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.

• MeSH
• chloroplasty chemie   ultrastruktura   MeSH
• konfokální mikroskopie metody   MeSH
• listy rostlin chemie   ultrastruktura   MeSH
• mikroskopie fluorescenční multifotonová metody   MeSH
• počítačové zpracování obrazu metody   MeSH
• Selaginellaceae chemie   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• fascie chemie   ultrastruktura   MeSH
• konfokální mikroskopie metody   MeSH
• kůže chemie   ultrastruktura   MeSH
• mikroskopie fluorescenční multifotonová metody   MeSH
• polarizační mikroskopie metody   MeSH
• prasata MeSH
• svaly chemie   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Higher harmonic contributions in the movement of an oscillating atomic force microscopy (AFM) cantilever are generated by nonlinear tip-sample interactions, yielding additional information on structure and physical properties such as sample stiffness. Higher harmonic amplitudes are strongly enhanced in liquid compared to the operation in air, and were previously reported to result in better structural resolution in highly organized lattices of proteins in bacterial S-layers and viral capsids [J. Preiner, J. Tang, V. Pastushenko, P. Hinterdorfer, Phys. Rev. Lett. 99 (2007) 046102]. We compared first and second harmonics AFM imaging of live and fixed human lung epithelial cells, and microvascular endothelial cells from mouse myocardium (MyEnd). Phase-distance cycles revealed that the second harmonic phase is 8 times more sensitive than the first harmonic phase with respect to variations in the distance between cantilever and sample surface. Frequency spectra were acquired at different positions on living and fixed cells with second harmonic amplitude values correlating with the sample stiffness. We conclude that variations in sample stiffness and corresponding changes in the cantilever-sample distance, latter effect caused by the finite feedback response, result in second harmonic images with improved contrast and information that is not attainable in the fundamental frequency of an oscillating cantilever.

• MeSH
• endoteliální buňky ultrastruktura   MeSH
• epitelové buňky ultrastruktura   MeSH
• eukaryotické buňky ultrastruktura   MeSH
• lidé MeSH
• mikroskopie atomárních sil metody   MeSH
• myokard cytologie   MeSH
• myši MeSH
• plíce cytologie   MeSH
• pružnost MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

A laser-scanning microscope using second harmonic generation (SHG) as a probe is shown to produce high-resolution images of duckweed Lemna minuta leaves. These leaves are multi-cell layer thick. Second harmonic light is generated by a tightly focused short pulse laser beam and is collected by two objectives yielding forward and backward second harmonic digital images. This measurement shows that the signal of the second harmonic imaging in the forward and backward directions depends on the thickness of the chloroplast and that the forward-SH image was brighter than the backward-SH image. The image intensity also depended on the orientation of the chloroplast in relation to the illuminating polarization direction. Their light-induced re-orientation which was affected by the intensity of the illumination could be observed during the experiments. The novelty of this work is to establish new compact technique in which one can use the SH imaging to investigate the true architecture of the sensitive samples, the unknown samples and the samples which is not producing auto-fluorescence. Moreover, investigation of new or unknown samples needs a long time for looking at details of the sample. Thereby the sample will be exposed for long time to the laser radiation that will cause photobleaching and photodamage. Since the SHG does not undergo photobleaching and photodamage this will be the promising technique for investigating the sensitive and new unknown samples. Then one can move to acquire fluorescence images after complete investigation of the true architecture of the sample. The other advantage of SHG is that it has the ability to image highly ordered structural proteins without any exogenous labels. The SHG is an intrinsic and a coherent process. Imaging of intrinsic compounds avoids the complications of slicing and labeling, and samples can be investigated under physiological conditions.

