Microscope and types of microscope.pptx

Microscope and types of microscope.pptx

1. Types of microscope (based on source of energy to generate an image) Light microscope –in which light is used to detect the small object Electron microscope – in this, short beam of electrons are used to generate the image. Light microscope is again broadly classified into two categories (based on the number of lenses) Simple microscope- it consists of only one biconvex lens Compound microscope- consist of two separate lens systems such as objective and ocular (eye piece)

2. The Light Microscope many types bright-field microscope dark-field microscope phase-contrast microscope Differential interference contrast microscope (DIC) fluorescence microscopes

3. Fluorescence Microscopy Fluorescent molecules absorb light at one wavelength and emit it at another , longer wavelength . If we illuminate such a compound at its absorbing wavelength and then view it through a filter that allows only light of the emitted wavelength to pass, it will glow against a dark background This microscope is similar to an ordinary light microscope except that the illuminating light is passed through two sets of filters- one to filter the light before it reaches the specimen and One to filter the light obtained from the specimen. The first filter passes only the wavelengths that excite the particular fluorescent dye, while the second filter blocks out this light and passes only those wavelengths emitted when the dye fluoresces

4. Pholobleaching Continuous illumination of fluorophoros leads to loss of fluorescence from the sample resulting in fading of the image

5. Application Fluorescence microscopy is most often used to detect specific proteins or other molecules in cells and tissues by fusion with GFP, YFP/RFP. A very powerful and widely used technique is to couple fluorescent dyes ( fluorescein ,, rhodamine ) ) to antibody molecules, that bind selectively to the particular macromolecules they recognize in cells or in the extracellular matrix. Cell in Mitosis stage Spindles microtububles with a green Fluorescent antibody Centromeres with a Red fluorescent antibody DNA- DAPI

6. Electron Microscopy Uses short beam of electrons and magnetic condenser lenses to produce the image. It is possible to resolve objects as small as 0.001 nm. transmission electron microscope (TEM ) scanning electron microscope (SEM)

7. transmission electron microscope (TEM) T TEM uses electrons as energy source and much lower wavelength to get good resolution. In TEM, beam of electron are transmitted through the specimen, magnified and focused by an objective. Drawback Sample preparation is laborious and time consuming process Application: tomographic reconstruction of small cells or thin section of larger cells

8. scanning electron microscope (SEM) A scanning electron microscope (SEM) directly produces an image of the three dimensional structure of the surface of a specimen. TEM uses the electrons that have passed through the specimen to form an image , the SEM uses electrons that are scattered or emitted from the specimen's surface . Thus only surface features can be examined and used to study whole cells and tissues rather than subcellular organelles A beam of electrons is produced at the top of the microscope by an electron gun. The beam travels through electromagnetic fields and lenses, which focus the beam down toward the sample. Once the beam hits the sample, electrons and X-rays are ejected from the sample and they are collected by detector converted into a signal. This produces the final image.

9. Application of SEM Produces images in 3D format