Do you love science?
If the answer is yes, then you need a microscope. Why, you ask? Because you can have a glimpse of the world otherwise unseen. And also you can attach a Raspberry Pi and an Ipad (or any other sort of screen).
In the early days of microscopy, the microscope wasn't used as a scientific instrument very much. It was used as a toy for rich grown-ups, to be used to amaze friends at parties.
If you look into a microscope without our understanding of life, you will not see what we see today. You will not see cells or bacteria; you will see a strange unknown world.
Seeing is understanding but you will notice that since the reality depends on its obervation, it gets more imprecise the closer you look. (adapted from Heisenberg)
1. We will take an old microscope and polish it up. I'll try to address the most common problems handling worn microscopes. Afterwards, you can use it as an ordinary microscope if you already have a light source.
2. We'll build a light source for the microscope. Now it's a real microscope. With light.
3. We'll attach a camera using a cheap adapter to make stunning microphotographies.
And with this, you'll be able to soon follow my next instructable in which we'll: (a) create an attachment for a Raspberry Pi camera and prepare the Raspberry Pi. Now you can use the Raspberry Pi to make pictures. (b) Notice how microscopes have a shallow depth of field? We can remedy this by "stacking" images on top of each other using a Python script.
I took an old German ROW microscope, because it's quite cheap yet has all the function you normally desire but you may use any other microscope as well. I'll have some tips for you which microscopes work well.
Step 1: Choosing a Microscope
If you're choosing a microscope, look for an older model, but not too old. Say a good 60s/70s model will be plenty good. The model we're using is a little older, but I wouldn't go much older than this. Zeiss, Leitz, Wild, Nikon, Olympus these are all brands you can trust. (In this order.) Feel free to shop for Eastern European / Russian models like ROW, Biolam, or Lomo as well. In the UK, you may also consider Brunel.
(The queen of all microscopes if of course the Zeiss Standard. If you have a Zeiss Standard you can buy adaptors for almost anything you can think of. It's the ultimate modular microscope and it will last a century.)
- We are using a monocular microscope, but you can alternatively get a binocular (two eyepieces) or a trinocular (two eyepieces plus an extra one for a camera). Stereo microscopes are something different altogether. By all means get one. However, this tutorial is primarily for the first kind.
- Get a microscope that has at least a condensor lens that has changeable position. You'll need to adapt your illumination if you want to make nice pictures.
- Don't look for very high magnification factors. I find the most interesting pictures don't require a magnification factor of much more than 100x. Also, high magnification factors often require oil immersion (lens is then marked 'oil', like our lens here. It's messy, you need to buy oil. If you're not explicitly interested in things within cells, it's not really worth it. Get another medium sized lens instead.
- My recommendation: 4X, 10X, 25X and 40X objectives — and either 5X or 10X widefield eyepieces
- if you want more, Stollenberg recommends: 10x/NA 0.25 or 0.3; 20x/NA 0.45; 40x/NA 0.65; 100x/NA 1.25 or 1.3 and
However, for our purposes, almost any microscope will do.
Step 2: Anatomy of a Microscope
Anatomy of a Microscope
This step is about the different parts of a microscope. (If you already know or want to get your hands dirty, save this bit for later.)
The type of microscope we'll use is an optical compound microscope designed for viewing samples by 'transmitted light'. That means that light passes through the sample instead of being reflected from it.
A compound microscope uses a combination of an objective lens and en eyepiece. The objective lens focuses a real image of the object inside the tube of the microscope which is then magnified by the eyepiece to give an enlarged image of the object. This image appears inverted. By using a combination of two optics, higher magnification can be achieved and objectives can be exchanged so that several different magnification factors are available in a single device.
Magnification factors of objective lenses range from 3x to 100x, that of eyepieces from about 5x to about 15x. The total magnification thus ranges from about 15x to 1500x. However, you'll rarely use magnification factors that are very high, because at high magnification, the image is not very sharp and has little contrast.
