2/4/15. Brightfield Microscopy! It s all about Magnification..! or is it?!
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1 Brightfield Microscopy It s all about Magnification.. or is it? 1
2 What actually does go into chosing a microscope Choice depends on what you need the microscope to do. Do you want to magnify stained specimens? Do you want high magnification? What are the resolution requirements? What does that mean magnification and resolution? Magnification versus Resolution Magnification Think of it as experimenting on a computer by typing the letter A and magnifying it. What happens? Resolution Accepting each pixel as a part of the puzzle, take some away. What happens? 2
3 Magnification versus Resolution - magnification in a good dissecting scope can be similar - however, as magnification increases, resolution does not => empty magnification Dissecting Microscope Compound Microscope If magnification is the same, why does this occur? Solving Resolution differences in lens systems in the dissecting (stereo-) microscope compared to the compound microscope SIMPLE MICROSCOPE 3
4 The Compound Microscope Take the time and see whether you can still identify the different parts of the microscope. I know you have already done that before, but during this class you will really have to know the microscope in the dark. The Optical Path in the Brightfield Microscope = that s why the NA has to match between objective and condenser 4
5 The Numerical Aperture = dimensionless number of an optical system characterizing the angles over which the system can transmit light - How do you compensate for the change in numerical aperture when you change objective magnifications? Answers: How to address and compensate for changes in the NA between different objective magnifications? To adjust for the NA adjust the settings above the condenser (numbered lines) to the same numerical value as the NA Thickness and the texture of the condenser lens. The Iris Diaphragm can be adjusted to the correct measurement to match the aperture of the objective lens. The iris diaphragm knob would have to be adjusted to the specific numerical aperture that would be on the objective lens in use. What you have to do is to open and close the diaphragm of the iris to condense light through the condenser. 5
6 Correct Answer The NA on the condenser should be adjusted so that it equals the NA on objective lens used to view the specimen. This should allow the appropriate cone of light light to be gathered from the light source to focus on the image. - objective and condenser NA match = increased resolution - objective NA increases with increased magnification, therefore condenser NA needs to be matched = match condenser setting to NA on objective The Objectives Achromatic = chromatic aberration, generally doublet Apochromatic = best correction, spherical/chromatic aberration, generally triplet Semi-plan = flatter lenses (80% flat field) Plan = flatter lenses (95% flat field, generally triplet) 6
7 When to use Brightfield Microscopy Brightfield = stained specimens amplitude specimens Unstained specimen Amplitude specimen Two Compound Microscope Options - upright versus inverted How to chose and why 7
8 Again.. what do you want to look at? If you have regular specimens, i.e. a stained specimen mounted on a slide. If you are looking at 96-well plates, petri dishes or culture dishes. Does that mean you cannot use regular slides on an inverted microscope? of course you can You just have to adjust your settings: - have slide upside down - buy specific cover slips - buy specific objective lenses 8
9 Koehler Illumination - set up and adjust microscope => combination of contrast & resolution - condenser diaphragm controls light => condenser+objective aperture = realized NA - higher magnification = closer to specimen = large NA = smaller lens opening Koehler Illumination: Getting started Focus sample in brightfield Close field diaphragm (light source) If image view not centered, align using condenser screws (silver screws) Focus edges of the internal, centered image view by moving the condenser (black condenser screws) Open the field diaphragm (light source) 9
10 Exercises today: (work in pairs) 1. Find/identify the important components of the different microscopes (i.e. Inverted versus upright microscope). Are you able to name the parts for the purpose of an exam? 2. Understand where and how to set Koehler illumination in the different microscopes (i.e. inverted, upright, polarizing, DIC). 3. Are you capable of adjusting the NA on the associated parts of the different microscopes? Are all microscopes capable to compensate for objective changes in NA? 4. Observe differences when you open up and or stop down the condenser entirely (disregarding the NA compensation) = make slides of material you brought for exercises 3&4 10
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