The current and the futuristic scenario for studying basic science has evolved to virtual microscopy, thus providing an immense aid in easing down the process of learning 1415. Despite of this evolution, experiments related to understanding of resolution and contrast are difficult to materialise. Moreover, in reality, while focusing an object under a microscope either contrast or resolution is achieved at the cost of other 4. In this study, we tried to quantify and learn the bargain between contrast and resolution by the means of hands-on experiment. Upon reminiscing the background of our study, many questions would emerge in our minds as:
Q: On which of the entity does the quality of the image formed in the microscope depend: Resolution or Magnification?
A: Pondering on the answer, we often mistake magnification and resolution as one, though they are quite different as mentioned above. The resolution can be computed using the given formula.
Resolution (r) = 1.22λ/ [NA (obj) + NA (cond)] (Equation-1)
Here, λ is the wavelength of light that influences the resolution as a shorter wavelength provides greater resolution and vice versa. NA (obj) equals the objective numerical aperture, and NA (cond) is the condenser numerical aperture. However, several similar equations have been derived which show a relationship between resolution, wavelength, and numerical aperture, differing by a multiplication factor 16.
Where the numerical aperture is the ability of the microscope lens to gather light and resolve the fine specimen detail. It depends upon the refractive index of the imaging medium (medium between the front lens of the objective and specimen) and one-half angular aperture of the objective. Therefore, the higher the refractive index of the medium, the higher would be the numerical aperture and consequently the resolution power. In addition, the correction for optical aberration has an impact on the numerical aperture of an objective. Higher the correction for chromatic and spherical aberrations larger is the numerical aperture for respective magnifications 17,18.
Furthermore, resolution cannot be defined without the reference of contrast with which it shares a reciprocal relationship. When viewing in the microscope, a specimen is imperceptible by the human eye, as the specimen itself has little or no contrast. Unlike resolution and magnification, it is unfeasible to derive contrast as it is multifactorial. These hurdles lead to the discovery and evolution of staining. An adequate contrast is a key to decipher the complete microscopic details of a magnified and well-resolved specimen. Hence, the elements influencing resolution are the type of specimen, coherence of illumination, degree of aberration correction, and other factors such as contrast-enhancing methodology either in the optical system of the microscope or in the specimen 19.
Summarizing it, magnification as a sole component of an optical system yields an enlarged image without delineation of minute structures so that the enlarged image is perceived by the human eye. However, resolution makes it more meaningful by adding minute details in it. Thus, we can interpret that for formation of a quality image at eyepiece, both magnification and resolution hold equal ground.
Q. Which sources of light are used in the microscope and why?
A: The visible spectrum ranges from 400–700 nm, of which the human eye is more sensitive to 550 nm, which corresponds to the green colour. So, microscopists prefer using the sources that emit this spectrum of light (400–700 nm, centered at about 500 nm) for bright field microscopy 19. The most common example is the tungsten bulb. While other examples are mercury arc lamps, xenon arc lamps, metal halide lamps, LED’s, etc. 20.
In addition, the NA inscribed on the objectives used for calculation of resolution is determined by the manufacturers keeping this wavelength in mind.
Q: How to derive magnification and resolution in a classroom?
A: Magnification power is determined by multiplying the magnification values of the objective and the eyepiece that are imprinted on the microscope.
Generally, resolution is estimated by measuring either the minimum resolvable distance between two adjacent structures in an image, or alternatively, it can be estimated from the intensity profile analysis such as simple full width-half-maximum (FWHM) or more complex fitting 21 of sub-resolved sized structures. Techniques such as Fourier ring correlation (FRC) 22 and Fourier shell correlation (FSC) have been used for decades to estimate image resolution in electron cryomicroscopy 23. FRC was also adopted in the field of optical nanoscopy to address the issues with the traditional resolution assessment methods 24–26. However, this would require sophisticated designed tools or models, which take a lot of expertise and time. Hence, for a resource poor setups, the method used in this study would be the most feasible.
While crudely, resolution is derived using above equation-1 where the wavelength is taken as 550 nm and NA is taken from the imprinted value on the eyepiece as explained above.
Q. Why there is a preferential use of blue filters instead of green, even though human eyes are more adapted to green colour?
A: Incandescent tungsten-halogen lamps display colour shifts with fluctuation in light intensity. When the intensity of the lamp is low the spectrum of the light is more towards the longer wavelength of light while when the light intensity is high the spectrum is more towards the shorter wavelength. This can result in a significant shift of colour in the spectrum, which is ignored while visualizing the images through the microscope but is exasperating while capturing the micrographs. In micrographs, at lower intensities, the image has a more yellowish-orange background, while at higher intensities the image appears more natural 27. When a blue filter is applied over the lamp, light with a longer wavelength is absorbed, giving a much cooler and natural image. Hereafter, it is not necessary to correct the temperature of the picture. In addition, as mentioned above, light with a lower wavelength increases resolution. On the above-mentioned grounds, some microscopes come with an inbuilt blue filter placed under the condenser.
At this point, it is worth mentioning here that various studies have depicted the hazards of blue light 28. So, looking at the pros and cons of blue light, its use becomes quite subjective.
