Application of Modern Microscope Concept in Observation of Microscopic World
From ancient times to the present, human beings have been pursuing higher and farther truths. From ocean voyages to space exploration, people have been conquering grand goals one after another. However, the macroscopic world that people see with the naked eye is not the whole world, and the human eye cannot see it clearly. It also attracts countless people to explore and pursue.
Regardless of macroscopic or microscopic things, our observations are based on the attributes of three-dimensional space, that is, XYZ three-dimensional, and the observation of changes in the shape of things needs to introduce another measurement factor - time T, so the most complete way to observe things must be Simultaneous recording of XYZT, that is, long-term photography of shape + time, is also the ultimate function of the microscope.
After more than 300 years of development, modern microscopes have proposed concepts such as resolution, depth of field, and field of view, and have continuously proposed solutions. Microscopes have initially met our needs for observing the microscopic world and helped us record the space and time of the microscopic world.
The most important thing in microscopic world observation is the resolution of details, and the concept of resolution was born from this. Resolution refers to the minimum distance between two points that can be distinguished by the human eye, and is only valid in the XY dimension. According to the Rayleigh criterion, Rayleigh Criterion, the limit that normal people can distinguish is two points of 0.2mm at a distance of 25cm. When we use a microscope, we can see two points at a smaller distance, which improves the resolution of our observation. With the continuous deepening of modern research, people's requirements for resolution are also constantly increasing, and scientists are also constantly improving the resolution of microscopes. For example, electron microscopes have increased the resolution to the nanometer level, enabling the observation of viruses. Ultra-high microscopic imaging technology improves the resolution of the microscope from 200 nanometers to tens of nanometers, realizing the observation of living cell organelles.
The improvement of resolution also brings new problems, that is, the reduction of field of view and depth of field. When using the ordinary central illumination method (the photopic illumination method that makes the light evenly pass through the specimen), the resolution distance of the microscope is d=0.61 λ/NA, the wavelength range of visible light is 400-700nm, the average wavelength is 550nm, and the wavelength is a fixed constant. Therefore, increasing the NA value can get a smaller D value, that is, the distance between two points that can be distinguished Smaller, allowing people to see smaller objects clearly.
The NA value is the numerical aperture, which describes the size of the lens light-receiving cone angle, NA = n * sinα, that is, the product of the refractive index (n) of the medium between the lens and the object to be inspected and the sine of the half of the aperture angle (2α). n is the light refractive index of the medium between the objective lens and the sample. When the microscope object space medium is air, the refractive index n = 1. Using a medium with a higher refractive index than air can significantly increase the NA value. The water immersion medium is distilled water, and the refractive index The ratio is 1.33; the oil immersion objective medium is cedar oil or other transparent oils, and its refractive index is generally around 1.52, which is close to the refractive index of the lens and slide glass. Therefore, the NA value of the oil lens is higher than that of the air lens.
Aperture angle, also known as "mirror mouth angle", is the angle formed by the object point on the optical axis of the lens and the effective diameter of the front lens of the objective lens. Increasing the mirror mouth angle can increase the sine value, and its actual upper limit is about 72 degrees (the sine value is 0.95), multiplied by the refractive index of cedar oil 1.52, it can be obtained that the maximum NA value is about 1.45, and substituted into the resolution calculation formula, it can be obtained that the limit XY plane resolution of a conventional microscope is about 0.2um.
The NA value also directly affects the brightness of the microscope's field of view (B). From the formula B∝N.A.2/M2 we can deduce that the brightness increases with the increase of the numerical aperture (N.A.) or the decrease of the objective lens magnification (M).
Theoretically, we should pursue the highest possible NA value to obtain better XY plane resolution and field of view brightness. However, everything has two sides. The improvement of XY plane resolution will reduce the Z-axis depth of field and observation field of view.
Microscopes generally view the view vertically downward. When the convex position and the concave position on the surface of the object observed within the diameter of the field of view can be seen clearly, then the height difference between the convex point and the concave point is the depth of field. Well, for microscopes, the larger the depth of field, the better. The larger the depth of field, the better and more three-dimensional clarity images can be obtained when observing the surface of uneven objects. The large depth of field helps us observe the microscopic world in the vertical direction. That is, the Z-axis information in the XYZ three-dimensional form.
Depth of field is the depth of front and rear space corresponding to the clear image on the image plane: dtot=(λ*n)/NA + n/(M∗NA) * e, dtot: depth of field, NA: numerical aperture, M: total Magnification, λ: wavelength of light, (usually λ=0.55um), n: refractive index of the medium between the sample and the objective lens (air: n=1, oil: n=1.52) According to this formula, we can know that Z Axis depth of field is inversely proportional to XY plane NA value.
In addition to the depth of field, the field of view is also affected by the NA value. The spatial range that can be seen when the instrument is fixedly looking at a point is the field of view. Its calculation is directly related to the magnification of the objective lens. The actual diameter of the field of view seen by observation is equal to the diameter of the field of view Divided by the magnification of the objective lens, the eyepiece will indicate the corresponding field of view, such as 10/18, that is, the magnification is 10 times, and the diameter of the field of view is 18mm. Therefore, when the eyepiece is determined, the larger the magnification, the smaller the observed field of view.
The XY plane resolution is the analysis of local details, and the field of view determines our observation range of the sample. The larger the field of view, the better, but limited by the current technology, we must use high-power objective lenses to obtain good NA values, therefore, visual field and NA values have an indirect negative correlation.
