Biological samples are quite complex structures with a lot of stuff in them. Looking for something very specific (for example looking at neurons inside C.elegans larvae) could be almost impossible many a times. Bright Field microscopy cannot reveal this.
Fluorescence microscopy comes in very handy in such situations. Here a component of interest is specifically labeled with a fluorescent molecule called a fluorophore (such as green fluorescent protein (GFP), DAPI etc:). Then by observing the fluorescence of the label the component of interest can be observed. Fluorescence Microscopy collects light only from the component of interest and not from other structures surrounding it (by filtering in the fluorescence wavelengths and filtering our all others).
Fluorescence is a quantum mechanical process that can be explained using the Jablonski Diagram shown in Figure 27.
The excitation process excites the ground state (S0) electron of the fluorophore to its lowest excited singlet state (S1). Excitation process happens very quickly in about few femtoseconds (fs). Since the S1 state is vibrationally broadened (has a number of vibrational states) excitation can occur over a range of wavelengths. This gives rise to the absorption spectrum of the fluorophore. Once the electron is in one of the higher vibrational levels of S1, it non-radiatively relaxes to the lowest vibrational level of the singlet excited state. This non-radiative decay happens in few picoseconds (ps).
The electron waits at the bottom of the singlet excited state for about few nanoseconds. Fluorescence occurs when the electron returns to the ground state with the emission of a photon. The ground state is vibrationally broadened as well. The electron can return to any of these vibrational levels before non-radiatively decaying to the lowest vibrational level of the ground state. This allows for a range of emitted photon energies (wavelengths), observed as the fluorescence emission spectrum.
Since the electron has lost some of its energy in non-radiative processes in the excited and ground states, the emitted photon has energy lower or in other words wavelength longer than the excitation photon. The difference between the peak excitation wavelength and the peak emission wavelength is called the Stoke's shift.
The total time for the whole process (time for excitation+non-radiative decay+emission) is the fluorescnce lifetime. This is fs+ps+ns which is essentially ns. So, the fluorescence lifetime ends up being the time taken for the electron to decay from the bottom of the excited state to the ground state in the fluorescence emission process.
The basic ray diagram of a fluorescence microscope is given in Figure 28.
Fluorescence Microscopy is all about separating the emission wavelength from all other wavelendths inlcuding the exciation light and any stray light. This is achieved with the help of the following optical components:
- Excitation Filter
- Dichroic Mirror
- Emission Filter
An epi-fluorescence configuration where the objective also acts as the condenser is used. The light from a broad-band intense light source like a Mercury Arc-Lamp is passed through the excitation filter. This filter transmits the excitation wavelength (band) towards the dichroic mirror. The dichroic mirror reflects the light onto the objective. After interacting with the sample the excitation light excites florescence in regions of the sample that has the specific fluorophore.
Fluorescence is mostly isotropic. The fluorescence light that emitted in the backward direction is collected by the objective and relayed to the dichroic mirror. The dichroic mirror is designed to transmit the emission wavelength and it, in turn, relays the light to the emission filter. The dichroic mirror behaves differently for the excitation and emission wavelengths. It reflects the excitation wavelength and transmits the emission wavelength. The emission filter, filters out any residual excitation or stray light and transmits the emission wavelength towards the detector.
The excitation light is way stronger than the emission light. The excitation filter should be designed to handle the entire power of the illuminaiton source usually in 100s of mWatts, of which few 10s of mWatts at the excitation band of the fluorophore is transmitted by the excitation filter . The fluorescence emission is usually in micro-watts, so the emission filter needs a very high transmission at the emission bandwidth and a sharp roll off outside this bandwidth.
In commercial fluorescence microscopes the three components are mounted in a single cube called the Filter Cube. This makes it convenient to change between fluorophores by changing the filter cube as a whole and not worry about individual components.
Configuring the fluorescence microscope in the epi-illumination configuration has a number of advantages:
- Objective doubles as the condenser. Alignment is easier.
- Resolution depends on both Objective and Condenser NAs. Since an Objective usually has a high NA and since it is doubling as the condenser the resulting resolution is high.
- Easier to separate the excitation and emission wavelengths. Most of the intense excitation light is transmitted through the sample and only a small fraction is scattered back. Hence separating excitation and emission wavelengths in the backward direction (epi-configuration) is easier.
Like in case of bright field imaging we need to setup the epi-fluorescence microscope to achieve Köhler Illumination. There is a small problem here. The Aperture Diaphragm needs to be placed at the back focal plane of the condenser. Since the objective doubles as the condenser, placing it there means both the illumination and collection NAs are affected. Reducing the Aperture Diaphragm to decrease the NA of the condenser will lead to decrease in NA of the objective as it collects the emitted light. To overcome this problem Köhler Illumination in epi-illumination uses a configuration as shown in Figure 28.
Figure 28 shows the schematic of an epi-fluorescence microscope that uses an arc lamp as the excitation source. Unlike in the case of Köhler Illumination for bright field imaging, the Aperture Diaphragm comes first in the light path and then the Field Diaphragm.
Consider a single point at the center of arc-gap. Light from this point is collimated by the Collector Lens and then focused by Lens B. Focal point of Lens B creates an image of that point on the arc-gap. As we discussed for Köhler Illumination in transmitted illumination configuration the focal plane of Lens B is a conjugate plane of the light source. We can place the aperture diaphragm here. Lens C is placed at its focal distance from Aperture Diaphragm. This hence produces a collimated beam of light that is directed into the Field Lens.
The Field Aperture is kept at the focal distance away from the Field Lens. This creates an image of the Filed Aperture on the sample plane. The Field Lens focuses the collimated beam of light reflecting through the dichroic mirror onto the back focal plane of the objective. This creates an image of the arc-gap point at the back focal plane of the objective. The objective then collimates this light into a beam pencil to impinge on the sample plane. Like what we discussed earlier the different points on the light source create beam pencils at different angles that intersect at the focal point of the objective (acting as condenser). This creates the cone of light, condensing it on the sample plane.
Note that we have essentially moved the position of the Aperture Diaphragm from the back focal plane of the objective to a point before it. We can now control the NA of illumination without affecting the NA of light collection by the objective.