Light microscopy remains very important to pathologists today. Surprisingly, the functions and uses of light microscopes have remained essentially unchanged over the past century. Choosing the right instrument is critical to obtaining high-quality cytology images, ease of use and comfort, and maintenance of microscope functionality.
Inspectors should have two basic knowledge:
- ①How to set microscope parameters to obtain the best image.
- ②How to keep the instrument in good condition.
The purpose of this article is to provide a practical guide on how to choose the type of microscope, the specifics of microscopic examination, and daily use and maintenance tips.
Everything in this article refers to upright or compound microscopes. There are other types of microscopes, such as dissecting or surgical microscopes, and much of the information discussed here applies to these microscopes as well; however, some specific technical differences are not discussed here. This article will focus on bright-field techniques for examining stained specimens on slides.
Chapter One Microscope Choice
Buying a microscope isn’t much different than buying any big-ticket item (a TV, or even a car). Although there are different models, it is best to buy from a supplier that provides complete information, best understanding of the product, and comprehensive after-sales support. Things to pay attention to when purchasing a microscope include:
Does the supplier provide microscope installation and training services?
Warranty duration and level
After-sales support: Does the manufacturer have people available to help, or regional representatives who can call or come to your home?
Chapter Two Microscope parts
1 microscopic lens
There are four types of microscope lenses: monocular eyepieces, binocular eyepieces, trinoculars, and ergonomically adapted ones.
Monocular microscopes are very low-cost and not suitable for long-term use. Therefore, a binocular microscope is the minimum requirement for a laboratory or medical facility. If you need to add a camera to the microscope, you will need a trinocular tube. The ergonomic eyepiece can adjust the lens according to the height of different users to obtain the most comfortable and safe viewing angle and extend the use time of the microscope.
Figure 1.1 Structure of an optical microscope
Note: Aperture refers to the entity that limits the light beam in the optical system. It can be the edge of a lens, a frame, or a specially designed perforated screen. Its function can be divided into two aspects, limiting the beam or limiting the size of the field of view (imaging range). The aperture that limits the most light beam in an optical system is called the aperture aperture, and the aperture that limits the field of view (size) the most is called the field aperture.
2 microscope stands
Microscope stands typically have a coaxial coarse and fine focusing helix. The illumination system can be mounted within the stand or in a light box located at the back of the microscope. More complex stands are often designed for specific clinical uses, with low-mounted stages and focus control systems to reduce hand and wrist fatigue. Some stands are also equipped with a safety lock that adjusts the focus height to prevent the slide from hitting the objective lens, thereby avoiding damaging the specimen or damaging the objective lens.
There are two mechanical versions of the microscope stand with different ways of mounting the components. According to its lighting type, it is divided into:
- Koehler illumination microscope
This clinical/research-grade microscope produces extremely uniform illumination of the sample, and the light source (such as halogen filament or LED light) does not show up in the resulting image.
- Critical illumination microscopy
This lower-cost version of microscope illumination relies on a ground glass diffuser. The main problem is insufficient uniformity of illumination, with the light source being visible in the image.
3 eyepieces
Microscopes are always equipped with a pair of 10x eyepieces (other magnifications such as 15x are rarely provided). Magnification is recorded on each eyepiece. These names might look like this:
10×/20
WF (widefield)10/22
WF 10×/22
The number (e.g. /20, 22) after the eyepiece magnification (e.g. 10×) indicates the field of view number, which indicates the size of the field of view (in millimeters) that can be observed with a given objective lens. The larger the field of view number, the larger the sample area that can be observed.
It is not recommended to purchase a microscope with a field of view less than 20㎜; ideally, a field of view of 22㎜ can fully and comfortably observe the sample. Some eyepieces can also display images with glasses on, indicating that they are high-eyepoint eyepieces, allowing for comfortable viewing even when wearing glasses. Additionally, some eyepieces have measurement graduations (eyepiece graticules, reticles, or graticules) inserted into them.
4 objective lenses
There are three different types and special variations of objectives. According to the nature, it can be roughly divided into:
- Achromatic lenses: regular quality
- Fluorite Lenses: Research/Clinical Grade
- Apochromatic lenses: highest quality and resolution
The quality of an objective lens depends on the degree of chromatic aberration correction as well as differences in spherical aberration and numerical aperture. Understanding the specifications shown on an objective lens is important to ensure proper use of the lens.
