Introduction
Video Explanation
Lab Prep
Supplemental

Lab 1 Microscopy Questions - as with lectures, it is labeled in canvas as a quiz, but just think of them as companion questions / worksheet. They are meant to guide you through your work. You are given unlimited attempts, and only the best score counts.
The material in the canvas module should all be covered in lab. I've left this hear in case you need a refresher or if you were absent. This is how the canvas labs will typically be.

Learning Objectives

Be able to use a microscope.
Be able to properly store a microscope.
Be able to Identify the parts and functions of a microscope.
Be able to describe the concepts of Magnification, Illumination, Field of View & Depth of Field.
Be able to prepare a wet mount

Introduction

This lab will focus on common lab techniques, focusing on pipetting and microscopy.

You are used to seeing and observing the world on the large scale. Yet there is an unseen world around you, teeming with a great diversity of life forms that become apparent when viewed through the lens of a microscope. Today you will explore this unseen world, learn the fundamentals of microscopy, and then conduct an investigation with the microscope.

You will use a compound microscope, which uses a system of two lenses to magnify a specimen. One lens, the eyepiece or ocular lens, can provide a modest amount of magnification (10x). When this lens is used in combination with one of several objective lenses, the total magnification is the product of each individual lens (i.e. 10x ocular x 40x objective = 400x total magnification).

Be careful with your wording. If you say 40x or 100x, make sure to specify objective or total. 100x could mean oil lens and therefore 1000x total, or it could mean 100x total.

A compound microscope also has another lens called a condenser. This is found below the stage and is not involved with magnification. Instead, this focuses / concentrates light on the specimen. The condenser may have an attached iris / diaphragm, which alters the amount of light on the specimen. Together the condenser and diaphragm play a role in adjusting contrast.

Occasionally, you will examine the large components of a specimen, and in this case a stereoscope, or dissecting scope, is more practical. This microscope is much lower magnifying power (typically about 2 - 40x). The dissecting scope is also useful when you need to manipulate a specimen while viewing it. Organs/tissues, whole specimens, and wet samples can be viewed under this microscope without first being mounted on a slide.

Major Microscope Components

Power Switch
Light dimmer - controls light intensity
Light Source
Stage - where you place the slide
Slide Clip - holds slide in place
Stage Move - 2 knobs: Forward/back & left/right
Objective Lenses - Provide magnification
- 4x (scanning)
- 10x (low power)
- 40x (high power)
- 100x (Oil Immersion)
Eyepieces (Ocular Lenses) - For viewing AND 10x magnification
Coarse Focus Knob - Moves stage up & down to adjust focus in large increments / quickly
Fine Focus Knob - Adjust focus in small increments / slowly, gives greater focusing control
Condenser - Lens that focuses light onto slide,
Iris / Diaphragm - blocks amount of light

Orientation

When images pass through a lense it get's flipped.
flipped image
See more on how lenses work on Khan Academy.

Light intensity, Field of view & Depth of Field

As you increase the magnification (switch from 4x to 10x to 40x to 100x objectives) you will notice 3 phenomena. The illumination (light intensity), the Field of View, and the Depth of Field will all decrease.

As you increase the magnification, you are essentially taking a small area and expanding it. So whatever light was concentrated is now spread out. Therefore it will appear dimmer / the light intensity will decrease. You are also seeing fewer things / objects in total, so your field of view has decreased.

Illumination and Field

The Depth of Field refers to how much you can see at the same time. As you increase in magnification you can see less and less of the background and foreground. You can only focus 1 plane at a time.

Depth of Field

Scale

When using a microscope, it's important to understand scale and when it's appropriate to use a microscope, what kind of microscope you should be using an what magnification you should be using.

Mammoth

Check out the this cool website on Cell Size and Scale to get some idea on how big an object is. Typically for a very small organism, such as a flea, or for a tissue (collection of many cells) where you are looking at their shape as a whole, you would use the 4x or 10x objective. For looking at individual cells you would use the 40x or 100x objective lenses. The oil immersion lens requires special procedures, and so is more appropriate for very small cells, such as bacteria.

Anything smaller would require a more sophisticated apparatus. For example, a scanning electron microscope would be good for viruses or organelles. On the other hand, larger specimens would be examined using a dissecting microscope.

As it's name implies, a dissecting microscope is often used for dissecting. Unlike the typical compound microscope you will be using, dissecting scope has the correct orientation, making it easier to handle. However, it typically can only magnify up to 70x. It also only views the surface whereas the compound microscope transmits light through the specimen. More on the differences.

You will try out the dissecting scope, but you will primarily focus on the compound microscope.

FYI my "e" that I draw in the video is wrong. I fixed it for the notes in the file bank. It should be upside and backwards.

