POSES - Series 4

Practical Rules of Thumb for Microscopy

Building an optical system in general can be very tricky and exhaustive, regardless it is a simple system or a sophisticated one. Imaging sensor and illumination technologies indeed have advanced toward vast, limitless development and innovation; yet the limits in design and manufacturability are still very challenging for their realization. Similarly for optical components, there are many thread-offs to be considered to achieve their best performances.

But, afraid not, as below are some rules of thumb which can be very useful as practical guides to setup a microscopy system. Lets check them out.

Rule #1: Know your object/sample

It is important to understand the properties of your object or sample to match with what your system can do. The differences in its material, surface finish, dimension range, tolerance, shape, profile, and so on can be critical inputs to determine what kind of techniques or setup to be employed. The objectives are principally to produce a high-quality image with good sharpness, contrast, and/or colour.

Rule #2: Know your system

After getting sufficient inputs from the object/sample to be measured, it is necessary to filter that information in order to define the specification or characteristics of the microscopy system to be built. It will ease the process of managing and selecting the microscopy system components over the broad range of their available options. In doing so, having fundamental understanding of the components involves in the microscopy system is also equally important, which are mainly objective lens, camera, and illumination source.

It is typically less trouble and more straightforward to define optical specifications first, as other specifications should be easier and more flexible to be adjusted later following the optical ones, including the selection of illumination source. Lens and camera are extremely essential to determine the image quality and can only be practically meaningful when the object is illuminated properly.

Rule #3: Trust no human

Human body consists of complex and ingenious mechanisms which can construct any inexistent information at hand per se. That includes coordination between eyes and brain as an advanced imaging system. How human perceives image (perspective, contrast, color, etc.) can be very subjective and varying, which can be even more complicated with the degradation of vision condition (like myopia or astigmatism) and its vision compensation/correction. To eliminate these issues, it is important to implement analytic software to maintain image quality and system performance, as well as to introduce relatively consistent assessment on the image.

Rule #4: Too near not always good

It is very common that user desires to have higher detail from the sample/object observed using microscopy system. To do so, higher magnification is preferred, which implies the objective lens with shorter working distance (WD) and smaller field of view (FOV). To facilitate the flexibility of observation with larger FOV (~lower magnification) and higher magnification (~smaller FOV), some microscope system is equipped with multiple objective lenses attached on a turret. To be noted, in order to avoid the change of focusing position, it is highly recommended to use objective lenses with same parfocal length.

Another solution that may slightly improve the FOV is the use of camera with larger sensor. One constraint to be aware of is that the diagonal of the camera’s sensor shall not be larger than the 25 mm theoretically, which is associated with the typical diameter of main optical path throughout the whole microscopy system. To minimise user concern about it, microscope manufacturers always give the recommended camera size to be used with their systems, (commonly with 2/3” format, some recent ones with 1” and 1.1” format).

Rule #5: Too resolved not always good?

Another common desire user has is to have higher resolution image, which is defined by the numerical aperture (NA) of the objective lens. User also expect the larger depth of field (DOF) to allow the observation at different depth without losing resolution, which also depends on objective lens’ NA. Unfortunately, maximizing the resolution and DOF works on oppositely, where one cannot have higher resolution and larger DOF at the same times. More sophisticated and expensive solution may be available by using the premium quality objective lens, including the one with liquid lens.

Rule #6: No single lens for everything

Objective lenses have been designed with some constraints, leading their characteristics and specifications. Even without any constraints implemented, there are some limitations due to the laws of physics those determine the working principle and performance of objective lens. Furthermore, some special design input may be added on to the objective lens based on the microscopy technique applied. Therefore, it is always wise to define suitable criteria to build the microscope system and allow some possible options of objective lenses to serve the functionalities and applications.

Rule #7: Colour Counts

Colour represents of the light wavelength on its visible range, which can play a critical role in some applications. Colour also provides certain level of constraints in the design of optical components, including the objective lens and camera which operate as colour or monochromatic devices. Some implications of colour to the optical components are multiple focal points as the diffraction is a function of wavelength and different contrast levels as the intensity is a function of wavelength.

It is highly recommended to understand the operating wavelength range of the application in order to properly select the optical components to build the microscope system. Every objective lens and camera sensor usually include the wavelength characteristics in their specifications. Having filter on the optical path can be fruitful to minimize the focal points consistency or to separate different information with colour sensitivity.

Rule #8: Be strict, yet adaptable

Optical system and camera, including the microscopy system, usually need to be installed and operated under control environment to maintain the consistency and reliability of the results. Control environment covers some aspects, such as: humidity, temperature, ambient light. Humidity and temperature may affect the camera’s efficiency to generate minimum noise on the output image, change the dimension of metal components on the moving stage for scanning due to heat expansion, or change the condition of the sample on the stage leading to alteration of its structure and/or colour. Ambient light may contribute to low contrast as the background is getting higher or even disrupt the output image as the unwanted background is inseparably overlapping with the main image. The implementation of the control mechanisms can be straightforward, relatively simple, and relatively cheap, which will be definitely advantageous to improve and maintain the performance of microscopy system.

However, providing flexibilities and alternatives in some design aspects of the microscopy system, are important from different perspectives. This consideration can be useful to deal with the update of the components or allowance of using components from different vendors as the technology keeps progressing and advancing. Other benefit is the possibility for customization to accommodate slightly different needs. On the top of this, providing flexibility does not necessarily mean sacrificing the performance of the microscope system. Eventually, the performance shall not be affected by the change and remain as same as possible. One of the common practices is to define strict requirements for the optical performances; then, organize other requirements and performances with flexibility (mainly mechanical and electronical ones) around the optical requirements defined earlier.?