Some basics first

There are several options for connecting a camera to a microscope. The camera system can either be connected via a dedicated photo tube on a trinocular head, or can be connected to one of the microscope’s eyepieces. Depending on the set-up, there can be either intermediate optics between the camera and the microscope’s objective, or not.

• The image produced by the microscope objective can be directly picked up by the sensor of a camera, without an eyepiece or other intermediate optics. Here objective of a microscope produces a real image directly on the camera’s sensor. The objective produces a relatively large image, compared to the small sensor of many cameras. Unless the sensor is large, there may be quite much empty magnification and the brightness of the image is low.
• The image produced by the microscope objective can also be passed through a reduction lens before reaching the camera sensor. This way the image produced by the microscope objective is reduced in size to better match the small sensor size of the digital camera. The reduction lens produces a real image on the camera sensor. Without the reduction lens the image would be magnified too much. The reduction lens also results in a brighter image. This is an improvement to the first point from above. Eyepiece cameras for microscopes use this system. The reduction lens is not a compensating photo eyepiece and therefore does not correct lens errors produced by the objectives.
• The image produced by the microscope objective is first passed through a regular eyepiece. A virtual image is produced this way, which can not be used to directly make a picture. A camera (with its own objective) then picks up the virtual image and projects it on the sensor. The camera works like the eye, which converts a virtual image to a real image. This system is used in afocal photography, in which a regular compact camera (with its own objective and all) is attached in front of the eyepiece.
• The image produced by the microscope objective is passed through a photo projection ocular (photo eyepiece), which then projects a real image on the sensor of an SLR camera. There are no camera objectives involved. The projection eyepiece corrects optical errors which are produced by the microscope objective. These photo projection eyepieces are compensating optical elements. This means that they are designed to correct various lens errors that the objectives produce, including field curvature and chromatic aberration. These projection oculars are therefore manufacturer dependent and must correspond to the objectives of the manufacturer. Besides image quality, another advantage is, that parfocality is maintained between the camera and the eyepieces (i.e. both images are in focus at the same, and there is no focus deviation).

Let’s now have a look at a few real life applications:

Connecting a webcam (home-made solution)

This was one of my earlier attempts of connecting a camera to a microscope. I completely dismounted a webcam and removed all of the optics. Leaving the webcam optics in place (used in afocal photography) would result in a too small image because most webcams have wide-angle optics. I then placed the electronics with the attached sensor into a separate plastic box and attached a short metal tube to the box for easy placement on the trinocular head. There were no intermediate optics involved and the image was directly projected from the microscope’s objective. Make sure that the blue filter is still in pace in front of the sensor (they are quite red sensitive), otherwise you have to use a blue “daylight” filter (for photography) on top of the halogen lamp of the microscope.

DIY webcam mounted without intermediate optics to the microscope.

The webcam sensor.

The electronics are held in place with some foam material.

The sensor is mounted directly to the main board.

Advantages:

• This was a low-cost solution and worked reasonably well. The plastic case was not expensive at all and the short metal tube I obtained from an old discarded telescope. I was lucky that the diameter was just right.
• The webcam produces a live image on the computer screen, which can be easily focused.
• The webcam was released through the computer and therefore there was no camera shake, resulting a steadier image.

Disadvantages:

• The resolution of the camera was quite low (640×480 pixels), but this can be resolved by using a different webcam.
• The far bigger disadvantage was, that the objective generated a relatively large image and the sensor of the camera was quite small. For this reason there was much empty magnification. Intermediate optics (reduction lenses) would have reduced the size of the image and would therefore also have made a brighter and sharper picture. These optics were not available to me, however.
• Some lack of parfocality can also be a problem. The focus of the camera and of the eyepieces are not the same. The objectives produced an image 10mm down in the phototube. This place was of course not accessible by the webcam. If the deviation is too large then this can become a problem for the objective to slide distance during focusing.
• Webcams often have a blue filter covering the sensor to reduce the effect of infra red light, which gives the whole image a reddish hue. If you remove this filter with the optics, then you need to add a blue filter over the microscope illumination or into the condenser

Connecting an analog video camera (home-made solution)

The principle here was the same as in the webcam solution. I ordered a color surveillance camera module, without case, only the electronics, and mounted this into an aluminum case, which a friend of mine made for me using the appropriate tools. I connected the camera both to a VCR recorder and a TV. Advantages and disadvantages were quite similar to the webcam solution.

