Microscope HowTo
From Physics 111-Lab Wiki
All pages in this lab
II. Staff Sign-off Sheet (BMC)
III. Simulating Brownian Motion
V. BMC Software
Contents |
Microscope How-To
 |
 |
The main piece of equipment is a Zeiss Aviovert 200 microscope. Starting from the top right of the diagram, the microscope has a variable intensity halogen light source. A condenser focuses light from the halogen bulb onto the sample under observation. A rotating turret in the condenser holds several irises and stops that are used for different contrast methods such as bright- and dark-field. (As shown, the condenser iris is selected, as would be the case for normal bright-field illumination.)
The objective nosepiece rotates to select one of three objective lenses. The microscope in the lab is outfitted with four objectives: 5x, 10x, 20x, and 40x. Although they are depicted as simple lenses in the diagram, the objectives are actually sophisticated, compound optical elements. More information about objectives can be found in references 6 and 7. Specifications of the objectives may be found at the Zeiss website.
Light enters the microscope body through the selected objective and is optionally split into two paths. A setting wheel selects whether 0, 50, or 100 percent of the light is diverted to the left sideport. (The sideport is essentially a big hole in the microscope cover.) The remaining light goes on toward the eyepieces. A CCD camera is mounted with its sensor at the focal plane located just outside the sideport. Images captured by the camera are transferred to the PC via a Firewire connection.
A binocular tube splits the image headed toward the eyepieces in two for direct observation with both eyes. The eyepieces are 10x, providing overall magnification of 50x, 100x, 200x, or 400x depending on which objective is selected.
Look over the detailed schematic of the Axiovert 200 microscope. You will notice some differences between the microscope in the drawing and the one in the lab – most significantly in the condenser (described below). To become familiar with the microscope controls, follow the instructions below to make a slide with a combination of 10µm and 0.44µm polystyrene spheres and then view it in both transmitted light and dark-field illumination. But first some general warnings:
* The microscope is a delicate instrument. It is never necessary to force any adjustment. * Wear safety goggles when you work with glass slides or liquids. Pay particular attention when handling coverslips – they can break into very sharp pieces with unexpected violence. * Do not remove any parts from the microscope (except for eyepieces) or attempt to clean the optics. * If the microscope needs cleaning, ask a GSI or Don for help. * Do not use canned air on the microscope. It can leave a difficult-to-remove residue. * Feel free to use it on your slides. I personally find it to be ineffective. * To lengthen the lifetime of the halogen lamp, turn off the light source with the HAL on/off switch (Schematic-7) when it is not in use.
How to pipet
Forward Technique: Fill a clean reagent reservoir with the liquid to be dispensed.
- Depress the push button to the first stop.
- Dip the tip under the surface of the liquid in the reservoir to a depth of about 1 cm and slowly release the push button. Withdraw the tip from the liquid touching it against the edge of the reservoir to remove excess liquid.
- Deliver the liquid by gently depressing the push button to the first stop. AFter a delay of about one second, continue to depress the push button all the way to the second stop. This action will empty the tip.
- Release the push button to the ready position.
If necessary, change the tip and continue pipetting.
- Adjust the volume of fluid dispensed by rotating the thumbwheel until the desired volume
Never allow liquid to enter the body of the micropipette
- To avoid contaminating the nanoparticle suspensions, wear gloves and put a fresh filter tip on the pipette each time you dip it in a new solution.
- Shake bottle for loosening.
Make a viscous solution
A solution will consist of three parts, water, which makes up most of the solution, a solute (either PVP or glycerol) to provide the viscosity, and the beads which we will then observe.
Water can be located in the large squeeze bottle marked "Deionized Water," be sure to use this rather than tap water as tap water will fill your slide with various unwanted particulate matter.
The Solutes can be found in the upper cabinet and are also clearly marked. Use the long and thin metal scoops/spatulas to measure out the glycerol and the large-volume pipette to measure out the glycerol. Note that the glycerol is extremely sticky (ie viscous) and will not be measured precisely using the pipette as it will stick to both the inside and outside of the filter tip. To measure both the PVP and glycerol it is best to use the scale located at the lab station.
Beads are located (and should be kept) in the refrigerator. Each of the vials is clearly marked with the size of bead that it contains. Note that these vials are often extremely concentrated and you may wish to create your own diluted solution to work with. The densities vary between the differently sized particles.