The following will discuss the key elements of the objective lens and the identification of the technical specifications of the lens barrel.
Figure 1.2 Microscope objective lens
- Plan
Inexpensive microscope objectives do not provide flat field images. This means that the field of view boundaries of such images may look a bit blurry. Objective lenses that can ensure a flat field of view should be marked plan, plano, or PL.
- Gain
The magnification of the objective lens multiplied by the magnification of the eyepiece (usually 10 times) is the magnification observed by the eye. A 40x objective lens and a 10x eyepiece provide a 400x magnification. The most commonly used objective lens magnifications in biological microscopes are 4×, 10×, 20×, 40×/50× and 100×.
- Numerical aperture
This term refers to the resolution of the objective lens. It represents the ability to collect light at a fixed distance and resolve specimen details.
High-quality objectives (in order: achromatic – fluorite – apochromatic) have larger numerical apertures, which provide better image resolution. For numerical apertures of 1 or higher, the objective lens must be an immersion objective lens (oil lens).
- Manager
Lower quality and older microscopes have a fixed tube length, usually 160mm. Modern microscopes can correct the tube length at will, regardless of quality; accessories (such as double observation tubes) will be inserted between the scope and the user’s head, without changing the magnification of the image to the eye.
However, using both a fixed tube length objective and an arbitrarily corrected objective on the same microscope results in poor image quality and risks damaging the objective.
- Coverslip correction
Most dry objectives with a magnification of 20x and above are designed for observing samples with a cover glass, which acts as a lens. Therefore, if the coverslip is not positioned correctly, the image quality may be poor.
The thickness of the standard coverslip is 0.17mm.
To obtain the highest numerical aperture and resolution, oil immersion is required between the objective and specimen. If the objective is for oil immersion, write the word “Oil” on the barrel of the objective after the magnification/numerical aperture (e.g. Plan 100×/1.25 Oil).
Choosing high-quality oil is essential, as this determines the quality of the image. Never mix different oils as this may cause a reaction and increase the turbidity of the oil. Also avoid old, low-cost or particularly viscous yellow oils.
If a dry objective lens (e.g. 40×) is contaminated with oil, it can be wiped clean with a mild solvent (e.g. 30% ethanol) or a commercially available lens cleaning solution.
5 condenser
The condenser and its aperture provide a cone of light at the correct intensity and angle to achieve the objective’s optimal resolution. Different types of condensers have different degrees of correction and numerical apertures.
The most common condensers are:
- Abbe condenser
This condenser concentrates and controls light before entering the objective lens without optical correction. This condenser has two controls, one that brings it closer to or farther from the stage, and a variable aperture that controls the diameter of the beam. These controls can be used to adjust brightness, lighting uniformity, and contrast.
Abbe condensers are suitable for most brightfields but have limitations when using high-power objectives.
- Achromatic and aspherical condensers
The aspherical condenser can correct the spherical aberration of the concentrated beam, and the achromatic condenser can correct the chromatic aberration. This condenser helps achieve the best resolution and highest numerical aperture.
The 0.9 Abbe condenser can already meet the needs of conventional clinical microscopes and is also standard equipment for general microscopes. More advanced microscopes with fluorite objectives often have higher-corrected condensers.
6 microscope illuminators
Conventional microscopes have been equipped with halogen lamps for a long time. The only disadvantage of this lamp is that the high temperatures produced result in a shortened microscope life.
In the past few years, microscopes have begun to be equipped with LED lights, which emit bright white light without producing heat. However, the color characteristics of LED lights are not as good as halogen lights, especially older LED lights. Therefore, it may still take some time for the microscope to switch from halogen lamps to LED lamps, and the appearance of certain stains is slightly different under different light sources.
Third chapter Microscope settings
Microscope setup operations:
- ①Adjust the distance and center position between the condenser and eyepiece to achieve the best;
- ②Adjust the aperture on the microscope (see below).
Eyepiece
People often ignore eyepiece settings, so they often feel eye fatigue. Most microscope eyepieces have focusing capabilities as well as diopter markings. Start by pointing the eyepiece at the sample and keep adjusting until you see a clear image.