A surveilance camera in a case made of aluminum. No intermediate optics.

The camera body is made of one piece.

The camera sensor can be seen.

The main board (with sensor) is held in place by teflon rings (white ring inside).

Advantages

• Fast video display on a TV is possible. This makes it suitable for use in classrooms that have TV screens. There is no need to convey the signal over a computer.
• The speed is in real time and faster than when using slower webcams.

Disadvantages

• Unlike webcams, a separate power supply is necessary.
• Analog video is now somewhat outdated and the resolution is lower than with modern webcams.
• The surveillance camera must be compatible with the TV system (NTSC, PAL or SECAM).
• Capturing still images on a computer (or simply viewing the images on a computer) requires an analog video input, which not every computer has.

Connecting an SLR

“Big name” microscope manufactuerers all have dedicated imaging solutions for their microscopy systems. This one was the most expensive solution, but also the one giving the best results. The image from the microscope’s objective is further processed by a special photo-projection ocular, which then projects it directly on the sensor of an SLR (single-lens-reflex) camera. The projection eyepiece corrects all remaining lens errors from the objectives. I have worked a lot with this system, and the resolution of the pictures is very high. I now have an 18 megapixel SLR camera and this resolution is much higher than the resolution produced by the microscope and specimen.

The photo eyepiece adapter is mounted on the phototube of the trinocular head and holds the photo ocular.

The Canon EOS 600D has a swing-out LCD screen. This makes it possible to focus while sitting.

Advantages:

• The best image quality is produced. The projection ocular corrects chromatic aberration and also generates a flat field of view (so that the sides of the image are not out of focus).
• SLRs have a high dynamic range (both bright and dark areas can be captured at the same time, without much information loss).
• Some SLRs allow for the capturing of RAW images. These are able to capture an even higher color depth for a high dynamic range.
• SLRs have a high resolution, often much higher than dedicated microscope cameras (for the same cost).
• The sensor of SLRs is quite large, therefore the signal to noise ratio is high. This makes it suitable for low-light photography or high ISO photography and very short exposure times.
• It is possible to couple the SLR with a flash system for the microscope, for microflashing. I never tried this, though.
• The SLR can also be controlled from the computer for automatic photography (called tethered shooing).
• Some modern digital SLRs also allow for video recording. This is a major advantage, if needed.

Disadvantages:

• Generally a high cost of both SLR camera and associated adaptor tubes, projection oculars, etc. The projection eyepiece and phototube are manufacturer specific and can be relatively expensive, especially when the components are not manufactured anymore. I read somewhere, that there are also non-manufacturer specific adapter tubes available for SLR cameras, but one has to do some research for this. For best results, the projection ocular must match the objectives and sensor size of the camera.
• A dedicated trinocular head with phototube is necessary. It is not possible to connect the SLR and phototube in front of an eyepiece tube (which extends 45 degrees horizontally), due to its weight.
• Camera shake during the release can be much higher than with other cameras and can blur the image a bit, but there are solutions for this (long exposure time, using mirror lock up).
• Live-view on the computer screen is not always possible, this depends on the SLR.
• The photo projection ocular should be from the same manufacturer as the microscope objectives, otherwise lens errors are not properly corrected.
• My system uses a photo projection ocular which was designed for analog 36mm film cameras. My current digital camera has a smaller sensor size and therefore there is extra magnification and the field of view is not as wide. Stitching images together with panorama software can be necessary. The field of view is much wider than in the webcam and analog video solution (from above), however. Different projection oculars for smaller sensors do exist, they are not manufactured anymore, and are thus relatively expensive.
• SLRs can not be used for focal photography, the lens diameter is too large.
• Not every microscope manufacturer may offer SLR adapter tubes and corresponding compensating photo eyepieces and it may be necessary to use manufacturer independent products. These may not deliver the highest image quality, however.

Connecting compact camera (afocal photography)

Fortunately it is possible to make good quality pictures by using regular digital compact cameras as well. This solution is more cost effective. The front lens of the compact camera must be sufficiency small, which is usually the case. The camera is mounted on a tripod and the picture is taken through the eyepiece of the microscope. It is necessary to zoom in and it is also necessary to adjust the camera-eyepiece distance properly. There are some adapters available, which allow one to clamp a compact camera to the base of the eyepiece, without any modifications to the camera. These clamps use the tripod connector of the camera.

