almost disappearing. Many cancer cells, some in cavity‐like shape full of mucus surrounded by some collagen fibers, occupied the submucosa, which are comparable to hematoxylin‐eosin (HE) stained images. Second harmonic generation and two‐photon excitation fluorescence (SHG/TPEF) intensity ratio can be used further to quantitatively evaluate normality and abnormality. The fast Fourier transform (FFT) images show that the normal collagen fibrils are dense and in random order, and the cancerous collagen is certainly organized. The exploratory results show that it has potential for the development of multiphoton mini‐endoscopy in real‐time early diagnosis of CRC. SCANNING 35: 277‐282, 2013. © 2012 Wiley Periodicals, Inc.     

Scanning-less wide-field single-photon counting device for fluorescence intensity, lifetime and time-resolved anisotropy imaging microscopy

A scanning‐less single‐photon counting system for FLIM and fluorescence anisotropy wide‐field imaging is described and characterized in this paper. The two polarizations of the fluorescence are divided by a Glan prism and acquired at the same time by the Q_A detector. Fluorescence decay profiles can be reconstructed for any desired area up to each pixel and used to calculate time‐resolved fluorescence anisotropy decays.     

High second harmonic generation signal from muscles and fascia pig's muscles using the two-photon laser scanning microscope

I have provided update to our two photon laser scanning microscope by adding new technique which enables us to simultaneously measured the second harmonic generation signals in the forward and backward directions; in the meantime, one can measure the two photon excitations fluorescence if the materials produce fluorescence. In the present work, the fascia muscles, muscles of pig and pig's skin were used. I found that these materials produced high second harmonic generation signal in both directions. These measurements show that the second harmonic generation strongly depends on the state of the polarization of the laser light and the orientation of the dipole moment in the molecules that interact with the laser light. It is therefore advantageous to control the laser's state of polarization, to maximize second harmonic generation. The novelty of this work is to establish new multi-functional technique by combing three platforms of laser scanning microscopy – the fluorescence microscopy, harmonic generation microscopy and polarizing microscopy in which one can use the second harmonic imaging to investigate the true architecture of the sensitive samples and the samples which do not produce auto-fluorescence. Moreover investigation of the new sample needs to look at all details of the true architecture of the sample. Thereby the sample will be exposed to the laser radiation more than the well-known sample, and that will cause photo-bleaching and photo-damage. Since the second harmonic generation does not undergo from photo-bleaching and photo-damage it will be the promising technique for investigating the sensitive and new samples. Then one can move to acquire fluorescence images after good investigation of the true architecture of the sample by the SH imaging.     

The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates

The utility of fluorescence lifetime imaging microscopy (FLIM) for identifying bacteria in complex mineral matrices was investigated. Baseline signals from unlabelled Bacillus subtilis and Euglena gracilis, and Bacillus subtilis labelled with SYTO 9 were obtained using two-photon excitation at 730, 750 and 800 nm, identifying characteristic lifetimes of photosynthetic pigments, unpigmented cellular autofluorescence, and SYTO 9. Labelled and unlabelled B. subtilis were seeded onto marble and gypsum samples containing endolithic photosynthetic cyanobacteria and the ability to distinguish cells from mineral autofluorescence and nonspecific dye staining was examined in parallel with ordinary multichannel confocal imaging. It was found that FLIM enabled discrimination of SYTO 9 labelled cells from background, but that the lifetime of SYTO 9 was shorter in cells on minerals than in pure culture under our conditions. Photosynthetic microorganisms were easily observed using both FLIM and confocal. Unlabelled, nonpigmented bacteria showed weak signals that were difficult to distinguish from background when minerals were present, though cellular autofluorescence consistent with NAD(P)H could be seen in pure cultures, and phasor analysis permitted detection on rocks. Gypsum and marble samples showed similar autofluorescence profiles, with little autofluorescence in the yellow-to-red range. Lifetime or time-gated imaging may prove a useful tool for environmental microbiology. LAY DESCRIPTION : The standard method of bacterial enumeration is to label the cells with a fluorescent dye and count them under high-power fluorescence microscopy. However, this can be difficult when the cells are embedded in soil and rock due to fluorescence from the surrounding minerals and dye binding to ambiguous features of the substrate. The use of fluorescence lifetime imaging (FLIM) can disambiguate these signals and allow for improved detection of bacteria in environmental samples.     

