Light microscope

light microscope and its types, princple, uses application, advantages and limitation

1. Light microscope Dr. R asika Deshmukh (MBBS, MD)

2. The evolution of the Microbiology field put to perspective the need to identify, view, observe and understand microorganisms, including their structural morphologies and mechanisms. 

3. History  The first simple microscope was discovered by two Dutch scientists, Zaccharias Janssen and his father, Hans who made spectacles, were the first to experiment with their lenses by combining lenses in a tube and observed that the objects that were nearby, appeared closer and larger.

4. History  Antony Van Lewnehoueek an amateur Microbiologist made the first simple microscope, that enabled him to observe the presence of tiny living organisms in pond water that appeared like dots. His simple microscope was made up of a double convex glass lens that was held between two silver plates.

5. History

6. What is a light microscope?  Instrument or tool, that uses visible light to detect and magnify very small objects and enlarge them. Use lenses to focus light on the specimen, magnifying it thus producing an image

7. Principles of Light Microscopy For light microscopy, visible light is passed through the specimen and then through a series of lenses that bend the light in a manner that results in magnification of the organisms present in the specimen . The total magnification achieved is the product of the lenses used.

8. Key component to light microscopy Magnification Objective lens magnifies objects 100× (times) the ocular lens magnifies 10×. Using these two lenses in combination, organisms in the specimen are magnified 1000× their actual size when viewed through the ocular lens.

9.  Contrast Needed to make objects stand out from the background Contrast is most commonly achieved by staining techniques that highlight organisms and allow them to be differentiated from one another and from background material and debris.

10. Resolution To optimize visualization, other factors besides magnification must be considered. Resolution, defined as the extent to which detail in the magnified object is maintained, is also essential.

11. Types of microscopes Depending on the number of lenses, there are two types of microscopes Simple light microscope (it has low magnification because it uses a single lens) Compound light microscope (it has a higher magnification compared to the simple microscope because it uses at least two sets of lenses, an objective lens, and an eyepiece).

12. Light microscope The modern types of Light Microscopes include: Bright field light microscope Dark field light microscope Phase contrast light microscope Fluorescence light microscope

13. Bright field microscope most basic optical Microscope produces a dark image against a bright background provide a high-resolution image

14. Parts of a bright-field microscope

15. Objective lens: magnifies the image by 10x, 20x, 40x and 100x Ocular lens: magnifies the image by 10x Condenser: focuses a beam of light onto the specimen Diaphragm: controls the diameter of the beam of light that passes through the condenser Two focusing knobs: focus on the image and sharpen the image.

16. Applications of Microscopy in Diagnostic Microbiology Rapid preliminary organism identification by direct visualization in patient specimens Rapid final identification of certain organisms by direct visualization in patient specimens Detection of different organisms present in the same specimen Detection of organisms not easily cultivated in the laboratory Evaluation of patient specimens for the presence of cells indicative of inflammation (i.e., phagocytes) or contamination (i.e., squamous epithelial cells) Determination of an organism’s clinical significance; bacterial contaminants usually are not present in patient specimens at sufficiently high numbers (×10 5  cells/ mL ) to be seen by light microscopy Provide preculture information about which organisms might be expected to grow so that appropriate cultivation techniques are used Determine which tests and methods should be used for identification and characterization of cultivated organisms Provide a method for investigating unusual or unexpected laboratory test results

17. DARK GROUND MICROSCOPE Background is dark Object or organism appears bright

18. PRINCIPLE OF DARK GROUND MICROSCOPE To view a specimen in dark field, an opaque disc is placed underneath the condenser lens, so that  only light that is scattered by objects on the slide can reach the eye Instead of coming up through the specimen, the light is reflected by particles on the slide.

19. Application of Dark ground microscopy demonstration of very thin bacteria not visible under ordinary illumination  method for rapid demonstration of  Treponema pallidum   demonstration of the motility of flagellated bacteria and protozoa. Darkfield is used to study marine organisms such as algae, plankton, diatoms, insects, yeast and protozoa  Darkfield is used to study mounted cells and tissues. It is more useful in examining external details, such as outlines, edges, grain boundaries and surface defects than internal structure.

20. Bright field vs Dark field Microscope Bright field microscope Darkfield microscope Bright field microscope formed a dark image against bright field. Darkfield Microscope formed a bright image against dark background Stained, fixed, and live specimens are observed. We can see the living and unstained cells. In a bright-field microscope, only the scattered lights are able to enter the objective lens and transmitted lights or unscattered light rays are omitted, that’s why the viewer sees a dark image against the brightfield . The only light that has been reflected or reflected by the specimen forms an image. Organism a ppear as dark Organism appear as bright Background appear as bright Background appear as dark.

21. Bright field vs Dark field Microscope Bright field Microscope Dark field Microscope It shows us the morphological as well as the internal structure of the specimen. It show us External structure of specimen in great detail. Bright fields are inexpensive Dark fields are expensive. Specimen preparation or staining is a complex and lengthy process. Takes too much time. No need stain. Opaque disc is Absent Opaque disc is Present It is easy to use. operating procedure is not easy as compared to bright field microscope.

22. Phase contrast microscope

23. Application

24. The Fluorescent Microscope dye molecule  added to the specimen. 

25. Advantages helps in the study of  cell behaviour . highlights the  biomolecule  of interest. highlight the image of particular  structural components  within the microscopic organisms. highly  sensitive  technique that can detect around 50 molecules/µm3. analyze or track the physiochemical properties of multiple biomolecules simultaneously.

26. Disadvantages It only allows the observation of specific structures inside a cell tagged with the fluorescent dye. The  photobleaching  due to the electron excitation during the process of fluorescence may affect reactive molecules of the fluorescent dyes. As a result, the reactive dyes might lose their chemical property of fluorescence emission intensity. The cells are susceptible to the  phototoxic effect  after staining with fluorescent dyes, as the fluorophore molecules absorb the high energy photons from the short-wavelength light.

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