Oil immersion lens to see an object we need contrast

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Oil-immersion lens
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To see an object, we need contrast between the object and the background. In bright-field microscopy, objects absorb or scatter light more than the surrounding material, so they look dark against a light background. BUT most bacteria don’t absorb or scatter enough light to be clearly visible in bright-field. Saccharomyces cerevisiae Baker’s yeast, 8-10 μm
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Create contrast by staining the sample
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Gram stain – a differential stain that can be used to distinguish between different organisms in the same specimen.
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Gram stain – continued
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Create contrast optically- phase contrast microscopy Objects in a specimen have different (higher) refractive index than the surrounding medium Light waves that interact with the specimen are deflected along a different path than rays that hit the surrounding medium, or that pass through the specimen without interacting. Specialized lenses reduce the intensity of the undeflected light, then “subtract” the deflected rays from the undeflected rays to generate an image of objects that would be very difficult to see in bright-field microscopy. Objects with a refractive index higher than the background appear dark. Caulobacter crescentus , scale bar = 1 μm
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Transmission electron microscopy (TEM) Uses electrons as radiation and electromagnets as lenses. Wavelength of an electron is a function of the potential difference through which it is accelerated. λ ≈ 1.23/V 1/2 (where λ is in nm and V is in volts) A typical TEM uses 50,000 V, so these electrons have λ = .0055 nm (compared to green light = 540 nm) Electron microscope objectives have very small NA, because
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