Flashlight
- A) Kohler lighting setup steps
- ① Place the specimen slide with good contrast on the stage and use a 10× objective lens. Align the specimen using focus controls;
- ②Move the button under the condenser and stage until it is about 0.5cm away from the top;
- ③Close the field of view aperture, and the blades of the aperture are visible at this time (may not be in the center of the field of view);
- ④Move the condenser up and down until the aperture blades can focus;
- ⑤Use a knurled screw or an Allen wrench (usually on the base of the condenser, left or right) to center the aperture;
- ⑥Open the field of view aperture until it is just beyond the field of view;
This way the condenser will be at the correct height and in the center of the light path, providing optimal illumination of the sample and obtaining the best image quality.
Figure 1.3 Adjustment of Kohler lighting system
- B) Critical Lighting Setup Steps
This setup is relatively difficult because there is no field aperture in the focused image. In this case, the aperture aperture (i.e. condenser aperture) needs to be used instead. The condenser aperture is usually invisible. You need to remove the eyepiece and observe inside the lens tube (keep a distance of 10-15cm from the lens tube).
Figure 1.4 Adjustment of critical lighting system
Adjustment steps
- ① Focus on the specimen on the stage and raise the condenser to the top or the highest point of the lifting range;
- ② Remove one eyepiece and observe the tube until only the edge of the condenser aperture is visible, but no image of the specimen is visible;
- ③Use the front centripetal screw on the condenser to center the condenser.
condenser aperture
The condenser aperture is one of the most important parts of a microscope setup.
If the condenser aperture distance is too close, the resolution of the image will be significantly reduced. The practical problem in this situation is that after viewing the specimen image clearly under a low magnification lens (such as a 10x objective lens), the aperture is relatively closed when viewed under a higher magnification. Therefore, the condenser aperture should be opened when using a higher magnification objective.
The condenser adjusts the cone of light entering the objective and also reduces contrast through glare, preventing too much light from focusing on the specimen. If closed too much, the resolution of the image will be limited.
Specific operations
- ① Observe the specimen and fully open the condenser diaphragm.
- ②Close the condenser gently until the light begins to decrease.
A good microscope will have the correct condenser position marked on it, so the user knows exactly how to adjust the knobs.
Chapter Four Microscope maintenance
Generally speaking, modern microscopes require very little maintenance. The key to maintaining a microscope is to cover it when not in use, because dust is the enemy of optical microscopes.
Another major problem is contamination of dry versus oil immersion objectives. More modern and advanced microscope objectives have a flat front for easy cleaning. Older objectives are concave, making cleaning more difficult. Cleaners should be used in accordance with the manufacturer’s recommendations. If the manufacturer does not specify, you can use dilute ethanol solution or special camera lens cleaner.
Chapter Five Take microscope images
There are two camera systems available: dedicated microscope cameras and digital single-lens reflex cameras (DSLRs).
Dedicated microscope camera
The advantage of a dedicated microscope camera is that it can provide a full screen live image and has many functional settings such as white balance and correct exposure. This allows us to see exactly what the camera is capturing.
In addition, almost all microscope-specific cameras have supporting software, including image processing, manual calibration and graticules. The only drawback is that it requires a specialized computer to run. Of course, there are also cameras on the market that support HDMI interfaces. They can run directly on the monitor, have complete setup and control software, and can store images to SD cards.
Commercial DSLR camera
Most modern DSLR cameras have some form of output to a TV screen or PC. The images obtained may be excellent, but these cameras require user operation to obtain correct and repeatable results.
One of the major issues with using a DSLR camera is that a high-quality interface is required to connect the microscope, which comes at additional expense.
Camera/microscope interface
To obtain good results, it is important to use the correct interface between the microscope and camera.
Most microscope-specific cameras use a so-called C-mount. The C interface has different amplification factors, usually 1×/0.5×/0.4×/0.35×.
The sensors of early cameras were very large, and the 1× C-mount was sufficient for a wide field of view. However, as technology advances, camera sensors become smaller and smaller, and the magnification factor becomes larger, so the field of view obtained by the camera sensor becomes smaller. For this reason, a zoom lens is required to observe the entire field of view of the slide.