Confocal microscope presentation pt

It helps for Bsc first years to understand the basics of Confocal Microscopy

1. Confocal Microscopy

2. INTRODUCTION An optical imaging technique for increasing optical resolution and contrast of a micrograph. Radiations emitted from laser cause sample to fluoresce. Uses pinhole screen to produce high resolution images. Eliminates out of focus. So images have better contrast and are less hazy. A series of thin slices of the specimen are assembled to generate a 3dimensinal image. Is an updated version of fluorescence microscopy.

3. HISTORY Two investigators at Cambridge, Brad Amos and John White attempted to look at the mitotic divisions in the first few divisions in embryos of C. elegans . They were doing antitubulin immunofluorescence and were trying to determine the cleavage planes of the cells,  But were frustrated in their attempt in that the majority of the fluorescence they observed was out of focus. They looked at the technique called confocal imaging which was first proposed by Nipkow and pioneered by a postdoc at Harvard named Minsky . He made the first stage scanning confocal microscope in 1957. By illuminating single point at a time, Minsky avoided most of the unwanted scattered light. For buiiding the image, Minsky scanned the specimen by moving the stage rather than light rays.

4. COMPONENTS OF CONFOCAL MICROSCOPY Laser Photomultiplier Filters

5. LASER

6. LASER Light Amplification by Stimulated Emission of Radiation (Laser). Lasers are used because they are an intense coherent monochromatic source of light, capable of being expanded to fill an aperture or focused to a spot. The laser beam is usually linearly polarized. The main drawback in using lasers is that to cover a large excitation range you will need several lasers. However a single laser line may not optimally excite your molecule.

7. PHOTOMULIPLIER

8. PHOTOMULTIPLIER A photomultiplier tube, useful for light detection of very weak signals,it is a photo emissive device in which the absorption of a photon results in the emission of an electron. These detectors work by amplifying the electrons generated by a photocathode exposed to a photon flux.

9. FILTERS Microscopy Filters are used in a variety of microscopy applications for increasing contrast, blocking ambient light, removing harmful ultraviolet or infrared light, or for selectively transmitting only wanted wavelengths. Most of the microscopes, are based on optical filters. A typical system has three basic filters: an excitation filter (or exciter), a dichroic beamsplitter (or dichromatic mirror), and an emission filter (or barrier filter). An excitation filter is a high quality optical-glass filter commonly used in fluorescence microscopy and spectroscopic applications for selection of the excitation wavelength of light from a light source. Barrier filters are filters which are designed to suppress or block (absorb) the excitation wavelengths and permit only selected emission wavelengths to pass toward the eye or other detector. A third version of the beam splitter is a  dichroic  mirrored prism assembly which uses  dichroic  optical coatings to divide an incoming light beam into a number of spectrally distinct output beams. The dichroic beam splitter controls which wavelengths of light go to their respective filter.

10. DICHORIC MIRROR

11. INSTRUMENTAL DESIGN Ray diagram for confocal microscope

12. CONFOCAL MICROSCOPE Schematic diagram confocal microscope

13. PRINCIPLE In confocal microscopy two pinholes are typically used: A pinhole is placed in front of the illumination source to allow transmission only through a small area This illumination pinhole is imaged onto the focal plane of the specimen, i.e. only a point of the specimen is illuminated at one time. Fluorescence excited in this manner at the focal plane is imaged onto a confocal pinhole placed right in front of the detector. Only fluorescence excited within the focal plane of the specimen will go through the detector pinhole. Scanning of small sections is done and joined them together for better view.

14. WORKING MECHANISM Confocal microscope incorporates 2 ideas: 1. Point-by-point illumination of the specimen. 2. Rejection of out of focus of light. Light source of very high intensity is used—Zirconium arc lamp in Minsky’s design & laser light source in modern design. a)Laser provides intense blue excitation light. b)The light reflects off a dichoric mirror , which directs it to an assembly of vertically and horizontally scanning mirrors. c)These motor driven mirrors scan the laser beam across the specimen. d) The specimen is scanned by moving the stage back & forth in the vertical & horizontal directions and optics are kept stationary.

15. Contd …. Dye in the specimen is excited by the laser light & fluoresces. The fluorescent (green) light is descanned by the same mirrors that are used to scan the excitation (blue) light from the laser beam Then it passes through the dichoric mirror Then it is focused on to pinhole. The light passing through the pinhole is measured by the detector such as photomultiplier tube. For visualization, detector is attached to the computer, which builds up the image at the rate of 0.1-1 second for single image

16. LIGHT PATHWAY Light from excitation filter passes through objective lens and light absorbed by specimen. Light emitted goes back through objective lens, barrier filter, then detector.

17. x y z OPTICAL SECTIONING AND 3-D RECONSTRUCTION

18. x y z OPTICAL SECTIONING AND 3-D RECONSTRUCTION

19. x y z = OPTICAL SECTIONING AND 3-D RECONSTRUCTION

20. 3-D Imaging with Confocal Microscopy

21. ADVANTAGES The specimen is everywhere illuminated axially, rather than at different angles, thereby avoiding optical aberrations. Entire field of view is illuminated uniformly. The field of view can be made larger than that of the static objective by controlling the amplitude of the stage movements. Image formed are of better resolution. Cells can be live or fixed.  Serial optical sections can be collected. Taking a series of optical slices from different focus levels in the specimen generates a 3D data set.

22. DRAWBACKS Resolution : It has inherent resolution limitation due to diffraction. Maximum best resolution of confocal microscopy is typically about 200nm. Pin hole size : Strength of optical sectioning depends on the size of the pinhole. Intensity of the incident light Fluorophores : a)The fluorophore should tag the correct part of the specimen. b) Fluorophore should be sensitive enough for the given excitation wave length. c)It should not significantly alter the dynamics of the organism in the living specimen. Photobleaching : photochemical alteration of a dye or a fluorophore molecule such that it permanently is unable to fluorescence.

23. APPLICATIONS 1.Confocal microscopy allows analysis of fluorescent labelled thick specimens without physical sectioning. Zebra fish embryo wholemount Neurons (green) and Cell adhesion molecule (red)

24. APPLICATIONS 2. Three-Dimensional Reconstruction of Specimen . 3D shadow projection

25. 3. Improved Resolution APPLICATIONS An Intestine Section Kidney cells (fluorescence vs Confocal microscope

26. 4. More Colour Possibilities Because the images are detected by a computer rather than by eye, it is possible to detect more color differences. APPLICATIONS

27. IMAGE TAKEN BY CONFOCAL MICROSCOPE Colour coded image of actin filaments in a cancer cell

28. IMAGE TAKEN BY CONFOCAL MICROSCOPE Pancreatic islets of mouse

29. IMAGE TAKEN BY CONFOCAL MICROSCOPE Drosophila brain with triple antibodies

30. IMAGE TAKEN BY CONFOCAL MICROSCOPE Hepatocyte sphenoid

31. Confocal vs. Widefield Confocal Widefield Tissue culture cell with 60x / 1.4NA objective CONFOCAL VS. WIDEFIELD Tissue culture cell with 60X /1.4NA Objective

32. CONFOCAL VS. WIDEFIELD 20 micrometer rat intestine section recorded with 60 x/1.4NA Objective

33. EXAMPLES Drosophila S2 cell expressing GFP-H2B and mChery-tubulin [ Nico suurname and Ron vale ]. S.Cerevisae expressing mitochondrially targeted RFP [ Sussane Rafeilski ,Marshall lab ].

34. THANK YOU