Multi-Photon Microscopy

Alison Gurney, Dept. of Physiology & Pharmacology, University of Strathclyde.

Multiphoton microscopy is a new method for imaging cells and tissues at high resolution using the light microscope. It exploits the two-photon effect whereby the simultaneous absorption of two infrared photons activates fluorescence in a molecule normally excited by UV or visible light. Fluorescence is excited only in the focal volume, thus reducing photobleaching and phototoxicity. The main advantages of multiphoton microscopy are that cells survive longer during the imaging process and images can be resolved at greater depths within tissues. The principles and advantages of multiphoton microscopy will be outlined and compared with imaging using the confocal microscope.

Ultrastructural eggshell quality assessment: A way forward for captive breeding systems ?

Edmond, A., Solomon, S.E. & Bain, M.M., Glasgow University Veterinary School.

Ultrastructural eggshell quality assessment has been used successfully within the poultry industry to monitor the effects of diet, environment and stress on the process of shell mineralisation. The correct eggshell structure is of great importance in allowing it to perform its role as an incubation chamber. Eggshell assessment techniques mainly using scanning electron microscopy are discussed. These procedures have recently been used to assess the shell quality of several captive bred falcon species and captive bred Houbara Bustard (Chlamydotis undulata). Some of the problems currently experienced within these captive breeding systems are described. The aim of this study was to elucidate, in the first instance, the morphology of the eggshells and to determine whether current eggshell quality techniques could be used to provide relevant information for these systems. Small scale studies are described looking at the normal shell and the changes which take place during incubation. Problems associated with using these techniques on the eggs of wild bird species are considered. Studies such as this can elucidate fundamental problems in diet or management systems and as such are entirely non-invasive.

This work was funded by the National Avian Research Centre (Abu Dhabi) and the International Foundation for Conservation and Development of Wildlife (IFCDW).

Non-invasive imaging of plant tissues using the confocal laser scanning microscope (CLSM).

Karl J. Oparka, Unit of Cell Biology, SCRI, Dundee.

Conventionally, the imaging of plant cells using the CLSM is conducted at high magnification using high numerical aperture lenses. This allows high resolution while maintaining a narrow confocal imaging plane. In our work, predominantly centered around the imaging of plant-transport processes, we frequently require non-invasive approaches that create minimal disturbance of plant-cell functions while retaining optimal resolution of cellular structure. By using long-working distance lenses, it is possible to obtain CLSM images of high quality of living biological specimens at extremely low magnification. Although the confocal plane is large, the out-of-focus flare is substantially reduced. I will describe a number of case histories in which intact plant systems have been imaged successfully using this approach.

Functional dynamics of plasmodesmata in sink-source transition leaves.

Alison G. Roberts, Petra Boevink, Simon Santa Cruz, Norbert Sauer† and Karl J. Oparka,Unit of Cell Biology, Department of Virology, SCRI, Invergowrie. †Department of Molecular Plant Physiology, University of Erlangen.

Plasmodesmata are known to exist in many forms but are commonly characterised as simple or complex. Simple plasmodesmata consist of a single channel while complex plasmodesmata have a central cavity and multiple pores on one or both sides of the cell wall.

Until recently, plasmodesmata were thought to have a relatively low size exclusion limit that permitted only molecules of less than 1 kDa to pass freely between cells. However, it has recently been shown that the size exclusion limit of some plasmodesmata is considerably larger than I kDa, and that plants are able to transport proteins and nucleic acids through plasmodesmata.

Transgenic plants expressing green fluorescent protein (GFP) under control of a companion cell-specific promoter, or expressing GFP targeted to plasmodesmata using a tobacco mosaic virus movement protein fusion, were studied. During the sink-source transition in tobacco leaves, simple plasmodesmata were observed to give rise to more branched forms as the leaf matured. Simple plasmodesmata in sink leaves allowed the unrestricted movement of the 27 kDa GFP while in source tissues, GFP was restricted to the companion cells. Biolistic bombardment and microinjection experiments using GFP-fusion proteins of different sizes demonstrated that proteins of up to 50 kDa could move through simple plasmodesmata but that this plasmodesmatal permeability decreased during the developmental switch from sink to source.

Further studies have shown that large numbers of simple plasmodesmata are lost during the sink-source transition, while branched plasmodesmata increased in frequency. The branching of plasmodesmata was also found to occur asynchronously in different cell layers.

COSMIC - a new interdisciplinary facility for advanced spectroscopy, micromanipulation and imaging.

Wilson C. K. Poon, Department of Physics and Astronomy, University of Edinburgh.