• MeSH
• Araceae ultrastruktura   MeSH
• konfokální mikroskopie metody   MeSH
• listy rostlin ultrastruktura   MeSH
• počítačové zpracování obrazu metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

INTRODUCTION: Remodeling of human placental membranes (amniochorionic or fetalmembrane) throughout gestation, a necessity to accommodate increasing uterine volume, involves continuous alterations (replacement of cells and remodeling of extracellular matrix). Methodologic limitations have obscured microscopic determination of cellular and layer-level alterations. This study used a combination of advanced imaging by multiphoton autofluorescence microscopy (MPAM) and second harmonic generation (SHG) microscopy along with tissue optical clearing to characterize the 3Dimensional multilayer organization of placental membranes. METHODS: Placental membranes biopsies (6 mm) collected from term, not-in-labor cesarean deliveries (n = 7) were fixed in 10% formalin (native) or treated with 2,2'-thiodiethanol to render them transparent for deeper imaging. Native and cleared tissues were imaged using MPAM (cellular autofluorescence) and SHG (fibrillar collagen). Depth z-stacks captured the amnion epithelium, underlying matrix layers, and in the cleared biopsies, the decidua layer. RESULTS: MPAM and SHG revealed fetal membrane epithelial topography and collagen organization in multiple matrix layers. Term amnion layers showed epithelial shedding and gaps. Optical clearing provided full-depth imaging with improved visualization of collagen structure, mesenchymal cells in extracellular matrix layers, and decidua morphology. Layer thicknesses measured by imaging corroborated with histology. Mosaic tiling of MPAM/SHG image stacks allowed large area visualization of entire biopsies. CONCLUSION: MPAM-SHG microscopy allowed for study of this multi-layered tissue and revealed shedding, gap formation, and other structural changes. This approach could be used to study structural changes associated with membranes as well as other uterine tissues to better understand events in normal and abnormal parturition.

• Klíčová slova
• Amniochorion, Epithelial shedding, Membrane rupture, Multiphoton microscopy, Nonlinear optical microscopy, Optical clearing, Placenta, Pregnancy and parturition, Second harmonic generation microscopy,
• MeSH
• extracelulární matrix MeSH
• extraembryonální obaly anatomie a histologie   diagnostické zobrazování   MeSH
• histocytologické preparační techniky MeSH
• lidé MeSH
• mikroskopie fluorescenční multifotonová * MeSH
• mikroskopie generování druhé harmonické * MeSH
• placenta anatomie a histologie   diagnostické zobrazování   MeSH
• těhotenství MeSH
• zobrazování trojrozměrné MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

BACKGROUND: Second harmonic information reveals information about the structure of spatially oriented structures with an asymmetry. We study the second harmonic images of the grana and intergranal parts of chloroplasts in the leaves of the shade moss Plagiomnium affine. RESULTS: A two-photon microscope with blocking filters to suppress fluorescence generated both forward and backward second harmonic illumination to be captured. In the starch free chloroplasts of the single cell layered laminae strong second harmonic generation (SHG) from the granal regions was emitted. Upon illumination the chloroplasts changed their orientation affecting the SHG signal. Chloroplast is reoriented upon illumination. CONCLUSIONS: SHG signals were successfully obtained from the grana without any possible confusion from SHG starch grains due to their absence.

• MeSH
• časové faktory MeSH
• dvojitý lom MeSH
• fluorescence MeSH
• fotony MeSH
• konfokální mikroskopie přístrojové vybavení   MeSH
• mechy ultrastruktura   MeSH
• mikroskopie fluorescenční multifotonová přístrojové vybavení   MeSH
• osvětlení MeSH
• škrob ultrastruktura   MeSH
• tylakoidy ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• škrob MeSH

UNLABELLED: The effect of increasing the numerical aperture and changing the immersion imaging medium of the objective in the forward second harmonic imaging of the grana and intergranal parts of chloroplasts in the leaves of shade moss Plagiomnium affine plant were demonstrated. Also the enhancement of the forward second harmonic generation images and simultaneously records the forward and backward second harmonic signals were demonstrated. The second harmonic signals in the forward and backward directions are generated by a tightly focused linearly polarized femtosecond pulses laser. CONCLUSIONS: Forward second harmonic signals were successfully enhanced by capturing higher order of diffraction rays from the samples using high numerical aperture water immersion objective.