Objectives also have a parameter called numerical aperture (abbreviated as NA) engraved on them. Similar to the aperture in photography lenses, this is the parameter determining the resolving power of that objective and how much light is lets pass through it. It usually ranges from 0.1 for a 3x objective to 1.25 or even 1.4 for a 100x objective.
Low magnification objectives such as 8x, 10x or 15x are used for rough surveys, e.g. in botany, or entomology. Standard biological sets usually include 10x, 40x and 100x objectives, an additional 20x objective will often prove valuable.
Sterrenburg's primer on microscopy recommends a good routine set giving manageable magnification steps of about 2x would as follows: 10x/NA 0.25 or 0.3; 20x/NA 0.45; 40x/NA 0.65; 100x/NA 1.25 or 1.3
The majority of microscopes consists of the same structural components.
- Eyepiece (ocular lens)
- Objective turret, revolver, or revolving nose piece (to hold multiple objective lenses)
- Objective lenses
- Focus knobs (to move the stage)
- A: Coarse adjustment
- B: Fine adjustment
- A: Coarse adjustment
Our microscope has two different interchangeable condensers; one traditional and a dark field condenser. The function of the condenser is to fill the objective evenly with a cone of light. This cone of light is narrow for an objective of low aperture but needs to be very wide for one of high aperture. The condenser allow you to adjust the code width by adjusting the distance between the condenser and your sample. Parts of the objective that do not receive light cannot fully participate in image formation!
Without a condenser, the light coming from our illumination source would not distribute evenly, and may cast the image of the bulb filament onto our sample. It will also introduce artefacts such as glare and shadowing in the image. While there are other methods of diffusing the light such as opal glass diffusers also diffuse the filament image, these all reduce the intensity of the illumination. Our proper "Köhler illumination" however achieves even distribution by ensuring that the image of the light source is perfectly defocused at the sample plane.
One big disadvantage of optical transmission microscopy is the low contrast: you need to use very thin slices which more often than not let most of the light pass through. Thus, what you get is a bright image with very little contrast and it can sometimes be quite hard to make out some of the distinct features you are looking for. This is why people have come up with different methods to enhance the contrast of microscopic images. While staining (ie. adding dye and then washing the sample) is the easiest and most common, a few methods achieve remarkable results without changing your specimen, solely by manipulating light.
Darkfield condensers block part of the light that is coming directly from the light source before it hits the sample, but let an outer ring of illumination through through that illuminates the sample from the side. (Similar to how in a room with the curtains almost drawn during a sunny day except for a narrow slit, tiny dust particles become visible in the beam of sunlight.) Only the scattered light goes on to produce the image, while the directly transmitted light is blocked.
This technique is particularly suited for biological samples that are very transparent such as single cell organisms or other water borne organelles. While under normal light they might be barely visible fair objects on a bright white background, they appear as bright objects on a black background when darkfield microscopy is used.
While this microscope does have a darkfield condensor, we won't be using it this time. Soon to come though. In the meantime, get acquainted with the hardware at hand.
Now, let's get our hands dirty...
Book pictured is: "Das Mikroskop" by Dr. Ludwig Otto, 1952
"Microscopy Primer" by Frithjof A. S. Sterrenburg http://www.microscopy-uk.org.uk/index.html?http://...
Step 3: Prepare Disassembly and Cleaning
Cleaning a microscope is not trivial. You have to pick and choose which parts you want to disassemble and which you maybe just leave alone. Most screws are non-standard, lost screws might turn your valuable piece of historical, scientific equipment into a very nice doorstopper.
My rationale of cleaning was that I didn't want to overdo it. An older microscope is a piece of history that you want to preserve and make functional at the same time. Scratches and patina are signs of use and tell the story of the artifact.
- Something to de-greasethe microscope. I'm using
- Petroleum ether (aka gasoline, benzine).
- Mild dishwashing detergent
- thick bleach (this is optional - if will more likely than not dull your paint and might damage some material, but it does help with the grease)
- Don't use Acetone or other more aggressive solvents, though, it might kill your enamel.
