Light phase contrast and fluorescence microscopy

1. Light phase contrast and fluorescence microscopy by c.Keerthana

2. Light phase contrast 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. ANNULAR RING AND PHASE RING 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. Basic principle 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. CONSTRUCTIVE AND DESTRUCTIVE INTERFERENCES

6. PATH OF THE LIGHT Light source Annular ring Condenser Specimen plate (interference) objective lens phase ring Image plane

7. Working 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

8. Cont.. 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.

9. Positive phase contrast 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

10. Negative phase contrast 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

11. Fluorescence microscopy 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.

12. Parts of a fluorescence microscopy

13. How does fluorescence occurs 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.

14. How does it work? 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.

15. principle 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.

16. applications 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.

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