Differences and advantages and disadvantages between transmission fluorescence microscopy and reflection fluorescence microscopy
1. Transmission fluorescence microscope: The excitation light source is transmitted through the specimen material through a condenser to excite fluorescence. Commonly used dark field concentrators, or ordinary concentrators, can be used to adjust the reflector to convert excitation light and side light onto the specimen. This is a relatively old-fashioned fluorescence microscope. Its advantage is strong fluorescence at low magnification, while its disadvantage is that its fluorescence weakens with increasing magnification. Therefore, it is better for observing larger specimen materials.
2. Falling beam fluorescence microscope: This is a new type of fluorescence microscope that has developed in modern times. Unlike the previous one, the excitation light falls down from the objective lens onto the surface of the specimen, using the same objective lens as the illumination condenser and the objective lens for collecting fluorescence. A dual color beam separator needs to be added to the optical path, which forms a 45 degree angle with the photouranium. The excitation light is reflected into the objective lens and gathers on the sample. The fluorescence generated by the sample and the excitation light reflected from the surface of the objective lens and cover glass simultaneously enter the objective lens and return to the dual color beam separator, separating the excitation light and fluorescence. The residual excitation light is then absorbed by the blocking filter. If different combinations of excitation filters, dual color beam separators, and blocking filters are used, they can meet the needs of different fluorescent reaction products. The advantages of this fluorescent microscope are uniform field of view illumination, clear imaging, and stronger fluorescence with higher magnification.
The advantages and disadvantages of transmission fluorescence microscopy
Light source: High pressure mercury lamp or halogen lamp.
Spotlight mirror: Using a dark field spotlight to separate excitation and fluorescence.
Objective lens: Any type of objective lens can be used.
Specimen: Thin specimens are more suitable for transmission observation.
Advantages: ① Due to the use of a dark field condenser, the excitation light cannot enter the objective lens, making it easy to form a dark background and achieve good contrast, and any objective lens can be used for observation. ② When using a low magnification objective, it is brighter than a reflective fluorescence microscope.
Disadvantage: The center and height of the spotlight must be adjusted correctly, otherwise a bright fluorescent image cannot be obtained. Due to the focal length limitation of the dark field spotlight, thick glass slides cannot be used. The illumination area of the field of view should not be too large, otherwise it may cause the fluorescence color of the fluorescent specimen to fade. Thick or opaque specimens are not suitable.
The advantages and disadvantages of reflective fluorescence microscopy
Light source: High pressure mercury lamp or halogen lamp.
Focusing lens: As the objective lens serves as a focusing lens, there is no need for a focusing lens.
Objective lens: The objective lens used must be able to transmit sufficient ultraviolet light and not produce fluorescence itself, with no limitation on numerical aperture.
Specimen: Both thick and opaque specimens can be observed.
Advantages: ① As the objective lens also serves as a spotlight, it does not require much adjustment. ② By using the Kolar illumination method, the effects of aperture and field of view apertures can be utilized The numerical aperture of the objective lens is not limited, and high-resolution images can also be obtained at high magnification Thick or opaque specimens can also be observed, and thick cover slips can also be used Other observation methods can also be combined with reflected fluorescence, especially with phase difference method and transmission differential interference method It can avoid unnecessary fluorescence color fading phenomenon.
Disadvantage: When using a ground magnification objective, the image is relatively dark, so a high numerical aperture objective must be used. The filter system must effectively separate excitation luminescence and fluorescence.,