The triplod holding the camera can either be placed on the table or on the floor.

Proper centering and correct camera to eyepiece distance is important.

Advantages

• No trinocular head needed and one can use a camera that one already has.
• It is also possible to take pictures freehand, without tripod, if there is sufficient light for short exposure times (but this is a bit difficult sometimes).
• Due to the high resolution of compact cameras, much image information can be captured.
• It is also possible to zoom in and out.
• Many modern compact cameras also allow for the recording of (HD) video clips!

Disadvantages

• There is the possibility of vignetting if the system is not properly set up. For proper photography one also needs to use a tripod, which takes space.
• The theoretically best image quality can not be achieved (compared to connecting an SLR with projection eyepiece), but often the quality loss is irrelevant. The reason for this is because there are simply more lens elements in the light path (camera objective) and because the regular eyepieces are not designed for photography.
• The suitability of the compact camera for microscopy has to be tested first, not all compact cameras may work equally well (depends on diameter of camera objective). If the camera objective is larger than the exit pupil of the eyepiece, then there is the possibility of vignetting (solution: zoom in more).
• Field curvature may possibly be a problem. In this case the side of the image is out of focus, while the center is in focus. This effect can be reduced by zooming in.
• The set up with tripod is not very convenient. It is possible to make an adapter tube which is connected directly to the camera. This tube then can be inserted into the microscope like an eyepeice. It is possible to use compact cameras that have a filter thread and connect the adapter tube to this thread.

Using a mobile phone camera

Yes, this too works! The objective lens of the mobile phone is smaller than the image produced by the eyepiece, so this works. The camera focuses to infinity.

Shaky, a bit improvised, but possible. The correct eyepiece to camera distance is critical.

Advantages

• Fast, simple, no extra cost and no microscope adaptations are necessary.
• There is a microscopy iPhone app which calculates magnification, it is worth checking out.
• The pictures can be directly sent from the phone, if this is needed.

Disadvantages:

• Very steady hand needed to hold the camera at the correct distance from the eyepiece. A wrong camera to eyepiece distance will result in vignetting.
• The camera is usually wide-angle and covers the complete field of view. The image is in a circle. For this reason pixels are wasted.
• The solution is somewhat improvised (having to hold the mobile phone by hand) and I have not used it on a regular basis.

Connecting a dedicated microscope camera

This is my favorite solution, because of its high convenience. It is possible to do microscopic observations without looking through the eyepiece, only the computer monitor. This can be more relaxing at times. These cameras are connected directly to the computer and are controlled entirely over the computer. Full manual control is possible with the camera that I have. This is absolutely essential (especially when one wants to make panoramic images). There are reduction lenses in front of the camera, so the brightness of the image and field of view are increased (compared the the DIY webcam solution from before). And for those of you who do not have a trinocular head: these cameras can be mounted instead of an eyepiece!

The camera has a reduction lens and can be mounted on the phototube.

The camera can also be attached in place of an eyepiece.

The camera can also be attached in place of an eyepiece - another view.

Advantages

• Most convenient, relatively small (compared to connecting an SLR).
• The image sensor (in combination with the reduction lens) also covers a wide field of view but does not make the image appear in a circle (like when using mobile phone cameras).
• The camera can stay on the microscope and I do not have to detach it like I had to for my SLR.
• There is no shutter release shake and all of the pictures are stored on the computer’s hard disk. It is therefore possible to take many images for time-lapse photography, without having to worry about the memory card becoming full.
• No trinocular head needed! These cameras are light enough to be used instead of an eyepiece.
• These cameras often have a C-mount or CS-mount to which the reduction lens is connected. It is therefore possible to exchange the reduction lens to obtain a different magnification. Unfortunately, these reduction lenses are difficult to obtain alone, without camera.

Disadvantages

• Compared to the camera resolution, these systems are quite expensive (but not unaffordable). I paid over $260 (EUR 200) for a 3.1 megapixel microscope camera with reduction lens. For my uses, a higher resolution is not needed, however.
• It is also necessary to have a computer around and running for taking pictures, as the camera is not able to store images.
• Another disadvantage is that the USB cable connection is too slow to allow for high-quality video recording. Movements are not recorded smoothly. Using a lower resolution may resolve the problem.
• The reduction lens can not be compared to a compensating photo projection eyepiece, which corrects lens errors (and is manufacturer specific).

