显微镜光学
Graduate examination paper
1. Please outline different methods of obtaining contrast in microscopic optical imaging, and compare their metrits and demerits.
There are three methods of obtaining contrast in microsocpic optical imaging.
1) Chemical methods. Chemicals methods use stained tissues or dye the organism and together with color contrast filters or mounting medium with a refractive index substantially different from the specimen to increase the contrast in the image.
Merits:
It's effective in outlining the interested areas of the specimen and obtaing high contrast.
Demerits:
It's difficult to dye the specimen while the dying process can change the cell structures or even kill the organism, which makes it unsuitable for people who wants to see things alive under microscope.
2) Decreasing the condenser aperture diaphragm opening size. That is, when a microscope operates in brightfield mode, decreasing the condenser aperture diaphragm opening size can increase the spatical coherence of light and thus increase visibility of particles and edges in phase specimens. And contrast for amplitude objects can also be improved by proper adjustment of the condenser aperture.
Merits:
No extra spectial equipments are required in this method, and its easy to adjust artifically and freely while observing the specimen.
Demerits:
Loss of diffracted light is caused since the decreasing of opening size, so some portion of light representing the information of object is lost, therefore loss of resolution,sharpness, and superimposition of diffraction rings will appear.
3) Phase contrast microscope. It's a specially designed microscope that takes advantages of phase differences between the parts in a specimen and in the surrounding medium. By using different filtering rings between the objective and eyepieces, the background light could be phase-shifted to eliminate the phase difference between tha background light and scattered light, and be dimmed by another filtering ring to increase contrast to the scattered light.
Merits:
It can produce high contrast without doing anything to the sample, and can be used simultaneously with reflected light fluoresence to reveal areas of a specimen that do not fluoresce. It can be particularly effective when the specimens are thin and scattered in the field of view.
Demerits:
It's complex and expensive due to extra components.It does not work well with thick specimens and phase images are usually surrounded by halos that may obscure the boundries of details. Besides, it can also decrease the numerical aperture so that resolution will be decreased and is less bright than the ordinary brightfield observation.
4) DIC
It uses steep gradients in path length to generate contrast and image will display a presudo three-dimentional relief shading.
Merits:
It can produce images having significant three-dimentional character plus all the phase constrast merits.
Demerits:
In some cases, its images suffer from excessive brightness along edges having very steep optical gradients. and the boundaries of extended specimens are often the only features that can be recognized with this technique.
2. Please explain the concept of magnification in microscopic imaging.
2.1 Basic principles
In microscopic imaging, the magnification is based on the principle of image formation of single convex lens, that is when object is situated between one and two focal lengths in the front of the lens or just at the front focal plane of the lens. Those two basic situations are illustrated as below.
(1) Just as in the Figure2.1, image is formed at the other side of the lens and a magnified, inverted and real image of the object.And when the eye receives the image light, it will perceive as if it were at a distance of 25 centimeters, which there exists a magnified, upright, and visual image of the object. This princinple leads to the development of finity-corrected systems.
(2) Just as in the Figure2.2, image is formed only when man look in the direction of the light transimition,and its a magnified, upright and visual image of the object.This principle leads to the development of infinity-corrected systems.
2.2 Magnification in microsocpic imaging
In the finity-corrected systems, there are several components in a microsopy system and all are illustrated in Figure2.3. When light from the source passes throught the substage condenser, light will be concentrated onto the object, and transmitted light will projects the first image, which is a magnified, inverted and real image to the intermediated image plane, which is located about 10 millimeters below the top of the microscope body tube and with the internal diaphragm of the eyepiece or about the optical tube length of distance after the back focal plane of the object.Then, the light continues go in the direction that determined by the mechanical tube and will go through the eyepiece to form a second amplified, upright and real image on the retina of the eye. The eye of the observer sees this secondarily magnified images as if it were at a distance of 25 centimeters from the eye. In terms of infinity-corrected systems, there are extra tube lens between objective and eyepieces for the image formation on the intermediate image plane.
There are three reasons that determines the magnification value of the system.
