microscope techniques and their precautions.pptx

microscope techniques and their precautions

1. BASIC TO ADVANCE MICROSCOPY & IMAGE ANALYSIS TECHNIQUES SHUMAILA BATOOL 3 PH. D SCHOLAR THE WOMEN UNIVERSITY MULTAN

2. An optical instrument that uses a lens or a combination of lenses to produce magnified images of small objects, especially of objects too small to be seen by the unaided eye is called microscope . WHAT IS MICROSCOPE ?

3. HISTORY BASIC TO ADVANCE MICROSCOPE 1st century BC: Romans introduce glass lens 12th century AD: Italian Salvino D’Armate made the first magnifying eyepiece. 1590: Zechariah and Hans Jansen built a first compound microscope 1609: Galileo Galilei built the compound microscope with concave and a convex lens 1665: Almost 6 decades later, Robert Hooke reported a device in his book“Micrographia ” 1674: A. V. Leeuwenhoek used the device to grind better magnifying the glass 1860s: Ernst Abbe discovered the Abbe sine condition & his colleague made a microscope 1931: Ernst Ruska built the first electron microscope

4. TYPES OF MICROSCOPE There are following 3 main types of microscope Light Microscope Electron Microscope Scanning probe microscope

5. LIGHT MICROSCOPE Visualize an image by using a glass lens, and its magnification is determined by, the lens’s ability to bend light and focus it on the specimen, which forms an image.

6. Many types: 1. Bright-field microscope 2. Dark-field microscope 3. Phase-contrast microscope 4. Fluorescence microscopes 5. Differential interference contrast (DIC) 6. Confocal microscope 7. Two photon microscope 8. Scanning acoustic microscope TYPES OF LIGHT MICROSCOPE

7. THE BRIGHT FIELD MICROSCOPE Uses visible light as a source of illumination Cannot resolve structure smaller than about 0.2um Specimen appears against a bright background Inexpensive and easy to use

8. Function Usually used in samples with colorimetric staining or good contrast Hemlock Leaf

9. Uses a special condenser with an opaque disk that blocks light from entering the objective lens directly Light reflected by specimen enters the objective lens and the specimen appears light against a black background DARK FIELD MICROSCOPE

10. Function Fine structures can often not be seen in front of a bright background but it can seen with dark background

11. PHASE CONTRAST MICROSCOPE Use a special condenser containing an annular or ring shaped diaphragm The diaphragm allow direct light to pass through the condenser focusing light on the specimen and diffraction plate in the objective lens Direct and reflected light rays are brought to produce the image No staining required

12. Function To facilitate detailed examination of the internal structures of living things or specimen.

13. FLORERESCENCE MICROSCOPE Uses UV lights as a source of illumination that cause florescent compounds(green colored) in a specimen to emit light.

14. Function For florescent antibody techniques to rapidly detect and identify microbes in a tissue and clinical specimen.

15. CONFOCAL MICROSCOPE Uses a single photon of light to illuminate one plane of a specimen at a time.

16. Function To obtain two or three dimensional images of cells for biomedical applications. Image of pollen grain see here.

17. TWO PHOTON MICROSCOPE Uses two photons to illuminate a specimen

18. Function

19. SCANNING ACOUSTIC MICROSCOPE Uses a sound wave of specific frequency that travels through the specimen with a portion being reflected when it hits an interface with in material

20. Function To examine living cells attached to another surface Like cancer cells, artery plaque and biofilm

21. ELECTRON MICROSCOPE Beams of electrons are used to produce images wavelength of electron beam is much shorter than light, resulting in much higher resolution.

22. TYPES OF ELECTRON MICROSCOPE 1. Transmission electron microscopy (TEM) : It is principally quite similar to the compound light microscope, by sending an electron beam through a very thin slice of the specimen. 2. Scanning electron microscopy (SEM) : It visualizes details on the surfaces of cells and particles and gives a very nice 3D view .

23. DIFFERENCE BTWEEEN SEM & TEM SEM vs TEM

24. How to use a microscope

25. CARE ABOUT MICROSCOPE Always carry microscope with two hands Only use lens paper for cleaning Always store covered Keep objects clear of desk Store with scanning objects clear of desk

26. STEPS OF USING MICROSCOPE Make sure all backpacks and junk are out of the aisles and off the tops of desks. Place the microscope at the desk. Remove the cover. Switch on the microscope.

27. FOCUSING SPECIMEN Always start with the low power Objective. Place your specimen in the center of your field of view. Use the Coarse Knob to focus. Next, use the Fine knob until clear. Now, switch to High Power. At this point, ONLY use the Fine adjustment knob to focus specimens.

28. HOW TO PREPARE A SLIDE OF WET MOUNT? Gather a thin slice/piece of your specimen Place on slide Put ONE drop of water directly over the specimen Covered it by cover slip Remove extra water by tissue paper Observe under microscope

29. CLEANUP Store microscopes with the scanning objective in place. Wrap cords and cover microscopes.   Double check to make sure you didn't leave a slide Place microscopes in their designated location (probably a cabinet)

30. TROUBLESHOUTING Occasionally you may have trouble with working your microscope. Here are some common problems and solutions. 1. Image is too dark! Adjust the diaphragm, make sure your light is on. 2. There's a spot in my viewing field, even when I move the slide the spot stays in the same place! Your lens is dirty. Use lens paper, and only lens paper to carefully clean the objective and ocular lens. The ocular lens can be removed to clean the inside.  The spot is probably a spec of dust.

31. 3. I can't see anything under high power! Remember the steps, if you can't focus under scanning and then low power, you won't be able to focus anything under high power.  Start at scanning and walk through the steps again.  4. Only half of my viewing field is lit, it looks like there's a half-moon in there! You probably don't have your objective fully clicked into place.

32. Practical use of microscope