www.fluorescencemicroscopy.it

Transmitted Light Fluorescence Microscopy

Fluorescence microscopy with transmitted light (Diascopy) provides illumination of the sample from below. The optical path, in this case is that usual in the common microscope, with the lamp at the bottom and the light focused onto the sample through the condenser. An excitation filter, to select the desired wavelength, is placed in the holder of the microscope, a barrier filter (secondary) is placed over the revolver of the microscope to capture the residual excitation light and to select the right length wave that will reach the eye of the observer or the recording camera.
The sample is excited by light passing through the primary filter and the fluorescent emission is captured by the objective lens, before reaching the eyepiece it is filtered and then selected from the secondary filter. This mode eliminates the need for dichromatic mirrors.
The fluorescence with transmitted light or diascopic fluorescence, was born before the Epi-Fluorescence, but today has been largely abandoned due to two major problems. First, the excitation light from the condenser lens and passes through the objective lens can not be completely blocked by the transmit filter, this causes, consequently, a high level of background noise. In addition, the optical alignment and matching of condenser lens and objective lens are often difficult, a consequence of this is not optimal fluorescence.

Another possible configuration diascopic Fluorescence is one that includes the use of a dark field condenser (Darkfield condenser) in the optical path.
In this case the mirror will cast light on the sample condenser at oblique angles, preventing excitation wavelengths to enter into the objective.
Fluorescence in bright field is difficult to separate excitation light from the fluorescent light, because both types of light entering on the objective.
In fluorescence in dark-field, bright field condensers are replaced by condenser with high numerical aperture and immersion oil (darkfield condenser), this type of condenser directs the excitation light with a strong inclination towards the sample, because of the design of this type of condenser most of the excitation light never enters on the objective. The sample absorbs excitation light and emits only light at longer wavelength, is this light through the objective and then passes through the filter of the barrier to reach the eye of the observer. The resulting image is an object more or less intensely fluorescent on a dark background, all light is blocked by the spread of excitation barrier filter.

Although the equipment for fluorescence in dark-field transmitted light is relatively simple, the technique has significant disadvantages. The high numerical aperture of the immersion oil objectives or water is reduced by the use of the iris in order to prevent the excitation light enters directly into the objective(leading to loss of light intensity and resolution). The technique darkfield in transmitted light will also exclude the simultaneous use of fluorescence microscopy with Phase or Nomarski differential microscopy (DIC). This method also means that most of the light from the light source can not be used, as the excitation light radiating the majority of the sample outside the field of view is observed, thus reducing its effiicient.