A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

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.     

Compact non‐contact total emission detection for in vivo multiphoton excitation microscopy

We describe a compact, non‐contact design for a total emission detection (c‐TED) system for intra‐vital multiphoton imaging. To conform to a standard upright two‐photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non‐contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c‐TED device compared to heavily optimized objective‐based emission collection. The best light collection enhancement was seen from murine fat (5×–2× gains as a function of depth), whereas murine skeletal muscle and rat kidney showed gains of over two and just under twofold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a twofold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers, enabled by greater light collection efficiency, yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo‐damage, as well as the applicability of this device to other multiphoton imaging methods is discussed.     

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.     

In vivo monitoring of intracellular chloroplast movements in intact leaves of c4 plants using two‐photon microscopy

Dynamic changes in the spatial distribution of chloroplasts are essential for optimizing photosynthetic capacity under changing light conditions. Light‐induced movement of chloroplasts has been widely investigated, but most studies were conducted on isolated tissues or protoplasts. In this study, a two‐photon microscopy (TPM) system was adapted to monitor the intracellular 3‐dimensional (3D) movements of chloroplasts in intact leaves of plants during dark to light transitions. The TPM imaging was based on autofluorescence of chlorophyll generated by a femto‐second Ti:Sapphire laser. All chloroplasts did not exhibit the same motion in response to irradiation variation. In the sub‐epidermal mesophyll cells, chloroplasts generally moved away from the surface following blue light treatment, however many chloroplasts did not show any movement. Such spatial heterogeneity in chloroplast motility underlines the importance of monitoring intracellular orientation and movement of individual chloroplasts across intact leaves. Our investigation shows that the 3D imaging of chloroplasts using TPM can help to understand the changes in local photosynthetic capacity in intact leaves under changing environmental conditions. Microsc. Res. Tech. 77:806–813, 2014. © 2014 Wiley Periodicals, Inc.     

Two-photon fluorescence and second-harmonic generation imaging of collagen in human tissue based on multiphoton microscopy

Multiphoton microscopic imaging of collagen plays an important role in noninvasive diagnoses of human tissue. In this study, two-photon fluorescence and second-harmonic generation (SHG) imaging of collagen in human skin dermis and submucosa of colon and stomach tissues were investigated based on multiphoton microscopy (MPM). Our results show that multiphoton microscopic image of collagen bundles exhibits apparently different pattern in human tissues. The collagen bundles can simultaneously reveal its SHG and two-photon excited fluorescence images in the submucosa of colon and stomach, whereas it solely emit SHG signal in skin dermis. The intensity spectral information from tissues further demonstrated the above results. This indicates that collagen bundles have completely different space arrangement in these tissues. Our experimental results bring more detailed information of collagen for the application of MPM in human noninvasive imaging.     

Two‐photon microscopy assessment of the overall energy metabolism alteration of amoeba in hypertonic environment

In this study, a two‐photon fluorescence microscopic imaging technique is reported for assessment the effect of dynamic hypertonic environment on the overall energy metabolism alteration and adaptation of soil‐living amoeba Dictyostelium discoideum. For that purpose the fluorescence intensity of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) was monitored and quantified in order to evaluate the corresponded metabolic state of monolayer cultured cells. The two‐photon excitation of NADH with 720 nm near infrared irradiation produced blue fluorescence emission with maximum wavelength centered at 460 nm. The benefits of reported noninvasive microscopic technique are the significantly less cellular damage and avoiding the excitation of other biomolecules except of NADH. It enabled to acquire data for NADH levels of the observed cells on agar plate specimen and hypertonic nutrition media in a Petri dish. The method demonstrated also good sensitivity, reproducibility and the obtained results revealed that D. discoideum species form aggregation in hypertonic environment within several minutes with aim to survive. The formed aggregate had amorphous shape and it consisted from dozen amoeba cells, which kept their NADH amount in constant level for few hours. The reported imaging method might be applicable in various studies for characterization of metabolic events and assessment of the cell energy balance in hypertonic environment.     

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.     

Advanced imaging of tau pathology in Alzheimer Disease: New perspectives from super resolution microscopy and label‐free nanoscopy

Alzheimer's disease (AD) is the main cause of dementia in the elderly population. Over 30 million people worldwide are living with dementia and AD prevalence is projected to increase dramatically in the next two decades. In terms of neuropathology, AD is characterized by two major cerebral hallmarks: extracellular β‐amyloid (Aβ) plaques and intracellular Tau inclusions, which start accumulating in the brain 15‐20 years before the onset of symptoms. Within this context, the scientific community worldwide is undertaking a wide research effort to detect AD pathology at its earliest, before symptoms appear. Neuroimaging of Aβ by positron emission tomography (PET) is clinically available and is a promising modality for early detection of Aβ pathology and AD diagnosis. Substantive efforts are ongoing to develop advanced imaging techniques for early detection of Tau pathology. Here, we will briefly describe the key features of Tau pathology and its heterogeneity across various neurodegenerative diseases bearing cerebral Tau inclusions (i.e., tauopathies). We will outline the current status of research on Tau‐specific PET tracers and their clinical development. Finally, we will discuss the potential application of novel super‐resolution and label‐free techniques for investigating Tau pathology at the experimental level and their potential application for AD diagnosis. Microsc. Res. Tech. 79:677–683, 2016. © 2016 Wiley Periodicals, Inc.     