Other possibilities, which I did not try

• Using a camera system which was specially designed for the microscope. Bigger microscope companies offer these solutions. The camera has a mount which fits only to the microscopes of a particular manufacturer. Often the optics of the camera are also match the optics of the remaining microscope (compensating lens errors etc.). Sometimes these cameras also have other features that are not commonly found on digital cameras, such as stand-alone operation (no computer connection needed), LAN connectivity, or even a separate camera control panel. I would guess that these cameras are interesting for research institutions, who want to have a solution, which works out of the box.
• Removing the objective of a compact camera and attaching a custom-made adapter (with or without reduction lens) to the body of the camera. These adapters may need to be designed specifically for the camera. You may be interested in this link: Making Digital Camera Microscope Adapters
• Attaching a custom adapter to a compact camera for easy afocal photography. The camera’s objective stays on the camera. The adapter goes over the camera’s objective and can be connected to the eyepiece without the use of a tripod.
• Using a clamp to hold the camera for afocal photography. I have found one German shop specializing in amateur astronomy (!!) selling these holders for microscopes and telescopes. The holder is clamped to the tube of the microscope and also holds a compact camera. Check the following links to see what I mean (I am not affiliated with this shop):
  Link 1 | Link 2 | Link 3 | Amazon also has them

Do you have any further suggestions on how to connect a camera? USe the comments section below to share your thoughts!

Counting chamber: This one is called the Neubauer improved. There are other standards with different grids available as well.

Yeast cells in the hemocytometer. The grid is clearly visible.

Yeast cell suspension applied to the chamber. Notice that some of the cell suspension has gone into the overflow area.

One counting chambers has grids of different sizes. Consult the manual to find out the size.

Do not count cells on the top and right lines. Here it's necessary to count the in the big square because there are too few cells in individual small squares.

Counting chamber seen from the side.

Grid layout of the Neubauer Improved hemocytometer.

Purpose of the hemocytometer

The hemocytometer (or haemocytometer or counting chamber) is a specimen slide which is used to determine the concentration of cells in a liquid sample. It is frequently used to determine the concentration of blood cells (hence the name “hemo-”) but also the concentration of sperm cells in a sample. The cover glass, which is placed on the sample, does not simply float on the liquid, but is held in place at a specified height (usually 0.1mm). Additionally, a grid is etched into the glass of the hemocytometer. This grid, an arrangement of squares of different sizes, allows for an easy counting of cells. This way it is possible to determine the number of cells in a specified volume.

Preparing the sample

The fluid containing the cells must be appropriately prepared before applying it to the hemocytometer.

• Proper mixing: The fluid should be a homogenous suspension. Cells that stick together in clumps are difficult to count and they are not evenly distributed.
• Appropriate concentration: The concentration of the cells should neither be too high or too low. If the concentration is too high, then the cells overlap and are difficult to count. A low concentration of only a few cells per square results in a higher statistical error and it is then necessary to count more squares (which takes time). Suspensions that have a too high concentration should be diluted 1:10, 1:100 and 1:1000. A 1:10 dilution can be made by taking 1 part of the sample and mixing it with 9 parts water (or better saline of correct concentration to prevent bursting of the cells). The dilution must later be considered when calculating the final concentration.

Counting the cells

• Counting cells that are on a line: Cells that are on the line of a grid require special attention. Cells that touch the top and right lines of a square should not be counted, cells on the bottom and left side should be counted.
• Number of squares to count: The lower the concentration, the more squares should be counted. Otherwise one introduces statistical errors. How many squares? To find out one could calculate the cell concentration per ml based on the numbers obtained from 2 different squares. If the final result is very different, then this can be an indication of sampling error.

Calculating the cell density

Here it is necessary to do some simple math. The following numbers are needed: number of cells counted in a square, area of the square, height of the sample, dilution factor. The objective is to find the number of cells in 1ml of original solution.

• Step 1 – Averaging: If one did not count all of the cells in a large square (1mmx1mm) then it is necessary to average the results first before proceeding. For the purpose of this example, I use an average cell count of 123.456 cells.
• Step 2 – Computing the volume: It is necessary to determine the volume represented by the square. The width and height of the square (e.g. 0.25mm x 0.25mm) must be multiplied by the height of the sample (often printed on the hemocytometer, in this example it is 0.1mm): v = 0.25mm x 0.25mm x 0.1mm = 0.00625mm³ = 0.00625ul (where ul is microliters).
• Step 3 – Calculating the number of cells in 1 ml: if there are 123.456 cells in 0.00625ul, then how many cells are there in 1ml (=1000ul)? We do simple direct proportion:

  123.456cells/0.00625ul = X/1000ul
  (123.456cells*1000ul)/0.00625ul = X (the ul cancel out)
  X = 19 752 960 cells

• Step 4 – Correcting for dilution: If the sample was diluted before counting, then this must be taking into consideration as well. We assume that the sample was diluted 1:10. The final result is therefore 19 752 960 cells x 10 = 197 529 600 cells in 1 ml. That a lot of cells.

Things to watch out for

• Type of counting chambers: There are different types of counting chambers available, with different grid sizes. One counting chamber also has grids of different sizes. Take care that that you know the grid size and height (read the instruction manual) otherwise you’ll make calculation errors.
• Use the provided cover glasses: They are thicker than the standard 0.15mm cover glasses. They are therefore less flexible and the surface tension of the fluid will not deform them. This way the height of the fluid is standardized.
• Moving cells: Moving cells (such as sperm cells) are difficult to count. These cells must first be immobilized.
• Objective The hemocytometer is much thicker than a regular slide. Be careful that you do not crash the objective into the hemocytometer when focusing.

Different filters (patch stops) printed on overhead foil. The blue filter on the left is a commercial blue glass filter, on the bottom: the condenser with the 2 centering screws.

In a previous post, I already mentioned the making of patch stops from cardboard. For some background information (and more pictures) try these articles:

• Oblique Illumination
• Increasing Contrast using Optical Methods

Making Patch stops for dark-field illumination.

• Measure the diameter of the filter holder of your condenser.
• Using a program, such as PowerPoint or OpenOffice Impress to draw a circle, fill-color white, of the same diameter as the filter holder. You can adjust the size of the circle in the context menu.
• Draw a smaller black circle into the center. Copy-paste both circles and then change the size of the inner smaller circle. You want to make several filters to find the one that works best.
• Print the filters on overhead foil. Print with a laser printer. The overhead foils for laser printers are more heat resistant.
• Cut out the filters with a scissor
• Take a black marker and darken the black inner circle.
• For microscopy work, take two of these filters and place them on top of each other. This ensures that the central circle is completely black.
• Place the filter into the filter holder, completely open the condenser aperture diaphragm and the field diaphram (should you have one).
• Try out the different objectives and find the suitable filter/objective combination.

Making patch stops of oblique illumunation

The method is very similar to making patch stops for dark filed. In this case, light is only allowed to hit the specimen from one side only. This will produce a relief-like image.

• Draw a black and a white circle of the diameter of the condenser filter holder.
• Overlap the two circles, so that the white circle covers part of the black circle. The white circle should not reach the center of the black circle.
• Cut out and proceed as described for making a dark field patch stop.
• Again it is necessary to experiment to find the appropriate filter/objective combination.

Making Rheinberg filters

Maybe you want to show yellow specimens on a blue background. Take the dimensions of the dark-field patch stop and color the center yellow and the periphery blue (color printer!). You have to use intensive colors to achieve an effect. Try different color combinations.

A variety of different standards exist:

• Standard slide: 26 x 76 mm
• Geological slide: 75 x 50 mm
• Petrographic slide: 46 x 27 mm
• Thin sections slide: 48 x 28 mm

Microscope glass slides may be modified in a variety of ways:

• They may have a central indentation to carry several drops of liquid.
• They may have a frosted side to allow for easier writing with a marker.
• They may have polished corners to reduce the possibility of injury due to sharp corners.

Cover glasses (cover slips) exist in a wide range of different sizes, square, round, rectangular. Common sizes include:

• 18x18mm
• 20x20mm
• 22x22mm
• 24x24mm
• various rectangular sizes up to 24x60mm to cover nearly the whole slide.

Choose a cover glass that corresponds to the size of the specimen and the slide. The thickness of the cover glass is important, as it has a significant impact on the resolution of the image. The thickness should correspond to the thickness indicated on the objective lens. In many cases, the cover glass is 0.17mm thick, but there is often a small variation even in the same batch. For critical purposes, it may be necessary to measure the thickness of the individual cover glasses to find one close to the desired thickness (use a vernier caliper to determine the thickness).