Thus, a procedure to create a solution ready for observation may look like the following:
- Remove 1 micron bead vial from refrigerator
- Using a NEW filter tip extract 30μL from the vial and deposit into a new plastic vial
- Fill vial half full with Deionized Water
- This is our concentrated bead stock - set aside for now
- Take out a new plastic vial to contain the viscous solution
- Turn Scale on, place vial on scale
- Zero scale to cancel out the weight of the vial
- Place a moderate amount of solute (either PVP or Glycerol) into vial
- Weigh the amount of solute using the scale
- Calculate how much water is needed to obtain the desired viscosity using the table below
- Add the approprate amount of water to the vial
- This can be done either by measuring the volume with the pipette or by squirting water into the vial using the squeeze bottle and measuring the amount using the scale.
- If you use the pipette to transfer deionized water you may find it easier to squeeze a large amount of water into a glass beaker and then use that as your reservoir
- Now add 30μL of your bead stock to your viscous solution
- Cap vial using a plastic vial top and shake to ensure uniformity
Computing viscosity
Use this table to compute the required dilution of the solvent. The PVP in the lab is in pure powder form. The stock Glycerin is a thick liquid with >99% purity. It will require some care to measure the pure glycerin accurately, since it tends to stick to the sides of the pipette tip. For this reason, weighing the glycerin is probably a better technique than relying on the amount dispensed from the pipette alone.
PVP and Glycerol Viscosity versus Concentration Viscosity (cP) Percentage of PVP (by weight) Percentage of Glycerin (by weight) 1.66 0.50 18.0 2.50 1.00 32.2 4.65 2.00 44.2 13.2 4.00 64.5
You may either use these values or interpolate between them.
Make a slide
Now that we have a proper vial of viscous bead solution made up we need to transfer a sample of it onto a slide so that we can observe the beads' behavior.
- Take out a slide from its box and carefully rest in a position to minimize dust contamination.
- Place a clear reinforcement label on the center of a new slide to create a well. This will create a well for the solution and keep the liquid solution from drying out.
- Make sure that this label is well pressed down onto the slide to ensure that liquid isn't sucked out towards the open air. Rubbing the edge of another slide over the coverslip provides a good method of pushing down the well without contaminating the slide with oils from your hands.
- Use the pipette to transfer roughly 15μL of your bead solution into the center of the well
- Cover the slide with one of the small 18x18mm coverslips
- Rest one edge of the coverslip on the slide and then let go of the coverslip. Capillary action will adhere the coverslip to the slide
- Back off the objective first before placing the slide in the microscope stage.
- The entire carrier arm that holds the HAL 100 illuminator and the condenser tilts backward on a hinge to facilitate access to the microscope stage. Push back gently on the angled metal nosepiece just above the LCD display until the arm comes to a rest (Figures).
- Our microscope is an inverted microscope and as such the sample (ie, side with the coverslip) should be pointed downwards so that the sample is closest to the objective. You will not be able to focus on the sample using the 40x objective if this is not the case.
Figures
Picture of microscope with condenser tilted back
View the slide in transmitted light
Microscope images look best when properly illuminated. The procedure developed by August Köhler’s is nearly universally used to achieve uniform illumination with little reflection or glare and minimal sample heating. In this step, you will view your sample under Köhler illumination.
As shown in Figure 4, there are two irises in the illumination path of the microscope. The one on top (closest to the light source) is called the field iris. The condenser iris is a little farther down. The adjustments for both irises are highlighted in Figure 2. The condenser iris adjustment is located at the edge of the turret disk near the bottom of the condenser. You will adjust both of these irises to achieve the best image.
Fig. 2 is a picture of the actual condenser assembly you will find in the lab. The markings on this condenser are a bit confusing since it was designed to be used mainly in upright microscopes instead of inverted models such as the Axiovert 200.
It is necessary to go through this adjustment procedure every time you change objectives or samples.
- Use the On / Off Switch (Schematic-1) to turn the microscope on. Rotate the turret disk Schematic-19 on the condenser to the transmitted light position. (See Fig. 4 for a picture of the actual turret disk.)
- You will see an upside down number "2" just to the left of the condenser shaft when you have the correct setting. Tilt the condenser back and hold a piece of paper under the bottommost lens. If the spot gets brighter and darker as you rotate the field iris ring, you have the right setting.
- Rotate the side port setting wheel (Schematic-23) to the 100% visual setting (looks like two concentric circles).
- Open the condenser iris all the way using the Setting Wheel for Aperture Diaphragm on the Condenser Schematic-20. This is a thin, silver wheel on the turret.