Firstly, the visual image is amplified compared to the object because both the objective and eyepiece has magnifying power. For instance, if the magifying power of the objective is 50X, while that of eyepiece is 30X, the microsopy will give a visual magnification of 1500X.
Secondly, the tube length of the microscope also affects the magnification since certain tube length determines the types of objectives and eyepieces in consideration of the aberrations.
Thirdly, the tube lens which are inserted in the optical pathway to shorten microscopic mechanical tube length can introduce an extra magnification of tube factor to the system, which is usually around 1.25-1.5.
However, the range of the useful total magnification for an objective/eyepiece combination is defined by the numerical aperture of the system, which is usually set as from 500NA to 1000NA.
3. Please explain the concept of resoltion in microscopic imaging, and summary the key factors in determining resolution in Laser scanning confocal microscopy.
3.1 concept of resolution in microscopic imaging.
The resolution in microscopic imaging is defined as the smallest distance between two points on a specimen that can still be distinguished as two seperate entities. When a point source of light passes throught an objective, there will be a intensity point spread function mapped on the intermediate image plane, and when point sources all transmits light through the objective, the Airy diffraction patterns at the intermediate image plane will overlay parts upon each other if they are close to each other in the object two-dimentional plane. Two Airy disks are distinct if they are father apart than the the distance at which the principal maximum of one Airy disk coincides with the first minimum of the second Airy disk, and the minimum distance between two point light sources in two-dimentional plane to distinguish them can be calculated as the formular below.
r = 0.61λ/NA
And, as for a microscope system, the resolution is not only determined by the numerical aperture of the objective, but also the numerical aperture of the substage condenser, therefore, the formular above should be changed into below.
r = 1.22λ/(NA(obj) + NA(cond))
3.2 key factors in determining resolution in Laser scanning confocal microscopy.
In Laser scanning confocal microscopy, the r is reduced to :
r(lateral) = 0.4λ/NA
r(axial) = 1.4λη/NA*NA
1) NA(obj) and NA(cond)
The numerical aperture of the objective and substage condenser can both be calculated in the fomular below.
NA = n(sin(Θ))
In order to obtain higher resolutions, we should obtain larger NA, and therefore, high refractive index (n) of the medium between the front lens of the objective and the speciman, and between the objective and the substage condenser will lead to higher resolution. Besides, the alignment of the microscope optical system also affects the resolution. The adjustment of the aperture diaphragm can determine the formation of the light cone through the object and thus can determine the size of the angular aperture which is in relation to the inverted angular aperture(2Θ) after the light passes through the specimen. The wider the condenser diaphragm is opened, the bigger Θ is, so is the resolution, however, the condenser diaphragm should not be too wide to generate glare and lower contrast.
2) λ
It's obvious that the bigger the value of the wavelength(λ) of illumination light, the higher the resolution is, only under the circumstance that the select range of λ should be matched to the specimen to be observed.
3) contrast
Even the construction of a microscope can enable the resolution to reach the diffraction limits, there must be high contrast of the specimen to ensure distinguish of the two Airy disks that overlay with each other but is within the Rayleigh criterion.
4) sampling
In the laser scanning confocal microscopy, the relationship of the pixel size(or scanning line width) to the dameter of the Airy disk determines the nmber of pixels that are required to sample two adjacent Airy disks to achieve a certain contrast.
4. Please explain the physical process of fluorescent imaging, combing energy level and spectrum diagrams.
4.1 physical process of fluoresence
Fluorescence is a process that certain molecules absorb light of a certain frequency or kinds of frequecies, then raises their energy level to a brief excited state, and as they decay from this excited state, they emit fluorescent light, as it's showed in Figure 4.1. Basically, there are three steps during fluorescence. The first is excitation. When a photon which supplied by light source is aborbed by a fluorophore, the fluorophore creats an excited, unstable electronic singlet state s1. The second is excited state relaxation. The excited state lifetime is very short since its unstable on a high energy level, so the excited molecule will relax to a intermediate lower state and emit heat as the energy loss during a few nanoseconds. The third process is emission. When the fluorochrome molecule falls from the intermediate state to the ground state(its initial state before photon absorption), the energy loss will transfer into light and emit a photo with a frequence whose value is smaller than that of the absorption photon frequency.