- I'll be using WD-40 for now, but it's definitely not the best option: Microscope grease isn't only there for lubrication and to protect the surface. It's usually medium to high viscosity also provides the haptic feel of fine control. If you use WD-40, like me, your knobs will turn a lot more easily than desired. (Wild Microscopes use up to 30 different lubricants on a single scope!) Also, automotive grease will succumb to gravity and creep out of the crevasses.
- Suitable dampening grease: Micro-Tools Europe (Germany) and Micro-Tools USA sell small kits. Their hobby kit is moderately priced and contains high and low viscosity lubricants more than suitable for the job. (Nyogel 795A is a favorite among enthusiasts.
- Screwdrivers - See if you can find some that make a perfect fit with your screws. As these are hard to replace, you want to avoid cam-out at all costs. Get angled screwdrivers if you find screws that are hard to access.
- Face spanner (or spanner wrench)- this is not an essential tool if you don't want to touch the parts of the microscope that aren't usually disassembled by the user, but it you want to go all the way you need a set of those. Make sure that you get a set that includes very small sizes as most of them are used by car mechanics and machinists. I'm not using any for this tutorial, but I sure as hell decided to get one after making it.
- Polishing wheel - it's not necessary but useful and saves some time on the few placed you can reach with a polishing wheel. Do not use a metal brush wheel, it will scratch the metal.
- Toothbrush - for cleaning in between the teeth. (Of the microscope's gears of course.)
- Steam cleaner - this is not essential. You can use more chemical agents and lots of warm (!) water instead, but for me, this is the optimal tool for the job.
- Compressed air - this if quite important if you don't fully disassemble your microscope. I'm using air from a compressor, but a can of compressed air should do it just as well. If you use a can, buy two or three.
- Caliper (optional) - you can use it when making pictures as a reference to scale which will help if you lose or break a part. Often, screws differ only by a millimeter or so.
- Nitrile gloves - use different gloves if you want, but there's a lot of grease and nasty chemicals involved so get some protection either way.
- Aspirator brush - optional but helpful in removing dust from the lenses
Before you start:
One important thing: Always be knolling. If you keep your work surface organized, your brain will do a better job of keeping track and you're less likely to mess up.
Get a camera to make a picture every step of the way.
Disassemble all parts that move freely and/or have glass parts. Put them away, we'll start with cleaning the frame of the microscope. Note that our microscope has a single tube (monocular) without any internal glass parts. If you have a binocular tube, you will find that there are prisms inside the tube. You do NOT want to disassemble those.
You'll want to start by applying copious amounts of WD-40 to all the spaces that have grease already on them. This should loosen the old stuff and with a little wiggling, you're OK to go. If things are still stuck, apply heat. Slowly. Let it sit for a day to increase the effect.
"Restoring a stereo microscope base with reverse electrolysis". by Ian MacGregor, Burnaby, BC, Canada
"[Notes on refurbishing and the use of Nye special lubricants]". by Ian Walker. UK.
Step 4: Disassemble the Microscope
I assume you have taken all optical parts and put them aside. (Aside means out of harms way - grease!)
Also, take out all parts that slide in (slide holders) or have threads themselves. Then, focusing on one part at a time, take out the screws and put them aside. A magnetic tray helps.
You begin by removing the slide table, and then you'll remove the assembly holding the optics and then the assembly holding the condensor.
The assembly containing the eyepiece and objective turret consists of two sliders, a coarse focus slider and a fine focus slider. They may slide out if you move them far enough in the correct direction. Sometimes you'll have to detach another part first. In this example, the slider that holds the optics must be removed before you can access the stage. Then the stage needs to be removed before you can slide out the assembly that holds the condensor assembly.
Note that I did not further disassemble the condenser assembly. I chose not to do so to minimize the danger of breaking the iris, and because it was working just fine, but feel free to do so. Do not disassemble the condenser itself though and be careful cleaning it.
