Light phase contrast and fluorescence microscopy


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Light phase contrast and fluorescence microscopy

  1. 1. by c.Keerthana
  2. 2.  First described by Dutch physicist frits Zernike in 1934.  It is a type of light microscopy.  It is a contrast enhancing optical technique that produces high contrast images of transparent specimens.  Specimen- unstained and alive.
  3. 3.  ANNULAR RING : is between condenser and light source.  PHASE RING : is between objective lens and image plane.  SPL PROPERTY : both the rings allow partial light to pass through it and the rest is blocked.
  4. 4. Basic mechanism is interference of light beams.  INTERFENRENCE: Interaction of two light waves which leads to the formation of resultant wave.  TYPES OF INTERFERENCE: constructive interference destructive interference
  5. 5. Light source Annular ring Condenser Specimen plate (interference) objective lens phase ring Image plane
  6. 6.  Light passes through the condenser via annular ring.  After reaching the specimen plate two types of beams are formed.  IF THERE IS NO SPECIMEN IN LENS: 1.Surrounding wave (S) 2.particle wave (p) P=S NO INTERFERENCE
  7. 7.  IF LENS CONTAINS SAMPLE:  Light beam gets diffracted because of different density at different regions of sample. 1.surrounding wave (S) 2.diffaracted wave (D) P=S+D Either constructive interference or destructive interference may occur.
  8. 8.  Positive phase contrast produces Constructive interference. Thus, the image of the specimen obtained is  Inner region of the sample – darker  Outer region of the sample– bright  Surrounding lens – opaque
  9. 9.  Negative phase contrast microscopy produces destructive interference.  Thus, the image obtained is  Inner region of the sample – bright  Outer region of the sample– darker  Surrounding lens – opaque
  10. 10.  Fluorescence microscope is an one of the light microscope.  It refers to any microscope that uses fluorescence to generate an image.  It produces 3d image.  The technique is used to study specimens, which can be made to fluorescence.
  11. 11.  Fluorescence is a phenomenon that takes place when a substance absorbs light at a given wavelength and emits light at another wavelength.  Fluorescence occurs as an electron, which has been excited to a higher, and more unstable energy state, relaxes to its ground state and gives off a photon of light.
  12. 12.  The sample to be analyzed Is placed on a lens. And the sample is coated with a fluorescence material.  The light is illuminated through the lens with the higher energy source. The illumination light is absorbed by the fluorophores.  The sample causes them to emit a longer lower energy wavelength light.  This fluorescent light can be separated from the surrounding radiation with filters.
  13. 13. The light from the light source is passed through the excitation filter.  The specific wavelength of light is passed through the sample via dichronic filter.  The objective lens focuses the light to the specimen.  The light emitted from the specimen is filtered by barrier filter. 
  14. 14.  Imaging structural components of small specimens, such as cells.  Conducting viability studies on cell populations (are they alive or dead).  Imaging the genetic material within a cell (DNA and RNA).  Viewing specific cells within a larger population with techniques such as FISH.  To differentiate different type of cell.