Upconversion nanoparticles for differential imaging of plant cells and detection of fluorescent dyes

AbstractFemtosecond laser photoporation has become a popular method to deliver various kinds of molecules such as genes, proteins, and fluorescent dyes into single mammalian cells. However, this method is not easily applied to plant cells because their cell wall and turgor pressure prevent the delivery, especially for larger molecules than the mesh size of the cell wall. This work is the first demonstration of the efficient photoinjection of megadalton molecules into a cytoplasm of an intact single plant cell by employing a femtosecond laser amplifier under moderate enzyme treatment conditions. The intense femtosecond laser pulse effectively formed a pore on the cell wall and membrane of Tobacco BY-2, and 2 MDa dextran molecules were introduced through the pore. Along with the pore formation, induced mechanical tensile stresses on BY-2 cells were considered to increase permeability of the cell membrane and enhance the uptake of large molecules. Moreover, the moderate enzyme treatment partially degraded the cell wall thereby facilitating the increase of the molecular introduction efficiency.

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Conformation of Ribosomes from Escherichia Coli

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051062 ◽

1974 ◽

Vol 32 ◽

pp. 210-211

Author(s):

M. Boublik ◽

W. Hellmann ◽

F. Jenkins

Keyword(s):

Binding Sites ◽

Ribosomal Proteins ◽

Fluorescent Dyes ◽

Protein Biosynthesis ◽

Three Dimensional ◽

Spatial Arrangement ◽

Dimensional Structure ◽

Complete Understanding ◽

And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

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Analysis of 3-d molecular distribution with the digital imaging microscope

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102286 ◽

1988 ◽

Vol 46 ◽

pp. 40-41

Author(s):

W.A. Carrington ◽

F.S. Fay ◽

K.E. Fogarty ◽

L. Lifshitz

Keyword(s):

Image Restoration ◽

Digital Imaging ◽

Fluorescent Dyes ◽

Optical Axis ◽

Computer Hardware ◽

Computer Algorithms ◽

Low Contrast ◽

Specific Proteins ◽

Effective Use ◽

Single Living Cells

Advances in digital imaging microscopy and in the synthesis of fluorescent dyes allow the determination of 3D distribution of specific proteins, ions, GNA or DNA in single living cells. Effective use of this technology requires a combination of optical and computer hardware and software for image restoration, feature extraction and computer graphics.The digital imaging microscope consists of a conventional epifluorescence microscope with computer controlled focus, excitation and emission wavelength and duration of excitation. Images are recorded with a cooled (-80°C) CCD. 3D images are obtained as a series of optical sections at .25 - .5 μm intervals.A conventional microscope has substantial blurring along its optical axis. Out of focus contributions to a single optical section cause low contrast and flare; details are poorly resolved along the optical axis. We have developed new computer algorithms for reversing these distortions. These image restoration techniques and scanning confocal microscopes yield significantly better images; the results from the two are comparable.

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High-resolution measurement of birefringent fine structure in living cells using a new polarized light microscope

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136647 ◽

1995 ◽

Vol 53 ◽

pp. 54-55

Author(s):

Rudolf Oldenbourg

Keyword(s):

Light Microscope ◽

Fluorescent Dyes ◽

Polarized Light ◽

Processing System ◽

Video Camera ◽

Molecular Organization ◽

Computer Assisted ◽

Image Recording ◽

Birefringent Crystal ◽

Technological Advances

The recent renaissance of the light microsope is fueled in part by technological advances in components on the periphery of the microscope, such as the laser as illumination source, electronic image recording (video), computer assisted image analysis and the biochemistry of fluorescent dyes for labeling specimens. After great progress in these peripheral parts, it seems timely to examine the optics itself and ask how progress in the periphery facilitates the use of new optical components and of new optical designs inside the microscope. Some results of this fruitful reflection are presented in this symposium.We have considered the polarized light microscope, and developed a design that replaces the traditional compensator, typically a birefringent crystal plate, with a precision universal compensator made of two liquid crystal variable retarders. A video camera and digital image processing system provide fast measurements of specimen anisotropy (retardance magnitude and azimuth) at ALL POINTS of the image forming the field of view. The images document fine structural and molecular organization within a thin optical section of the specimen.

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SEM study of the morphological effects of kinetin and zeatin on the growth and development of the apical meristem in wheat

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141263 ◽

1995 ◽

Vol 53 ◽

pp. 978-979

Author(s):

G. M. Hutchins ◽

J. S. Gardner

Keyword(s):

Growth And Development ◽

Furfuryl Alcohol ◽

Apical Meristem ◽

Plant Cells ◽

Triticum Aestivum L ◽

Morphological Development ◽

Naturally Occurring ◽

Chinese Spring Wheat ◽

Sem Study ◽

Moist Environment

Cytokinins are plant hormones that play a large and incompletely understood role in the life-cycle of plants. The goal of this study was to determine what roles cytokinins play in the morphological development of wheat. To achieve any real success in altering the development and growth of wheat, the cytokinins must be applied directly to the apical meristem, or spike of the plant. It is in this region that the plant cells are actively undergoing mitosis. Kinetin and Zeatin were the two cytokinins chosen for this experiment. Kinetin is an artificial hormone that was originally extracted from old or heated DNA. Kinetin is easily made from the reaction of adenine and furfuryl alcohol. Zeatin is a naturally occurring hormone found in corn, wheat, and many other plants.Chinese Spring Wheat (Triticum aestivum L.) was used for this experiment. Prior to planting, the seeds were germinated in a moist environment for 72 hours.