- If you DO break a screw, take measurements with an accurate caliper. Most microscopes use the old DIN norm (DIN 13) which features screws that have names like M1- M1,2 - M1,4 - M1,7 - M2. Use google to find tables and references, then look online for a right model. Often, regulars is microscopy forums will be happy to help. (Include pictures and measurements with scale reference.)
- If things are stuck beyond removal, don't use excessive force. Use alternately heat (steam) and chemical agents and your patience and determination will be rewarded. High quality microscopes are usually made from brass or other soft metals. They easily break when using excessive force.
- If your frame is heavily oxidized, don't use rust remover as it might also damage alloys or the enamel. Use reverse electrolysis. Disassemble the oxidized parts as far as possible and remove all grease. Prepare a solution of 5% sodium carbonate (aka washing soda) in distilled water. Take a 12V battery charger as your power source and a sacrificial anode (carbon rods are ideal, but copper pipe will do fine but DON't use anything with chromium in it). Attach the frame to the negative terminal of the charger and the sacrificial anode to the positive terminal and submerge them in the solution. Take care that anode and cathode don't touch each other and leave them sit for a couple of hours.
Step 5: Cleaning the Frame
This step is simple. But it is dirty so put your camera away.
One by one, clean all parts with soap and steam, and use compressed air to dry it. This way, no residue will remain and the microscope will not rust.
Then, use car polish on the pain to buff it up a little.
Step 6: Reassemble and Enjoy Your Results...
Reassemble in reverse order.
Enjoy your shiny microscope.
Step 7: Cleaning the Lenses
Utmost care is warranted when cleaning the lenses. Be super careful, lenses are coated and scratch easily. Scratches in lenses will be visible in your image as areas that appear out of focus. On the other hand, scratches and dust in the eyepiece can be much more menacing because they will be visible as a sharp image overlayed onto your image in the microscope.
There are several methods of cleaning lenses that are better than mine, use at your own discretion.
- Distilled water
- Isopropyl alcohol (IPA) (95%)
- lens cleaning paper
- q-tips (for heavy dirt)
- a brush the uses air (well, I didn't end up using it because nothing beats compressed air at 6bar)
Cleaning an objective lens
- Before you touch the lens even with lens paper, try to clean it as best as you can first with compressed air, then with a lens brush, then with lens paper. Any dust particles under your tissue might scratch the lens.
- Then use lens paper. Lens paper is thin paper that you put on the lens when dry and then apply a drop of water (or IPA) to. Gently move around, then pull it off to remove any remaining dust particle.
- With regard to the front lens, stop here. It's clean now. If it doesn't appear clear, this is likely scratches. If unsure, maybe try steam? But don't blame me., I don't know how lenses like steam.
- You can see me cleaning the inaccessible inside of a lens with a Q-Tip. It's not the best of ideas, and most of the time (see later image) you can even disassemble the lenses into two parts
- The most important part is using a lot of IPA and drying it quickly with compressed air before (!) it has time to dry even a little bit. This process can be repeated until all the dirt and grease is completely gone.
- For eyepieces, I tend to use the q-tips, because eyepieces are usually much dirtier than objective lenses, especially on the side facing the eye.
- If there is dirt inside your eyepiece, you may want to disassemble it further. Yes, you'll maybe break your eyepiece, but face it: it's broken already. Dirt inside your eyepiece will be quite visible in the resulting image. If it's too bad, disassemble and buy a new one if you break it in the process. Eyepieces are quite cheap and most are standard size. The best eyepieces are 'widefield' eyepieces for use with glasses.
Step 8: Creating a Suitable Light Source With Plywood.
We'll use a flashlight for which we build a small stage that aligns the center of the flashlight with the center of the reflective mirror.