Condenser‐free contrast methods for transmitted‐light microscopy

Phase contrast microscopy allows the study of highly transparent yet detail‐rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser‐free yet highly effective method of obtaining phase contrast in transmitted‐light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light‐path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser‐free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser‐free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour‐contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser‐free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next‐generation transmitted‐light microscopy designs. The condenser‐free illumination method, using rings of independent or radially‐scanned emitters, may be exploited in future in other electromagnetic wavebands, including X‐rays or the infrared.     

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.     

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.     

Real-time noninvasive optical diagnosis for colorectal cancer using multiphoton microscopy

In contrast to colonoscopy biopsy, which contains several disadvantages such as bleeding, sampling error, crush artifact, and time-consuming pathological procedure, multiphoton microscopy (MPM) enables direct noninvasive visualization of tissue architecture and cell morphology in live tissues without the administration of exogenous contrast agents. We performed a proof-of-principle study to evaluate the feasibility of using MPM to make real-time noninvasive optical diagnosis of colorectal cancer by investigating 30 fresh, unfixed, and unstained full-thickness colorectal specimens. We found that MPM images demonstrated irregular tubular structures, reduced stroma, and cellular and nuclear pleomorphism in the cancerous tissues. Cancer cells, characterized by irregular size and shape, enlarged nuclei, and increased nuclear-cytoplasmic ratio, were clearly observed in MPM images, which were comparable to golden standard hematoxylin-eosin staining images. Our findings showed that MPM had the potential to make real-time noninvasive optical diagnosis of colorectal cancer. With miniaturization and integration of colonoscopy, MPM has a promising future in real-time noninvasive "optical biopsy" for colorectal cancer.     

Calcium silicate cement‐induced remineralisation of totally demineralised dentine in comparison with glass ionomer cement: tetracycline labelling and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy, in combination with tetracycline labelling, was used to observe the remineralising potentials of a calcium silicate‐based restorative material (Biodentine^TM) and a glass ionomer cement (GIC:?Fuji?IX) on totally demineralised dentine. Forty demineralised dentine discs were stored with either cement in three different solutions: phosphate buffered saline (PBS) with tetracycline, phosphate‐free tetracycline, and tetracycline‐free PBS. Additional samples of demineralised dentine were stored alone in the first solution. After 8‐week storage at 37 °C, dentine samples were imaged using two‐photon fluorescence microscopy and Raman spectroscopy. Samples were later embedded in PMMA and polished block surfaces studied by 20 kV BSE imaging in an SEM to study variations in mineral concentration. The highest fluorescence intensity was exhibited by the dentine stored with Biodentine^TM in the PBS/tetracycline solution. These samples also showed microscopic features of matrix remineralisation including a mineralisation front and intra‐ and intertubular mineralisation. In the other solutions, dentine exhibited much weaker fluorescence with none of these features detectable. Raman spectra confirmed the formation of calcium phosphate mineral with Raman peaks similar to apatite, while no mineral formation was detected in the dentine stored in cement‐free or PBS‐free media, or with GIC. It could therefore be concluded that Biodentine^TM induced calcium phosphate mineral formation within the dentine matrix when stored in phosphate‐rich media, which was selectively detectable using the tetracycline labelling.     

Two‐photon laser scanning microscopy as a useful tool for imaging and evaluating macrophage‐, IL‐4 activated macrophage‐ and osteoclast‐based In Vitro degradation of beta‐tricalcium phosphate bone substitute material

Two‐photon microscopy is an innovative technology that has high potential to combine the examination of soft and hard tissues in vitro and in vivo. Calcium phosphates are widely used substitutes for bone tissue engineering, since they are degradable and consequently replaced by newly formed tissue. It is well known that osteoclasts are responsible for the resorption processes during bone remodelling. We hypothesize that also macrophages are actively involved in the resorption process of calcium phosphate scaffolds and addressed this question in in vitro culture systems by two‐photon laser scanning microscopy. Beta‐tricalcium phosphate specimens were incubated with (1) macrophages, (2) interleukin‐4 activated macrophages, and (3) osteoclasts for up to 21 days. Interestingly, macrophages degraded beta‐tricalcium phosphate specimens in an equivalent fashion compared to osteoclasts and significantly more than IL‐4 activated macrophages. An average of ～32% of the macrophages was partially filled with ceramic material while this was 18% for osteoclasts and 9% for IL‐4 activated macrophages. For the first time by applying two‐photon microscopy, our studies show the previously unrecognized potential of macrophages to phagocytose ceramic material, which is expected to have implication on osteoconductive scaffold design. Microsc. Res. Tech. 77:143–152, 2014. © 2013 Wiley Periodicals, Inc.