Q. Should we use a single monochrome light to obtain the best results in our microscope?
A: Practically yes but for perception by the human eye, we need contrast to identify the object of interest, so a polychrome source of light and staining the specimen is the option that we would have. While for image processing assisted with machine learning or deep learning algorithms a single light will be better, as it will avoid many optical aberrations 29 30.
Q. Why do we use a combination of Stain?
A: In the bright field microscope, a light source that emits visible spectrum is used. Hence, if the objects are colourful, they would be perceived well. Imagine watching a black and white television over a coloured one, which one would be more appealing and informative? It’s just the same with microscopy, where we can better understand this concept by observing an unstained and a stained slide. If we talk about the present study, here the Leishman stain has methylene blue and eosin that stains the acidic and basic structures as per its strength; it imparts an overall shade of purple and violet on a slide. Hence, from a perceptive point of view, a single stain fails to impart enough contrast compared to two. Thus, to have better differentiation in a specimen concoction of stains is necessary 31.
Q. Can wrapping gelatin paper over the source of light improve the image quality?
A: The results suggest that the answer will depend upon an individual’s preference to opt for either contrast or resolution, as the ideal sequence for contrast and resolution are variable, as shown in table no 1 & 2. Hence, the selection of filters is purely subjective. To add, since the properties of gelatin paper related to the amount of spectrum of light that can pass through it or be blocked by it, is not known it cannot be relied upon for improving the image quality as far as the reproducibility of the results is concerned. So, we can say gelatin paper is just helping in changing the hue of an image, which can be used as subjective preference.
Q. Do the stain and the light used in a simple compound microscope provide the best resolution and contrast?
A: The results suggest that white light is best with Leishman stain, providing maximum contrast and resolution. The results distinctly indicates that there is considerable overlap for contrast and resolution and that humans have more tendencies for contrast, thus bargaining resolution.
As mentioned in the article above, we can either have resolution or contrast but not both. The hazard of blue light suggests avoiding blue light while maintaining contrast or increasing the contrast with minimal compromise of resolution. Hence, we propose there is a need for a stain that can give a green background while staining the specimen yellowish-white or any other contrasting colour. This is because after hazardous blue light shade, it is the green shade that provides maximum resolution and favours physiology of perception for green colour.
Since, many attempts made in past have had a great leap of success in increasing contrast and resolution (like phase contrast, fluorescence and all). The current study is only a small step in getting customized contrast and resolution in the existing usable simplest microscopes in many developing and least developed countries. It simultaneously suggests that a user can maintain good health of his eyes by avoiding prolonged and fatiguing exposure to the blue light.
Q. Is the setup of this study ideal? Are the results reliable?
A: No, neither the setup nor the results are completely ideal or reliable. While it can be said that the results and setups are good for half of the experiment, i.e., considering for the contrast the setup and results are very well reliable. While for resolution the method used is correct, but the setup is not the ideal one. Hence, though the setup is affordable and accessible, the offset is due to the use of low-cost filters and other optical components. Consequently, there is a need for standardization of setup considering the ideal setup protocols like for the illumination/intensity of light, capturing device and its filter (like CFA/IR cut) used, adjustment for condenser/iris/fine adjustment for perfect focusing (i.e., proper alignment of all the optical components) 3233. Such standardization would give more reproducible results with different devices like CCDs or mobiles.
4.1. Evaluation of Student Work
The entire exercise would be contemplated as self-evaluation. The data derived would depend upon the quest of a student to seek answers to the questions in his mind. Based on results, the student can compare the outcome of different filters as per the subjective preference and also conclude if the filter used was apt or not.
4.2. Wider Educational Applications
On a broader base, it is a confluence of two fields, viz. the medical field whose sole focus is to visualize a cell by emphasizing contrast and the technocrats/field of optical science that exclusively prioritizes on ameliorating systems resolution. Hence, this would be the most apt way to have an integrated learning. As recommended by Kent J. Crippen in his book, this would help in collaborative problem solving in the learning environment by organic integration of two different fields of science 1.
4.3. Additional Experimentations
Q. Is further accretion of data possible in this study?
A: Certainly, the data can be amassed using different sources (tungsten incandescent bulb, monochrome LED, CFL), filters, specimen (ZN stain, Gram stain), and observing devices (mobiles, CCD).
Q. Is there any scope for ancillary studies/experiments that can ameliorate the simple compound microscope?
A: The boundless possibility of a simple compound microscope is what makes it more intriguing. It is the need of the hour to have an affordable and easily tuneable technology that can add more meaning to the existing one.
Yes, there are other additional possibilities for the experiment as below: -
➢ Rotating filters: to reduce the hazards of blue colour on the eyes, a rotating filter can be used between condenser and mirror. That is, a device comprising two or more coloured filters rotating at the desired rate can be designed that can fit between the condenser and reflecting mirror. Example: fitting the device with green and blue filters alternately. At a higher speed, the device imparts an overall cyan colour, which can be evaluated for its quantification for contrast and resolution.
➢ Converting simple microscope to anaglyph microscope: two slightly separate images can be produced at the eyepiece with the use of the above device with coloured filter; or coloured polarised filter; or directly using monochrome lights, which can rapidly on and off alternately. The degree of separation of images can be adjusted by adjustable prism/or non-colour corrected glass at the eyepiece. These distinct images will be displayed on a screen affixed at the eyepiece, which can be visualized using 3D glasses.