Time-correlated single-photon counting fluorescence lifetime confocal imaging of decayed and sound dental structures with a white-light supercontinuum source

We report the demonstration of time‐correlated single‐photon counting (TCSPC) fluorescence lifetime imaging (FLIM) to ex vivo decayed and healthy dentinal tooth structures, using a white‐light supercontinuum excitation source. By using a 100 fs‐pulsed Ti:Sapphire laser with a low‐frequency chirp to pump a 30‐cm long section of photonic crystal fibre, a ps‐pulsed white‐light supercontinuum was created. Optical bandpass interference filters were then applied to this broad‐bandwidth source to select the 488‐nm excitation wavelength required to perform TCSPC FLIM of dental structures. Decayed dentine showed significantly shorter lifetimes, discriminating it from healthy tissue and hard, stained and thus affected but non‐infected material. The white‐light generation source provides a flexible method of producing variable‐bandwidth visible and ps‐pulsed light for TCSPC FLIM. The results from the dental tissue indicate a potential method of discriminating diseased tissue from sound, but stained tissue, which could be of crucial importance in limiting tissue resection during preparation for clinical restorations.     

Third‐harmonic generation imaging of breast tissue biopsies

We demonstrate for the first time the imaging of unstained breast tissue biopsies using third‐harmonic generation (THG) microscopy. As a label‐free imaging technique, THG microscopy is compared to phase contrast and polarized light microscopy which are standard imaging methods for breast tissues. A simple feature detection algorithm is applied to detect tumour‐associated lymphocyte rich regions in unstained breast biopsy tissue and compared with corresponding regions identified by a pathologist from bright‐field images of hematoxylin and eosin stained breast tissue. Our results suggest that THG imaging holds potential as a complementary technique for analysing breast tissue biopsies.     

Confocal imaging with bilateral scanning and array detectors

A novel arrangement for confocal microscopy is presented, in which the key elements are the use of an array detector such as a CCD for confocal image collection and the use of one double-sided scanning mirror element for bilaterally scanning the object and collecting the data on the CCD. The resulting arrangement is shown to be capable of confocal imaging with high photon efficiency under adjustable conditions of confocality and varying image acquisition rates, i.e. from slow speed up to real-time imaging. Either laser or conventional light sources may be utilized. In addition to CCD registration, direct observation by eye of the confocal image in fluorescence is also possible.     

Mapping piezoelectric-field distribution in gallium nitride with scanning second-harmonic generation microscopy

Taking advantage of the electric field-enhanced second-harmonic generation effect in bulk gallium nitride (GaN) and indium gallium nitride (InGaN) quantum wells, we demonstrated the piezoelectric field distribution mapping in bulk GaN and InGaN multiple-quantum-well (MQW) samples using scanning second-harmonic generation (SHG) microscopy. Scanning SHG microscopy and the accompanying third-harmonic generation (THG) microscopy of the bulk GaN sample were demonstrated using a femtosecond Cr:forsterite laser at a wavelength of 1230 nm. Taking advantage of the off-resonant electric field-enhanced SHG effect and the bandtail state-resonance THG effect, the second- and third-harmonic generation microscopic images obtained revealed the piezoelectric field and bandtail state distributions in a GaN sample. Combined with 720 nm wavelength excited two-photon fluorescence microscopy in the same sample, the increased defect density around the defect area was found to suppress bandedge photoluminescence, to increase yellow luminescence, to increase bandtail state density, and to decrease residue piezoelectric field intensity. Scanning SHG microscopy of the InGaN MQW sample was resonant excited with 800 nm femtosecond pulses from a Ti:sapphire laser in order to suppress SHG contribution from the bulk GaN substrate. Taking advantage of the strong piezoelectric field inside the InGaN quantum well, the wavelength resonant effect, and the electric field-enhanced SHG effect of InGaN quantum wells, resonant scanning SHG microscopy revealed the piezoelectric field distribution inside the wells. Combined with accompanying three-photon fluorescence microscopy from the bulk GaN substrate underneath the quantum wells, the direct correspondence between the piezoelectric field strength inside the quantum well and the substrate quality can be obtained. According to our study, the GaN substrate area with bright bandedge luminescence corresponds to the area with strong SHG signals indicating a higher stained-induced piezoelectric field. These scanning harmonic generation microscopies exhibit superior images of the piezoelectric field and defect state distributions in GaN and InGaN MQWs not available before. Combining with scanning multiphoton fluorescence microscopy, these techniques open new ways for the physical property study of this important material system and can provide interesting details that are not readily available by other microscopic techniques.