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Microscopy. UNITS OF MEASUREMENT. 1mm = 1000µm 1µm = 10 -3 mm (convert mm to µm by multiplying by 1000 = 3 zeros) Bacteria are about 1µm or smaller 1nm = 10 -6 mm (convert nm to mm by dividing by 1000000 = six zeros) Viruses are about 1nm 1000nm = 1µm
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UNITS OF MEASUREMENT • 1mm = 1000µm • 1µm = 10-3mm (convert mm to µm by multiplying by 1000 = 3 zeros) • Bacteria are about 1µm or smaller • 1nm = 10-6mm (convert nm to mm by dividing by 1000000 = six zeros) • Viruses are about 1nm • 1000nm = 1µm • 1000 viruses can fit into one bacterium • 0.001µm = 1 nm • Bacteria are so small, they are measured in µm. • Viruses are even smaller, so they are measured in nm.
VOCABULARY • Immersion oil: keeps light from bending and allows lens to be refracted. • Resolution: ability of two lenses to distinguish two points. • Parfocal: focused in all lenses. • Depth of field: how much of the background is in focus at the same time that the foreground is in focus. • Refractive Index: a measure of the light-bending ability of a medium • Numerical aperture: numerical aperture increases as depth of field decreases. • Resolution power: limits the useful magnification of the microscope resolving power.
RESOLUTION • The ability of the lenses to distinguish two points.
RESOLVING POWER • The distance between two closely adjacent objects where the objects still appear separate and distinct. The shorter the distance (the smaller the number), the better the resolving power (the sharper the image).
RESOLVING POWER • To calculate the resolving power (to see how close two objects can be so you can still see them): • D = distance (smaller number is better) • 0.61 = a constant number, does not change • NAobj = numerical aperture of the objective (larger number causes D to decrease, which is better) • λ = (lambda): the wavelength (nm) of the light going through the microscope. Convert nm to mm by dividing by 1000000 (six zeros) D = 0.61 λ / NAobj
PRISM • This is a triangular device that breaks up light into its various wavelengths so you can see all the colors of the rainbow (the visible spectrum). • The visible spectrum of colors starts with violet (350nm), and goes on to indigo, blue, green (550nm), yellow, orange, red (700nm).
Sample Problem • When we want to observe the color green (550nm) under an oil-immersion objective lens of a microscope, where the NAobj is 1.25, the resolution is as follows: • D = (0.61)(0.000550) / 1.25 • D = 0.0002684 mm convert this to microns (µm) by multiplying by 1000 • D = 0.27 µm
Sample Problem • The NAobj for the high-dry (400x) lens is 0.65 • What is the resolving power (D) of this objective when viewing a wavelength of 550nm? • D = 0.61 λ / NAobj • D = (0.61)(0.000550) / 0.65 • D = 5.1615mm • D = 0.52µm
Conclusions • Therefore, we can see an organism such as E. coli, which is 2µm long and 1µm wide because it is larger than the resolving power. However, we could not see Haemophilus influenza, which is 0.2µm long because it is smaller than our resolving power. • Therefore, the resolving power limits the useful magnification of the microscope. • Resolution determines the magnification.
REFRACTION • Refraction is the bending of light caused by the surrounding medium. • N = Refraction Index of the medium surrounding the lens • Air: N= 1 • Glass: N = 1.5 • Immersion Oil: N = 1.51 (about the same as glass)
TYPES OF MICROSCOPES • SIMPLE MICROSCOPE: Has only one lens, like an ocular (eyepiece) • COMPOUND MICROSCOPE: More than one lens, like an ocular and an objective. An example is the Brightfield microscope. • There are two main types of compound microscopes: Light Microscopes and Electron Microscopes.
Types of Compound Microscopes • Dissecting • Brightfield • Darkfield • Phase-contrast • Differential Interference contrast • Fluorescence
Fluorescence Microscopy • Uses UV light. • Fluorescent substances absorb UV light and emit visible light. • Cells may be stained with fluorescent dyes (fluorochromes). Figure 3.6b