- Select the 10x objective by rotating the objective nosepiece (Schematic-2) until the display reads 10x. Physically look at the body of the objective to make sure it is on the correct setting (10x should be setting #2).
* It is possible to make the tip of the objective collide with the slide. * If you crash an objective into the slide, you will very likely break the slide. This will release the liquid from the slide on to the objective and make it very dirty. Try very hard not to do this.
- You must set up Köhler illumination each time you change objectives.
- Focus on the sample using the coarse and fine focusing knobs (Schematic-6).
* When you get close to the proper focus, it is an excellent idea to engage the focus limiter at a setting slightly higher to reduce the chance of running in to a slide. * Avoid focusing on slower particles because they are close to the edges.
Adjusting the field and condenser irises
Do not skip this step. The microscope must be focused on a sample to properly set up Köhler illumination.
- Adjust the distance between the eyepieces on the binocular tube by rotating them toward or away from each other. When the distance between the eyepieces matches the distance between your pupils, you will see images in both eyes. You can make both eyepieces higher or lower by arranging them in a right-side-up or inverted V configuration. There is a scale on the binocular tube that will allow you to easily reestablish the correct setting after it has been changed by your annoying lab partner.
- The samples in this lab are difficult to focus on because they have very little contrast. If you have trouble focusing, try starting with the 10x objective. At higher magnification, it is sometimes helpful to focus on the edge of the slide first to get the setting close. Note: Stay away from slower particles for they are close to the edges.
- The eyepieces are designed to be used while wearing eyeglasses. If you do not wear glasses, don’t get too close to them.
- Close the field iris (Schematic-16) until you can see its outline in the eyepieces (Fig. 5a).
- Bring the field iris into focus by moving the condenser up or down using the condenser height adjustment knob (Fig. 5b). Do not adjust the focus of the objective. Make sure not to lower the condenser into the slide. Unfortunately, there is no limit setting on the condenser travel. If you crash into a slide, you will break it, making the objective and the condenser very dirty in the process, and possibly damaging them.
- Remove one eyepiece. Center the image of the iris using the condenser centering knobs (Schematic-18) until the image looks like Fig. 5c.
- If you do not see any light, open the field iris until you can see its edge. Use the condenser centering knobs to move the opening closer to center and then continue closing the field iris.
- Looking in the other eyepiece, open the field iris until its edges are just out of view (Fig. 5d).
- Remove the phase telescope and adjust the condenser iris until the size of the image is reduced to about 2/3 of its full diameter. Put the eyepiece back in (Fig. 5e).
- Use the image intensity switch on the front of the microscope to set a comfortable overall illumination level.
- Never use the field or condenser iris to adjust the brightness.
- Change the focus to see a very little portion of the particles for a movie. After you change the focus, let it stand. Have about 1-5 particles in view.
- Look at the 10μm spheres with the 20x and 40x objectives. You may have to refine the focus as you increase magnification. Remember to set up Köhler illumination each time you change objectives.
Why do the PS spheres appear bright in the middle?
Set-up darkfield illumination
 |
If you have been very diligent about setting up the illumination, you may be able to just barely discern the 100 nm gold particles frenetically wiggling about. After you change to dark-field illumination, these Lilliputian bullion nuggets will be easily visible as bright spots on a dark, black background. The effect is similar to looking at stars during the daytime versus night.
- Select the 20x objective and establish Köhler illumination.
- Rotate the condenser turret disk (Schematic-19) one position to the right. You should see an upside down number "2" just to the right of the condenser column.
- Remember that for bright field, the upside down number "2" is to the left of the condenser column; the upside down number "2" is to the right of the condenser column in dark field.
- To get an idea of how this effects sample illumination, tilt the condenser column back and hold a piece of white paper under the condenser. You should see a disk of light. In this position, an opaque disk blocks the light in the middle of the illumination field.
- Open the field iris all the way
- Increase the light intensity using the Toggle Switch for Illumination Intensity (Schematic-8). You will need to turn the light level up significantly in order to see the small amount of light scattered by the smaller nanoparticles (even though the PS spheres will be easily visible). Increase the illumination until you hear a beep, and the level will be approximately correct. (The beep indicates that the halogen light source is set to the correct level for color photography, incidentally.) Note: One beep indicates set point, three beeps indicate maximum light.
- Adjust the condenser slightly up or down to obtain an even, dark background. If you properly set Köhler illumination, the required adjustment should be very small.
- You should see something similar to Fig. 7.
Microscope schematic
