A microscope is a laboratory instrument used to examine objects that are too small to be seen by the naked eye

A microscope is a laboratory instrument used to examine objects that are too small to be seen by the naked eye

1. What is a microscope? Theoretically a microscope is an array of two lenses. Objective lens Tube lens Eyepiece lens Focal plane Image plane Classic compound microscope Image plane A microscope is a laboratory instrument used to examine objects that are too small to be seen by the naked eye.

2. M icro - = “small”; - scope  = “to look at” Photographs of cells are taken using a microscope, and these pictures are called  micrographs .

3. What is a microscope?

4. Fluorescence Microscopy 4 Fluorescence microscopy is an imaging technique that uses light to stimulate fluorophores (fluorescence), which then emit light at a different wavelength.  A fluorescent microscope uses a light beam, usually ultraviolet, to excite fluorescent molecules in a sample, which causes them to emit visible light. The emitted light can then be visualized by the microscope.  Fluorescence microscopy is used in many applications, including biology, medical research, and material science. It's a minimally invasive way to highlight specific components of complex biological specimens.

5. Fluorescence microscopy https://www.youtube.com/watch?v=SfzmW7EMdLE

6. Applications Used in the visualization of bacterial agents such as  Mycobacterium tuberculosis. Used to identify specific antibodies produced against bacterial antigens/pathogens in immunofluorescence techniques by labeling the antibodies with fluorochromes. Used in ecological studies to identify and observe microorganisms labeled by the fluorochromes It can also be used to differentiate between dead and live bacteria by the color they emit when treated with special stains

7. Sir George G. Stokes The phenomenon of fluorescence was known by the middle of the nineteenth century . British scientist Sir George G. Stokes first made the observation that the mineral fluorspar exhibits fluorescence when illuminated with ultraviolet light, and he coined the word " fluorescence" 7

8. Differences between Conventional and Fluorescent Microscope 8 The Conventional microscope uses visible light (400-700 nanometers) To illuminate and produce a magnified image of a sample. F luorescence microscope , uses a much higher intensity light source which excites a fluorescent species in a sample of interest. 10-100 times brighter than most incandescent lamps This fluorescent species in turn emits a lower energy light of a longer wavelength that produces the magnified image instead of the original light source.

9. What is Fluorescence? Fluorescence is light produced by a substance when it is stimulated by another light. Fluorescence is called " cold light " because it does not come from a hot source like an incandescent light bulb. 9

10. Fluorescence microscopy is a unique way of using a microscope to discover facts about specimens that often are not shown by standard bright field microscopy . In bright field microscopy, specimens are illuminated from outside, below or above, and dark objects are seen against a light background . In fluorescence microscopy, specimens are self-illuminated by internal light , so bright objects are seen in vivid color against a dark background . Bright objects against dark backgrounds are more easily seen. This characteristic of fluorescence microscopy makes it very sensitive and specific. 10 What is Fluorescence Microscopy?

11. The Fluorescence Microscope Exposes specimen to ultraviolet, violet, or blue light Specimens usually stained with fluorochromes Shows a bright image of the object resulting from the fluorescent light emitted by the specimen

12. 12 Most cellular components are colorless and cannot be clearly distinguished under a microscope. The basic premise of fluorescence microscopy is to stain the components with dyes . Fluorescent dyes, also known as fluorophores of fluorochromes, are molecules that absorb excitation light at a given wavelength (generally UV), and after a short delay emit light at a longer wavelength. The delay between absorption and emission is negligible, generally on the order of nanoseconds. The emission light can then be filtered from the excitation light to reveal the location of the fluorophores. Principle of Fluorescent Microscopy

13. Components of a Fluorescence Microscope Light source : Xenon arc lamps or mercury-vapor lamps are a common source of ultraviolet light; power LED and lasers are used in more advanced forms. A set of optical filters: Optical filters include a set of a compatible excitation filter, emission filter, and dichroic beam splitter; An excitation filter selects the wavelengths to excite a particular dye within the specimen. A dichroic beam splitter/ dichroic mirror reflects light in the excitation band and transmits light in the emission band, enabling the classic epifluorescence incident light illumination. An emission filter provides quality control by letting only the wavelengths of interest emitted by the fluorophore pass through. Darkfield condenser : It provides a black background against which the fluorescent objects glow. The filters are often plugged together in a filter cube (compound microscopes) or a flat holder (mainly stereo microscopes).

14. Principle of Fluorescent Microscopy Fluorescence microscopy uses a much higher intensity light to illuminate the sample. This light excites fluorescence species in the sample, which then emit light of a longer wavelength. The image produced is based on the second light source or the emission wavelength of the fluorescent species -- rather than from the light originally used to illuminate, and excite, the sample. 14

15. Works on Principles of Light Pathways Specifically, a dichroic mirror is used to separate the excitation and emission light paths. Within the objective, the excitation/emission share the same optics. In a fluorescence microscope, the dichroic mirror separates the light paths. 15

16. 16 Advantages of Fluorescent Microscopy Fluorescence microscopy is the most popular method for studying the dynamic behavior exhibited in live cell imaging. This stems from its ability to isolate individual proteins with a high degree of specificity amidst non-fluorescing material. The sensitivity is high enough to detect as few as 50 molecules per cubic micrometer. Different molecules can now be stained with different colors, allowing multiple types of molecule to be tracked simultaneously. These factors combine to give fluorescence microscopy a clear advantage over other optical imaging techniques, for both in vitro and in vivo imaging.

17. Types of Fluorescence Microscopes There are various types of fluorescence microscopes. Some of the common types are: Epifluorescence microscopes It is the most common type of fluorescence microscope. In this microscope, excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e., through the objective). Confocal microscope In this type of fluorescence microscope, high‐resolution imaging of thick specimens (without physical sectioning) can be analyzed using fluorescent-labeled dye. Multiphoton microscope In this type of microscope, multiphoton fluorescence excitation captures high-resolution three-dimensional images of specimens tagged with highly specific fluorophores. Total internal reflection fluorescence (TIRF) microscope Total internal reflection fluorescence microscopy (TIRFM) exploits the unique properties of an induced evanescent wave or field in a limited specimen region immediately adjacent to the interface between two media having different refractive indices.

18. Epifluorescence Microscopy Epifluorescence microscopy is a method of fluorescence microscopy that is widely used in life sciences The excitatory light is passed from above (or, for inverted microscopes, from below), through the objective lens and then onto the specimen instead of passing it first through the specimen. The fluorescence in the specimen gives rise to emitted light which is focused to the detector by the same objective that is used for the exc itation 18

19. Fluorescence Microscopy From Principles to Applications

20. The Specimens to be Stained Most specimens for fluorescence microscopy must be stained. Fluorescent stains are called " fluorochromes." Acridine orange , auramine O, and fluorescent antibody (FA) are the fluorochromes used most. 20

21. How to Use a Fluorescence Microscope 21 The object to be studied is marked with a molecule called a fluorophore (a dye). When the florescent light is activated, the light used for illumination is separated from the florescent molecule (the fluorophore), which is much weaker. This is done through an emission filter.

22. Step 1 22 Locate the light switch on the side of the microscope that turns on the light. Turn the microscope on. Write down the exact time you turn on the light. The florescent light is mercury-based, and a time log must be kept for exposure and use of the light.

23. Step 2 Locate the toggle switch on the right side of the microscope between the oculars and objectives. This switch controls the shutter for the mercury light to the objective lens. 23

24. Step 3 Select the appropriate dye for your object (this will depend entirely on what you are going to be studying). The most common dyes include I3 (for use with CTC, DTAF and fluorescein), A (for use with DAPI and f420), N21 (for use with Rhoda mine) and L3 (for use with fluorescein).

25. Step 4 Put the filter (dye) into the tray operated by the silver sliding knob. To remove the tray, simply pull the silver knob out. 25

26. Step 5 Select the lens you would like to use. The 63x objective lens will have the highest numerical aperture. The 100x objective lens will have the highest magnitude that can be used with the mercury-based florescent light source. 26

27. Step 6 Turn the light off when finished, and mark the time. Wait 30 minutes before turning the light back on, or the lamp could explode. It is a good idea to keep track of how many hours the lamp is in use and replace it according to the manufacture's guidelines. 27

28. Step 7 C lean off the microscope lens with lens paper, or if really dirty, use a cotton swap and glass cleaner. 28