• MeSH
• čočky MeSH
• fluorescenční mikroskopie metody   MeSH
• fotony MeSH
• konfokální mikroskopie metody   MeSH
• lasery MeSH
• listy rostlin ultrastruktura   MeSH
• mechy ultrastruktura   MeSH
• polarizační mikroskopie metody   MeSH
• tylakoidy ultrastruktura   MeSH
• vylepšení obrazu MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Collagen often acts as an extracellular and intracellular marker for in vitro experiments, and its quality defines tissue constructs. To validate collagen detection techniques, cardiac valve interstitial cells were isolated from pigs and cultured under two different conditions; with and without ascorbic acid. The culture with ascorbic acid reached higher cell growth and collagen deposition, although the expression levels of collagen gene stayed similar to the culture without ascorbic acid. The fluorescent microscopy was positive for collagen fibers in both the cultures. Visualization of only extracellular collagen returned a higher correlation coefficient when comparing the immunolabeling and second harmonic generation microscopy images in the culture with ascorbic acid. Lastly, it was proved that the hydroxyproline strongly contributes to the second-order susceptibility tensor of collagen molecules, and therefore the second harmonic generation signal is impaired in the culture without ascorbic acid.

• Klíčová slova
• ascorbic acid, cell culture, collagen, fluorescent microscopy, porcine VIC, second harmonic generation,
• MeSH
• barvení a značení MeSH
• buněčné kultury MeSH
• kolagen typu I analýza   genetika   metabolismus   MeSH
• kultivované buňky MeSH
• Leydigovy buňky chemie   metabolismus   MeSH
• prasata MeSH
• srdeční chlopně chemie   cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kolagen typu I MeSH

Extracellularly distributed collagen and chondrocytes seeded in gelatine and poly-ɛ-caprolactone scaffolds are visualized by two-photon excitation microscopy (TPEM) and second-harmonic generation (SHG) imaging in both forward and backward nondescanned modes. Joint application of TPEM and SHG imaging in combination with stereological measurements of collagen enables us not only to take high-resolution 3-D images, but also to quantitatively analyze the collagen volume and a spatial arrangement of cell-collagen-scaffold systems, which was previously impossible. This novel approach represents a powerful tool for the analysis of collagen-containing scaffolds with applications in cartilage tissue engineering.

• MeSH
• chondrocyty cytologie   metabolismus   transplantace   MeSH
• fluorescenční mikroskopie metody   MeSH
• interpretace obrazu počítačem metody   MeSH
• kolagen metabolismus   ultrastruktura   MeSH
• králíci MeSH
• kultivované buňky MeSH
• nelineární dynamika MeSH
• tkáňové podpůrné struktury * MeSH
• zobrazování trojrozměrné metody   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kolagen MeSH

We investigated possibilities of the combination of the one- and two-photon excitation microscopy for examination of the experimental melanoma tissue in vivo, in mice under general anesthesia, and ex vivo on freshly harvested specimens. Our aim was to obtain sufficiently informative images of unstained tumor tissues and their modifications after hyperthermia treatment. The mouse experimental melanoma structure was studied and compared with normal tissue from the same animal by using confocal and nonlinear microscopy techniques based on (i) one-photon excitation (1PE) fluorescence, (ii) 1PE reflectance, (iii) second harmonic generation imaging, and (iv) two-photon excitation autofluorescence. We checked different spectral conditions and other settings of image acquisition, as well as combinations of the above imaging modalities, to fully exploit the potential of these techniques in the evaluation of treated and untreated cancer tissue morphology. Our approach enabled to reveal the collagen fiber network in relation with the other tissues, and to identify invasive tumor cells. It also proved to be useful for the examination of interrelationships between functional and morphological aspects based on optical properties of the tissues, especially in studies of changes between the tumor and control tissue, as well as changes induced by physical treatments, e.g., delivery of microwave hyperthermia treatment. These differences were also evaluated quantitatively, when we found out that the maximum Euler-Poincaré characteristic reflects well the melanoma morphological structure. The results showed that the proposed investigative approach could be suitable also for a direct evaluation of tissue modifications induced by clinical interventions.

• MeSH
• indukovaná hypertermie * MeSH
• melanom experimentální patologie   radioterapie   MeSH
• mikroskopie * MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH