MMC Sperm analyzer: microscope, camera, PC requirements

Computer aided sperm analysis systems based on software, microscope and digital camera have certain advantages against automated "boxed" sperm analyzers (where all the optics and camera are put into one box):

You can select all hardware components of the sperm analyzer according to your budget available.
You can use your own microscope if it corresponds with the requirements for semen quality analysis.
The same digital camera and microscope can be used for any other applications besides sperm analysis like visualization of your samples, documentation, storage of images, manual or automated measurements (see MultiMedia Catalog database and MMC MultiMeter software).
Full-featured sperm analysis software provides much more functionality than a "boxed" automated analyzer (concentration, sperm motility patterns, sperm vitality analysis, morphology, DNA fragmentation, all on the same device). "Boxed" analyzers are usually doing sperm motility and rough morphology on unstained specimens, which does not comply with WHO requirements for semen analysis.
You can choose what kind of replaceable items you are going to use, which increase the self-cost of every analysis (for instance, use replaceable counting slides or reusable counting chamber; apply prestained slides or use Diff-Quik staining kits).
Full-featured sperm analysis software can be adjusted for using cheaper staining. For example, instead of working with expensive fluorescent dyes for vitality or DNA fragmentation analysis, we have adjusted the software for the use of cheap Eosin Y and relatively affordable staining kits based on Sperm Chromatin Dispersion. You can use negative phase contrast (motility) and simple bright field objectives (40x for sperm vitality analysis and DNA fragmentation, 100x oil for morphology). So, no expensive fluorescence equipment is required for your microscope due to software flexibility.

However, to build a computer assisted sperm analysis system, you need to consider certain hardware requirements. Herewith we will explain all the requirements, and what are the reasons behind them for you to understand the logic of selection of your hardware components for CASA system.

Microscope for Sperm Analysis

Trinocular transmitted light microscope is required. Trinocular microscope is a microscope, in which head there is a separate port for attaching a digital camera. This is required for convenient work with both binoculars and digital camera. Of course, the camera can be installed on an eyepiece adapter, but this will not allow you to use your binocular in a normal way, and will render your work very inconvenient.

10x objective for motility

We recommend 10x lens to be used for sperm concentration and motility analysis with MMC Sperm software. Why not your usual 20x? Because 10x objective provides more focus depth that 20x. Even when you use special counting chambers or slides with 10 micron depth, which should distribute sperms in one layer to get them all in focus simultaneously, some sperms can dive out of focus, and will not be detected by the software. 20x objective has less focus depth and hence intensifies this effect even more. Therefore, we recommend using 10x lens instead of 20x.

1х C-Mount adapter

The previous requirement (using 10x) is the reason for the next one: you should use 1x C-Mount adapter to install digital camera on your microscope instead of usual diminishing adapter tht provides more field of view. In case of sperm motility analysis, the field of view on 10x objective will be large. If you use diminishing adapter, sperms will appear too small, which is not convenient, and furthermore, the resolution of every object (the number of pixels per sperm head) will be not enough for precise detection. Always use 1x adapter. If you want to increase field of view on all other analysis routines (vitality, DNA, and especially morphology on 100x), buy a camera with larger sensor diagonal and use ROI (region of interest) in motility analysis if the field of view appears too large.

Microscope Contrast Techniques for Sperm Motility Analysis

MMC Sperm software can be adjusted for different contrast techniques available on your microscope. The software can detect dark objects on light background (bright field) as well as light objects on dark background (negative or positive phase contrast). Let’s consider advantages and disadvantages of every microscope contrasting method.

Bright field

To analyze sperm motility in bright field, you need to get the microscope slightly out of focus so that sperm heads appear as uniform dark objects on light background. However, when working in bright field, you will notice that the software might detect some extraneous objects having roughly the same shape and size as sperms, and classify them as spermatozoa. This leads to increase of detected sperm concentration of immotile sperms. When using phase contrast, this effect is significantly lessened, therefore, we recommend using phase contrast instead of bright field.

MMC Sperm: sperm motility analysis in bright field

Spermatozoa in bright field

Detection of sperm heads

Sperm tracks

Positive phase contrast

Positive phase contrast depicts spermatozoa as dark objects bearing a light spot in the center of the head on grey background. This contrasting technique allows you to cut off most of the extraneous objects, but since the light does not cover the head completely, the parameter describing sperm head shape is not working well enough, which leads to less recognition accuracy: the sperms will not be differentiated from debris well enough. Furthermore, positive phase contrast has a side effect: some kind of light halo is generated around sperm heads. These halos can also interfere with sperm detection.


Spermatozoa, positive phase contrast	Sperm head detection

Negative phase contrast

When using negative phase contrast, sperms appear as light objects on high contrasted dark background. The light covers sperm heads exactly preserving the shape. Most extraneous objects are covered by dark background and don’t interfere with sperm detection. Negative phase contras it the best contrasting technique for sperm motility analysis.

MMC Sperm: sperm motility analysis using negative phase contrast.

Spermatozoa, negative
phase contrast

Sperm head detection

Sperm tracks

Negative phase contrast: aids in elimination of extraneous objects, preserves the shape of sperm heads as light object on contrasted dark background.

Other sperm analysis modes are not so demanding in terms of microscope requirements. 40x bright field objective is used for sperm vitality and DNA fragmentation analysis. 100x oil immersion objective is required for sperm morphology analysis on specimens stained with Diff-Quik.

The following microscopes can be recommended for sperm motility analysis:

Some Chinese manufacturers provide negative phase contrast. For example, you can build your system on Motic BA310 microscope. Ask specifically for negative phase objectives.
Nikon, manufacturers specification of the negative phase contrast objective: CFI Achromat BM 10X, N.A. 0.25 WD=7.0mm, can be used on Nikon E200 (ECLIPSE E200) microscopes. Nikon E200 with negative phase contrast is a perfect solution but quite expensive.
Zeiss, specification of the negative phase contrast objective: A-Plan 10x/0.25 Ph1- M27 WD=4.2mm (negative phase contrast is marked with the minus sign after PH), can be used with Axio Lab.A1 series of microscope (not available for Primo Star series!). However, this lens has been discontinued recently.

MMC Sperm, bovine sperm motility analysis, negative phase contrast, Nikon E200

Bovine spermatozoa, negative phase contrast on Nikon E200 (ECLIPSE 200),
CFI Achromat BM 10X, N.A. 0.25 WD=7.0mm objective

Microscope beam splitter

Beam splitter is used to drive the light to digital camera instead of binocular. It is required that maximum light goes to the digital camera so that the camera sensor is able to provide 60 fps at minimum exposure time while analyzing sperm motility under 10x objective, and make high quality images with minimum digital gain in other sperm analysis techniques. Therefore we highly recommend 100/0 beam splitter (a switch that directs all the light to camera or to eyepieces). If your microscope manufacturer does not provide such version of beam splitter, consider using 50/50 splitter and maximum power light source. Beam splitter 80/20 (80% of light goes to binocular eyepieces) is not recommended.

Microscope light source

LED light source with maximum available power is recommended. This is a reliable, long-life light source. If only halogen lamps are available, make sure the power of the lam is at least 30 W, the more the better. Consider that cheap microscopes can be equipped with badly stabilized halogen lamp power units, which can lead to flickering effect that will be registered by the camera at frame rates higher than 50 fps. This will make sperm motility analysis at required 60 fps impossible. Therefore LED light source is highly recommended.

Microscope Setup

When a microscope is being used as a part of image analysis system, microscope setup becomes crucially important since any image analysis software is actually analyzing the provided image and not the specimen itself. Therefore, any flaws in microscope configuration can lead to wrong results. A computer aided sperm analysis system is no exception to this. Don't forget that the target of all setup procedures is a quality image on digital camera sensor. For this reason, all the steps of microscope setup should be performed with digital camera on and looking at the PC monitor instead of microscope binocular.

Kohler Illumination (Köhler Illumination, Koeler Illumination)

Place a specimen or stage micrometer under objective with minimal magnification. Get sharp image of the specimen or micrometer scale in focus.
Close microscope field diaphragm to its smallest opening and make sure that the light spot, which is formed by closed diaphragm blades, is in the center of the screen. If it is not the case, use condenser centering screws to move the spot into the center. It might be tricky, play with the screws to understand how the light spot is moving. Switch to 10x objective and check the centering again. If you have 20x, make sure the centering looks well at this magnification. At 40x, you already might not be able to see the edges of the closed diaphragm due to higher magnification.
Adjust condensor height: closed diaphragm blades must be in focus, and the diphraction halo should face the closed diaphragm blades, not the field of view. If a reusable counting chamber is used, its height can place the obejcts out of normal working distance of the condenser an destroy phase contrast. In this case, adjust condenser height looking at the specimen placed in your counting chamber.

Phase contrast setup

If phase rings are located inside the movable condenser, first adjust condenser centering, and only then adjust position of the phase contrast rings. They must be positioned with respect to centered condensor.
Adjust phase rings using centering telescope (the ring of light produced by the condenser should fit well into the dark ring of the objective). Then check the settings on actual specimen in your counting chamber or counting slide.

Aperture diaphragm

When working with phase contrast, aperture diaphragm should be opened.
When working with bright field, aperture diaphragm is used to adjust image contrast.

The video below shows the whole process of unboxing, assembling and setting up a coumpound biological microscope with specification for sperm analysis. Whatch it on YouTube to see timeline comments on every step.

Digital Camera for Sperm Analysis

Sperm motility analysis routine defines the requirements to digital cameras that should be used as part of computer assisted sperm analysis system MMC Sperm. For precise detection and tracking of spermatozoa the camera should be able to record video clips with frame rate of 60 fps at minimal resolution of 1.3 megapixel. At the same time, the camera should also provide high quality color images for other analysis routines. 60 fps at 1.3 Mp resolution, this is quite a big data flow. Recording video at such resolution and frame rate is only possible directly to PC operational memory. Camera setting for motility analysis are very much different from those for other routines: 60 fps at minimum exposure for motility, color is not important, and high quality color images with minimum digital gain and noise regardless framerate for other methods. For these reasons, we recommend only cameras with our direct drivers, which enable recording video directly to PC memory as well as saving and loading separate presets with all camera setting for every analysis routine (currently, motility, vitality, morphology, DNA fragmentation, should any new methods be added, all settings must be saved and loaded for them too). Ask for recommended cameras.

PC Requirements for Sperm Analysis

PC requirements are also mostly defined by sperm motility analysis. Processor and chipset should be fast enough to facilitate video recording to PC memory at 60 fps without losses, and fast processing of these video clips. Only motherboards based on Intel chipsets can be used for that purpose, preferably those with Z mark (e.g., Z270 or higher). Intel processors starting with i5, preferably, i7. Celeron and i3 processors usually are not fast enough for comfortable and fast workflow: frames are lost while recording, video processing becomes too slow. Minimum of 8Gb DDR4 operating memory is recommended. Windows 10, 64 bit operating system should be installed on your PC. SSD instead of HDD for installation of the OS and our software is preferable.

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