Hello classmates!
Continuing from the previous issue, today we will continue to introduce the other four of the seven observation methods of the Leica microscope.
4. Phase contrast observation
In the development of optical microscopes, the successful invention of phase contrast microscopy was an important achievement in modern microscopy technology. We know that the human eye can only distinguish between the wavelength (color) and amplitude (brightness) of light waves. For colorless and transparent biological specimens, when light passes through, the changes in wavelength and amplitude are not significant, making it difficult to observe the specimen in the open field.
Phase contrast observation: A phase contrast microscope uses the difference in the optical path of the object being examined for microscopic examination, which effectively utilizes the interference phenomenon of light to transform the indistinguishable phase difference of the human eye into a distinguishable amplitude difference. Even colorless and transparent substances can become clear and visible. This greatly facilitates the observation of living cells, so phase contrast microscopy is widely used in inverted microscopy.
Principle of phase contrast observation: A phase contrast microscope converts the optical path difference of visible light passing through the specimen into amplitude difference, thereby improving the contrast between various structures and making them clear and visible. The light refracts through the specimen, deviating from the original light path and being delayed by 1/4 λ (Wavelength), if further increased or decreased by 1/4 λ， Then the optical path difference becomes 1/2 λ， The interference between the two photosynthetic axes is strengthened, and the amplitude increases or decreases, increasing the contrast. In terms of structure, phase contrast microscopy has two unique features that are different from ordinary optical microscopes.
5. Differential interference observation
Differential interference contrast DIC: In the 1950s, French optician Normanski improved the Wollaston prism, and improved the DIC theory and the use of DIC in the microscope on the basis of Wollaston prism
Differential interference principle: By using a specially designed prism to decompose polarized light into beams that are perpendicular to each other and of equal intensity, the two points closest to the beam (smaller than the resolution of the microscope) have differences, giving the image a three-dimensional feel
DIC components: polarizer, 2 DIC prisms (1 in the condenser and 1 in the objective turntable)
Advantages:
1) It can create a three-dimensional sensation for the inspected object
2) More intuitive observation effect
3) No special objective required
4) Combined with fluorescence observation, it can better adjust the color changes of the background and objects to achieve the desired effect
Disadvantages:
1) Requires high light intensity
2) Birefringent substances cannot achieve DIC effect
3) Cannot be applied to observation of plastic container cultures
4) The detection sensitivity has directionality and the adjustment is complex
Main applications: fine structures of colorless transparent living specimens, colorless fluorescent specimens, stained specimens, microscope operations, etc
6. Fluorescence
Fluorescence:
1) The energy absorbed by electrons in matter changes from a low energy state to a high energy state, and then releases light when it returns to a low energy state, which is non temperature radiative light - cold light.
2) Microscopic fluorescence using light source excitation photochemical fluorescence
Fluorescence properties:
1) Absorbing light requires an excitation light source
2) Fluorescence wavelength>excitation wavelength (loss of thermal energy)
3) The fluorescence intensity is extremely lower than the intensity of excitation luminescence
4) There is varying degrees of attenuation
5) The fluorescence intensity depends on the excitation intensity, the concentration of the tested substance, and the fluorescence efficiency
Advantages:
1) High detection ability (amplification effect)
2) Low stimulation to cells (can be stained in vivo)
3) Can perform multiple staining
Purpose:
1) Observation of Object Structure - Fluorescein
2) The presence or absence of fluorescence, color comparison for substance discrimination - antibody fluorescent lamp
3) Determination of fluorescence quantity for qualitative and quantitative analysis of substances
7. Hoffman observation
Hoffman observation principle: Oblique light irradiates the specimen to produce refraction and diffraction, and the light passes through the objective density gradient regulator to generate different shadows, resulting in differences in brightness and contrast on the surface of the transparent specimen, increasing the observation contrast
Advantages:
1) Strong stereoscopic imaging effect, no halo
2) Applicable to plastic/glass Petri dish
3) Detectable birefringent substances
4) The working distance of the condenser is greater than DIC
5) Adjustable brightness changes between background and specimen
6) Objective lenses can be used for bright field, dark field, and fluorescence observation
Disadvantages: Debugging, slightly complex application, and high cost
Application: Microscopic manipulation, internal structure, external morphology observation
At this point, all seven observation methods of the Leica microscope have been introduced to the students.

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