What you'll need:
- scroll saw (or similar tool)
- cordless drill
- T10 torx drill bit (or equivalent)
- drill 3mm
- drill 2mm
- countersunk bit
- carpenter pen (optional)
- pen & paper
- some plywood (9mm)
- screws (T10, 3,5 x 16 mm)
- spray adhesive (the non-permanent kind)
- General tips on working with plywood: (I thank Tom Sachs for the lessons)
- Paint plywood before sanding and cutting. This showcases plywoods beautiful endgrain and prevents dripmarks from contaminating our scientific equipment.
- Use Torx screws to avoid cam-out, or use the philips head driver appropriate for your screw. (Cam-out is when your screwdriver skips out of a screw head.) Your shoulder, hand, bit, and screw should be aligned with the center of the earth, to avoid cam-out.
- We measure twice and cut once. We measure twice and cut once.
- When measuring consider the width of your marking tool.
- We cut to one side of our mark. Never down the middle.
- The scrollsaw removes about 1/3mm of material plus error. Account for this when making multiple cuts into the same sheet.
- After cutting, we phase the edge with sandpaper, to avoid injury.
- When screwing through plywood, we clear-drill AND countersink, to ease assembly.
First, we measure the distance between the table (underside of microscope) and the center of where the mirror is placed. As you can see, this is about 33 mm in our example.
Second, we measure the diameter of the flashlight at the points where we want to support it by our stage (about 30 mm here).
Consequently, we measure how far these two points are separated (80mm).
Finally, we draw the shapes of the parts we need. We carefully measure each dimension and use a compass to draw the crevasses for the flashlight. Be careful to consider the width of your material.
Once you're confident in your drawing, cut them out to use as patterns with some outer margin around the edges and apply some spray adhesive to the back. Let the adhesive dry and put the pattern onto the sheet of plywood.
Now you can easily cut the shapes from the plywood. Test fit them together, and sand the edges to prevent injury. Drill 3mm holes into the sides A and C, to ease assembly. Drill through and countersink. Now mark the locations of the holes on the side of piece C using the carpenter's pen. Then use the 2mm drill to drill holes into the side of piece C. (This is optional, but will guarantee that the plywood doesn't break from inserting the screws.)
One you're happy with the pieces, you can assemble them using the screws.
Test it and be happy.
- Building a proper 3W LED illumination: http://www.microscopy-uk.org.uk/mag/indexmag.html?...
- On lightguides for illumination from the top "A Fibre Optic Vertical Illuminator for Stereo Microscopes" by Ian MacGregor: http://www.microscopy-uk.org.uk/mag/indexmag.html?...
- On infrared light: "Simple experiments with video image capture using a near infra-red light source" by Dave Walker http://www.microscopy-uk.org.uk/mag/indexmag.html?...
- Tom Sachs on working with plywood: https://vimeo.com/44947985
Step 9: Adding a Camera Adapter
Of course you can just get a digital eyepiece off EBay, but where's the fun in that? Most of them are TWAIN compliant and will just run on most computers. Of course they lack the software. You can use Micam 2.0 which was just released to control settings of your webcam (like exposure etc.).
I decided to go with this nifty camera adapter for an old Nikon D70 DSLR. It is not the best for the purpose because it can't control the mirror. You want the mirror to be permanently up before you make a photo You can get these kinds of adapters for almost any DSLR on Ebay.
They use your eyepiece as an objective, so all you need to do is pick your favorite and assemble the adapter as shown in the pictures.
If you liked this instructable, let me know. In my next instructable, I will build a camera from a RaspberryPi camera module that can automatically combine several images (stacking) to improve the depth of field and overall sharpness of the pictures.
Find reviews of Ebay China eyepiece cameras here:
Step 10: Enjoy Your New Microscope.
Now you can go ahead and explore the world around you. It's so much bigger now. Can you feel how it expanded??
In my next instructable I will explore ways in which you can further modify and improve your new microscope. Let me know what you're most interested in.
- We'll be walking through some useful skills to have when preparing samples.
- We'll be adding an automated camera to achieve high depth of field using stacking of images made at different focal planes.
- We'll be adding dark field and polarized light microscopy to see even more things.
Thanks for your attention and have fun!!!