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En face dual fluorescence staining of fatty streaks allows observation of initiation and progression of atherosclerotic lesions

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140695 ◽

1995 ◽

Vol 53 ◽

pp. 864-865

Author(s):

Anne M. Klinkner ◽

Crystal R. Waites ◽

Peter J. Bugelski ◽

William D. Kerns

Keyword(s):

Fluorescent Dyes ◽

Nile Red ◽

Dimethyl Formamide ◽

High Cholesterol Diet ◽

Methods Development ◽

Atherosclerotic Lesions ◽

En Face ◽

Biochemical Evaluation ◽

Stock Solutions ◽

Dual Staining

A primary effort in the understanding of the progression of atherosclerotic disease has been methods development for visualization of the atherosclerotic plaque. We introduce a new method for the qualitative analysis of lipids in atherosclerotic fatty streaks which also retains those lipids for biochemical evaluation. An original aspect of the process is the ability to view an entire fatty streak en face, selectively stained for specific lipid classes within the lesion.New Zealand white rabbits were fed a high cholesterol diet(0.15%-0.3% for 14 wks). The aorta was removed and fixed in Carson's phosphate buffered formaldehyde followed by dual staining in the fluorescent dyes Nile red and filipin. Stock solutions of nile red(0.5mg/ml acetone) and filipin(2.5mg/ml dimethyl formamide) were prepared and kept at -20°C; all subsequent steps were at RT. 0.5cm × 1.0cm pieces of aorta were trimmed and adventitia removed. The pieces were then washed 3×15 min in PBS w/o CaMg, soaked in Nile red(NR)/filipin(Fl) stain(100(il NR stock + 200μl Fl stock in 10 ml PBS for 30 min, washed in PBS 3×30 min, rinsed with distilled water, mounted(Crystal Mount, Biomedia) and coverslipped and viewed by fluorescence microscopy.

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Calcium ion imaging in plant cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146503 ◽

1993 ◽

Vol 51 ◽

pp. 132-133

Author(s):

Peter K. Hepler ◽

Dale A. Callaham

Keyword(s):

Plant Cell Wall ◽

Cytoplasmic Membrane ◽

Fluorescent Dyes ◽

Free Acid ◽

Methyl Esters ◽

Free Calcium ◽

Local Concentration ◽

Ion Imaging ◽

Membrane Compartments ◽

Free Acid Form

Calcium ions (Ca) participate in many signal transduction processes, and for that reason it is important to determine where these ions are located within the living cell, and when and to what extent they change their local concentration. Of the different Ca-specific indicators, the fluorescent dyes, developed by Grynkiewicz et al. (1), have proved most efficacious, however, their use on plants has met with several problems (2). First, the dyes as acetoxy-methyl esters are often cleaved by extracellular esterases in the plant cell wall, and thus they do not enter the cell. Second, if the dye crosses the plasma membrane it may continue into non-cytoplasmic membrane compartments. Third, even if cleaved by esterases in the cytoplasm, or introduced as the free acid into the cytoplasmic compartment, the dyes often become quickly sequestered into vacuoles and organelles, or extruded from the cell. Finally, the free acid form of the dye readily complexes with proteins reducing its ability to detect free calcium. All these problems lead to an erroneous measurement of calcium (2).

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Confocal microscopy of the cytoskeleton in living plant cells following microinjection of fluorescent probes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146497 ◽

1993 ◽

Vol 51 ◽

pp. 130-131

Author(s):

Ann Cleary

Keyword(s):

Cell Division ◽

Fluorescent Probes ◽

Actin Filaments ◽

Intracellular Transport ◽

Cell Structure ◽

Plant Cells ◽

Cell Plate ◽

Cell Cortex ◽

Living Plant ◽

Rhodamine Phalloidin

Microinjection of fluorescent probes into living plant cells reveals new aspects of cell structure and function. Microtubules and actin filaments are dynamic components of the cytoskeleton and are involved in cell growth, division and intracellular transport. To date, cytoskeletal probes used in microinjection studies have included rhodamine-phalloidin for labelling actin filaments and fluorescently labelled animal tubulin for incorporation into microtubules. From a recent study of Tradescantia stamen hair cells it appears that actin may have a role in defining the plane of cell division. Unlike microtubules, actin is present in the cell cortex and delimits the division site throughout mitosis. Herein, I shall describe actin, its arrangement and putative role in cell plate placement, in another material, living cells of Tradescantia leaf epidermis.The epidermis is peeled from the abaxial surface of young leaves usually without disruption to cytoplasmic streaming or cell division. The peel is stuck to the base of a well slide using 0.1% polyethylenimine and bathed in a solution of 1% mannitol +/− 1 mM probenecid.

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