A new multidisciplinary facility, the Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre (COSMIC), is being set up in the University of Edinburgh. The Centre houses a concentration of lasers (pulsed and continuous wave, over a range of wavelengths) and advanced microscopes for research into a range of topics in physics, chemistry and biology. While some of the research will be single-disciplined, projects requiring input from more than one specialism receive particular emphasis. The intention from the beginning is to seek collaboration with colleagues throughout Scotland.

In this talk, I will give an outline of the thinking leading to the setting up of COSMIC, survey the equipment that will be available, and give some examples of the kinds of projects that such a facility will enable. I will also give an introduction to the technique of 'laser tweezers', and describe some of the innovations in this technique that we hope to implement in COSMIC.

Imaging intracellular localisation of drugs using fluorescent microscopy.

Richard W Horobin, IBLS, University of Glasgow.

As some drugs are fluorochromes, one way of looking at their intracellular sites of accumulation is to use fluorescent microscopy of living cells. Examples of drugs which may be studied in this way are the antipsychotic drugs, used to treat schizophrenia. These have a wide range of side effects, some life threatening. Some of these iatrogenic problems are of unknown etiology. One possibility is that neuroleptics can enter cells and exert direct toxic effects, as QSAR modeling and the biochemical literature indicate that antipsychotic drugs can accumulate in mitochondria and inhibit their function. To check localisation patterns of experimentally, cultured fibroblasts were exposed various antipsychotic drugs. Fluorescent microscopy demonstrated that the drugs entered cells very rapidly, and accumulated in several organelles, including the endoplasmic reticulum and the Golgi complex as well as in mitochondria. Uptake appeared to be by passive diffusion. Some implications of these observations will be discussed.

Fourier transform infra-red microscopy.

Alison Coats, Department of Chemistry, University of Aberdeen.

In the field of IR spectroscopy, many technological advances have occurred since the early days of dispersive instruments. However, it was the advent of the Michelson interferometer that truly revolutionised this technique. Without the mechanical limitations of older dispersive designs the new breed of spectrometer that emerged was not only faster and more stable than before, but was capable of far greater resolution.

The Nexus 870 is a state of the art research FTIR spectrometer with a modular design and up to date software control. Coupling this instrument to a Spectra-Tech Continuµm IR microscope creates an integrated system which provides a powerful and versatile FTIR tool with advanced scanning capabilities. The optics system within the microscope, in conjunction with a dedicated video link, enables continuous optical quality viewing of the sample during data collection. The IR spectrum of an area as small as 10 ?m can be measured using either transmission or reflection mode. Such a tool has proved to be ideal for the investigation of inclusions which previously were too small for accurate IR analysis. Furthermore, the microscope benefits from the addition of an automated mapping stage which enables random point, line and area maps to be created. Throughout the IR microsampling experiment sophisticated software integrates sample viewing, data collection and processing in a single package. Such a versatile instrument has many applications both in industry and research. Selected examples will be discussed.

Antigen Retrieval allows Improved Visualisation of V-ATPase Immuno-reactivity.

Susan Lindsay, Glasgow Caledonian University.

Sweat secretion involves water transport coupled to the movement of salt. A potentially dangerous loss of salt is prevented by reabsorptive processes in the sweat gland duct, resulting in the final sweat on the skin surface being hypotonic. Reddy & Quinton (1994) proposed that salt reabsorption at slow flow rates is driven by proton pumps in the cell membrane. Immonuhistochemistry has now shown the presence of such a proton pump (V-ATPase) in the cytoplasm and apical membrane of the luminal cells of the reabsorptive duct. However, it has been shown that formalin fixation of tissue masks epitopes, and reduces the sensitivity in the detection of antigens. Using a method for anitgen retrieval which involves microwave oven heating in a pressure cooker, improved visualisation of the V-ATPase was seen in the reabsorptive duct and secretory coil of the eccrine sweat gland.

Modern immunocytochemistry.

Peter Jackson, Department of Histopathology and Molecular Pathology, The General Infirmary at Leeds.

Tissue antigens in histological preparations can be lost, depleted, chemically altered or "masked" by the processes involved in coagulative and non coagulative fixation. Many antigens can only be demonstrated immunocytochemically by the use of frozen sections. However, the use of frozen sections have several inherent disadvantages when compared to their paraffin and resin counterparts and for most investigations the use of formalin fixed material is preferred. The development of antibodies which recognise antigens surviving in tissue sections which have endured formalin fixation and processing to paraffin wax or resin have enabled immunocytochemistry to become a diagnostic tool.

Complementing this work is the continuing development of heat mediated antigen retrieval techniques and highly sensitive labelling and amplification methods to visualise the immunocytochemical reactions. The presentation will discuss the various techniques and methods available, identify the problems involved and how they may be overcome in the production of high quality immunocytochemical staining in paraffin and resin sections for high resolution light microscopy.