4.2 physical process of fluoresence imaging
As is discribed in Figure 4.2, the excitation source(whether broad-wavelength sources or line sources)puts light into project light on the sample through the light projection pass and on the other side, light collection devices containing emission filter and detection and amplification through the light collection pass. The exicatation source need to be bright enough to produce sufficient fluoresent photons since the emission is actually very low, not to mention those can be collected and received into the eye. Another part is the Emission filter. Since the light throught the collection optics may include the scattered light of the exciation source, which does not contain the material information of the sample, thus can disrupt material detection and make error image formation, the fluoresece imaging system must filter these photons. The princinple used here is that the wavelength of excitation photon is shorter than that of the emission photon. Using some optical filter that allows only certain wavelength of light to pass and redirect the rest as is described in Figure 4.3 can reach this target. Besides, emitted light can also be filted to select only the range or band of wavelengths that is of interrest to the user. The other part is the detection and amplification devices. Photons that reached here should be amplified by PMT and collected by CCD and transfered into digital signal to form the image.
5. Please select a microscopic imaging system, draw its optical layout, and explain its imaging principle, the functions of main components and the key technology in the system. 
The confocal principle in epi-fluorescence laser scanning microscopy is diagrammatically presented in Figure 2. Coherent light emitted by the laser system (excitation source) passes through a pinhole aperture that is situated in a conjugate plane (confocal) with a scanning point on the specimen and a second pinhole aperture positioned in front of the detector (a photomultiplier tube). As the laser is reflected by a dichromatic mirror and scanned across the specimen in a defined focal plane, secondary fluorescence emitted from points on the specimen (in the same focal plane) pass back through the dichromatic mirror and are focused as a confocal point at the detector pinhole aperture. Refocusing the objective in a confocal microscope shifts the excitation and emission points on a specimen to a new plane that becomes confocal with the pinhole apertures of the light source and detector.
the confocal fluorescence microscope consists of multiple laser excitation sources, a scan head with optical and electronic components, electronic detectors (usually photomultipliers), and a computer for acquisition, processing, analysis, and display of images.
The scan head is at the heart of the confocal system and is responsible for rasterizing the excitation scans, as well as collecting the photon signals from the specimen that are required to assemble the final image. A typical scan head contains inputs from the external laser sources, fluorescence filter sets and dichromatic mirrors, a galvanometer-based raster scanning mirror system, variable pinhole apertures for generating the confocal image, and photomultiplier tube detectors tuned for different fluorescence wavelengths.
One of the most important components of the scanning unit is the pinhole aperture, which acts as a spatial filter at the conjugate image plane positioned directly in front of the photomultiplier. Several apertures of varying diameter are usually contained on a rotating turret that enables the operator to adjust pinhole size (and optical section thickness). Secondary fluorescence collected by the objective is descanned by the same galvanometer mirrors that form the raster pattern, and then passes through a barrier filter before reaching the pinhole aperture. The aperture serves to exclude fluorescence signals from out-of-focus features positioned above and below the focal plane, which are instead projected onto the aperture as Airy disks having a diameter much larger than those forming the image. These oversized disks are spread over a comparatively large area so that only a small fraction of light originating in planes away from the focal point passes through the aperture. The pinhole aperture also serves to eliminate much of the stray light passing through the optical system. Coupling of aperture-limited point scanning to a pinhole spatial filter at the conjugate image plane is an essential feature of the confocal microscope.
The entire depth of the specimen over a wide area is illuminated by the widefield microscope, while the sample is scanned with a finely focused spot of illumination that is centered in the focal plane in the confocal microscope.
In laser scanning confocal microscopy, the image of an extended specimen is generated by scanning the focused beam across a defined area in a raster pattern controlled by two high-speed oscillating mirrors driven by galvanometer motors. One of the mirrors moves the beam from left to right along the x lateral axis, while the other translates the beam in the y direction. After each single scan along the x axis, the beam is rapidly transported back to the starting point and shifted along the y axis to begin a new scan in a process termed flyback.