Dry-Lab

Microscopy - General Focusing Guidelines

will be provided during lab

Proper microscope alignment and set-up begins at the LOWEST objective lens magnification (4x objective). The stage should also be at its LOWEST level—use the coarse adjustment knob to achieve this.
The Iris/ diaphragm should initially be set to 4, to match the magnification. You can adjust this later at higher magnifications to your preference.
After the microscope has been plugged in adjust the light has been to roughly medium
Eyeball the location of your specimen on the slide (look directly at the slide with the naked eye). Try to find the approximate location of the sample on the slide. Place the slide on the stage and secure between the slide clips. Try to place the location of the specimen as directly over the light source as possible.
- ex: when looking at a population of cells under a cover slip, cells may cluster to certain areas (like edges), try to find an area with a high density (cloudy looking) to focus on.
- If your slide is well-placed, you might be able to see your specimen or a blurry outline even before you begin focusing.
- When handling slides, refrain from excessive movement, such as showing other people the slide. In order to minimize breakage, in general, limit equipment transport between storage areas to your benchtop only, preferably in secondary containers.
- Hold slides on the edges to reduce smears or other debris. You may need to wipe the slide with a kimwipe. (should be located on your benchtop)
While looking through the ocular lens, adjust the focus using the coarse adjustment knob.
- You may make rapid adjustments with the coarse knob at low power magnification. It will be nearly to the top, before it comes into view. In fact, you can go all the way to the top then start going backwards instead. The height of the 4x objective is short and is not in danger of being damaged.
- You know you are in focusing on the correct plane if moving the stage moves the specimen in your view. Try using the slide movers back and forth while observing through the ocular. You should see your specimen also move. If nothing is moving, you are probably focusing on the condenser or other microscope structure.
Now use the fine adjustment knob to make the image crisp and clear.
- You may need to adjust the ocular lenses to your individual comfort.
  - Adjust the distance / width of the ocular lenses such that a single (more or less) circle is visible. Your eyes should be roughly an inch or two away from the lens.
  - If the image is still unclear after your initial focusing, it's probably because the the separate eyes are at different focusing levels, or your eyes are are not quite the same.
- Pro-tip: To focus using both eyes, (it's ok to use one, but not quite as professional).
  - Focus using only one eye.
  - Once in focus, open the other eye.
  - Adjust the ocular lens of the second eye. It should rotate and alter the focus a little.
  - Adjust until both views are clear.
  - Sometimes, only one of the ocular lenses is adjustable, this then has to be your second eye. You should only need to do this once. However, because everyone's eyes are different, you will likely need to do this each time someone new uses the microscope.
- Other Notes:
  - There is typically a pointer in one of the ocular lenses. Use this to help indicate / point out structures when explaining or showing to other people. The pointer will be used later in demos or for testing purposes, so get used to looking for it.
  - Sometime your eyelashes will reflect back at you. There isn’t much you can do about this. However, it is due to the light shining so brightly on your face that the light travels back toward the sample casting a shadow on the image. Try moving closer or farther or altering the light intensity and you can sometime alleviate it.
Move the stage knobs to center your specimen.
- When you move up in magnification, the area you can view is smaller (though looks just as big). This means that objects on the periphery (edge) will no longer be visible.

Switch to the next higher magnification (if desired / indicated) by rotating the nosepiece so that the next objective lens (10x objective) is in place. It will click into place.
Use only the fine focus only to make any adjustments
- The lens should be very closely in focus (parafocal) and further focusing adjustments are typically minimal. Unfortunately, as this is a teaching lab with many novices using the equipment, some of the microscopes have been damaged / moved out of alignment and so the new view might not be as already focused as might be desirable. This is particularly true of the oil immersion lens.

Adjust the dimmer as necessary.
Repeat steps 7-10 using higher magnifications (40x objective).
If appropriate proceed to oil immersion lens (procedure as follows).

You will not do the oil immersion lens in this lab.

Microscope Care

Proper microscope is important to maintain the instrument in good working condition and can preserve its optimal use. Each lab will have its own maintenance routines. Often they are left in an isolated area to minimize wear and tear. Like a car, unnecessary handling will result in friction of mechanical elements and decrease the overall lifespan of the instrument. However, in labs with limited working space, sometimes instruments must be stored. Some precautions are universal and include:

Wipe down the oil immersion lens with lens paper and lens cleaning solution. NOT kimwipes! and any other part that might be soiled. Oil will start to harden and will distort the view in the future. It is also much harder to remove in the future.
Lower the stage - This prevents the objective lens from hitting the stage / slide.
Leave the 4x objective down - Similarly, this precaution, prevents accidental collision between the stage / slide and the objective lenses as the 4x is the shortest lens.
Turn the microscope off.
Turn the dimmer to the lowest setting. - This is mostly an residual artefact of older light bulbs. Suddenly turning on / off high powered bulbs at higher settings would often cause them to blow out. It's still a good idea to do though, as it will extend the life of the bulb. It is also a matter of consideration, because you are likely to have increased the intensity as you increased the magnification. However, when you start looking at the scanning objective, it will now be too bright.
set the iris to 4x - not strictly necessary, but this is to be considerate to the next operator, as this is the starting point for focusing
Center the stage so that the clips aren't hanging to far out - this helps prevent snagging the clips on anything, similar to when your headphone cords snag on outcropping door handles.

Others are specific to the particular lab and include:

Swivel the head backward
Coil up the power cord.
Put on a dust cover

Introduction

A video on microscopy with nice production quality.

Link

Magnification vs resolution —

Two elements of microscopy that can be at odds with each other. Resolution is the ability to see fine detail, or more formally, the ability to distinguish between two discreet points. Every microscope has a limit for resolution. Magnification, on the other hand, is the enlargement of the object’s image.