Confocal Microscopy II

In this write-up I will discuss some advanced topics related to confocal microscopy. 

Why do we need to use lasers in a confocal microscope?

Lasers have a lot of disadvantages when compared to using an incoherent light source like a mercury arc lamp.

  1. They are expensive
  2. They lase at a single wavelength, unlike an arc lamp which produces like 10 discrete spectral lines. 
  3. For multiple excitation wavelengths, we need multiple lasers and then make their beams colinear in a laser combiner. The lasers, the combiner, and associated electronics and controller could cost almost 25 to 30% of a confocal microscope's cost.
  4. They have more complex electrical and heat dissipation requirements
  5. Finally, the very nature of coherence is not good for imaging. Coherence creates unwanted interference patterns (speckles) resulting in non-uniform illumination.

So why lasers in a confocal microscope? Why not use in-coherent light sources like arc-lamps as excitation sources in confocal microscopy?

The answer lies in Figure 12, from our discussion on Köhler Illumination. Let's take a closer look at this figure again as shown in Figure 1567/1.

 

Figure 1567/1: Light from an extended source is not focused to a diffraction limited spot. An image of the source is created at the sample plane.

Incoherent light sources like a tungsten lamp or an arc lamp are not point sources of light. They are extended sources. If we consider a single point on the source, let's say at the center of it, it creates a diverging beam of light. This can very well be collimated by a collimating lens and then projected onto the back focal plane of the objective (see Figure 1567/1). The objective, in turn, will create a single focused diffraction limited spot on the sample plane. This is what we ideally need in a confocal microscope. 

But now all the other points on the extended light source which also create collimated beams of light after passing through the first lens.  But these individual collimated beams are not parallel to each other. See Figure 1567/1. After passing through the objective these collimated beams get focused at different spots, essentially creating an image of the source on the sample plane. So we do not have a single diffraction limited spot on the sample plane. In fact, a demagnified image of the filament/arc gap is created on the sample plane. The image is demagnified since the objective focal length is shorter than the focal length of the collimating lens and light is traveling from collimating lens towards the objective. 

In a point scanning imaging system like a confocal microscope, the resolution depends on how small the excitation spot is. Scanning an image of the light source in a raster pattern does not certainly help in achieving any resolution at all. 

So how do lasers help?

See Figure 1567/2. A laser by virtue of being coherent produces a single collimated beam of light. This single collimated beam of light is focused to a single diffraction limited spot. 

Figure 1567/2: A Laser produces a single collimated beam of light which is focused to a single diffraction limited spot.

For the objective, it would look like light coming into it from a single infinitesimally small point source at an infinite distance away from it. The objective is in-fact trying to create an image of this single point source, which ends up being a diffraction limited spot. It is in this sense that a laser is a point source of light. 

As discussed in my earlier write-up on confocal microscopy, modern confocal microscopes do not directly beam the laser light onto the objective back focal plane. Instead, a laser combiner combines laser beams from different laser sources into a single collinear beam of different wavelengths. This colinear beam is then launched into an optical fiber using a focusing lens. This is shown in Figure 1567/3. Light out of the fiber is diverging and is collimated by a collimating lens. This collimated beam is further passed through the scanning mirrors and expanded by the telescope formed by the scan lens and tube lens (microscope) combination. An expanded and collimated beam of about 1cm is projected onto the back focal plane of the objective. This helps slightly overfill the back aperture of the objective to engage the full numerical aperture of the objective.

Figure 1567/3: Excitation light path of a modern confocal microscope

It may, however, be noted that the collimating lens, scan lens, tube lens and objective combination is projecting an image of the tip of the core of the optical fiber onto the sample plane. A demagnified image of the tip of the core of the fiber is projected onto the sample plane.

Let us assume a magnification of 60X for the objective, tube lens combination, and a magnification of about 1.5 for the scan lens, collimating lens combination. So a total magnification of about 90X. Assuming a resolution limit of about 250nm and considering the fact that the image is demagnified,  a diffraction limited spot is only formed if the fiber diameter is less than 250x90=22.5micrometers.

This is the reason why modern confocal microscopes use the so-called single-mode fibers for launching the excitation light. Single mode fibers should ideally have a core diameter so small that they propagate only one mode of the laser (transverse modes are not propagated). The core diameters are usually less than 15 micrometers. This would qualify them to be single mode for optical communication applications (where this term is most commonly used) as they use much longer wavelengths there like 1.5 um. For wavelengths used in confocal microscopy (350nm to 800nm) these fibers may not single mode in its literal sense. However, they satisfy our core diameter criteria for achieving diffraction limited spot on the sample plane. 

There is an added advantage to launching excitation light into an optical fiber. If a diode laser which may not have a proper Gaussian beam profile, the near single mode optical fiber will cut-off a large number of higher order modes and the fiber output would be near Gaussian.

So why not launch the light from an extended source into an optical fiber and clean it up?

The extended source will create an image of the source at the fiber input and hardly any light will get coupled in! So we definitely need lasers. A laser with a slightly bad beam profile (slightly incoherent) would work but not something that is totally incoherent.

So what about the issue of coherent light creating speckles?

The point scanning confocal microscope is only detecting the intensity of emission light from the focal spot. The PMT or other single pixel photodetectors used in confocal microscopy donot measure the spatial distribution of intensity. So this is not an issue.

What is de-scanning?

Look at the block diagram of a confocal microscope as shown in Figure 33. The scan optics (scanning mirrors + associated lenses) is placed between the primary dichroic mirror and the objective. This means that in addition to the excitation light fluorescence emission passes through the scan optics as well. A lot the precious emission light is lost here. 

So why can't we place the scan optics upstream in the excitation path before the primary dichroic mirror and avoid emission light from passing through the san optics?

If what we need to do is only scan the focused laser beam across the sample plane this will certainly work. The problem arises when we pass the emitted light through the pinhole.

Lets us understand this with Figure 1567/4. The figure shows the block diagram of a non-descanned detection based scenario for a confocal microscope. Here the scan optics is placed between the excitation path, upstream of the dichroic mirror. The fluorescence emission does not pass through the scanning mirrors. 

Figure 1567/4: Scan optics in non-descanned configuration with both X and Y scan mirrors set at 0 degrees.

Figure 1567/4 shows one situation where the scan mirrors are not deflecting the light. Both the X and Y mirrors are set to 0 degrees deflection. In this scenario, the laser beam would ideally be focused exactly on the center of the field of view of the sample by the objective. The emitted light retraces the path of the excitation light and passes straight through the primary dichroic mirror and will strike the pinhole exactly at its center. The out of focus light will be blocked and the in-focus light will be passed through the pinhole. 

Now let us consider a situation where the X scanning mirror is deflecting the light towards the right at a certain angle. This situation is shown in Figure 1567/5.

Figure 1567/5: Scan optics in non-descanned configuration with Y scan mirror set at 0 degrees and X mirror deflected to the right by a certain angle.

Figure 1567/5 shows a situation where one of the scan mirrors is deflecting the light. The Y mirror is set to 0 degrees and X mirror is deflected off-axis to the right by a certain angle. In this scenario, the laser beam should be focused to a point to the right of the center of the field of view of the sample. The emitted light retraces the path and is deflected off-axis as well and after passing through the primary dichroic mirror does not hit the center of the pinhole. This means the pinhole is simply blocking most of the light, including the in-focus ones.

In a non-descanned configuration, as the excitation beam is scanned in a raster pattern,  the emission light gets scanned across the pinhole in a raster pattern as well. Light from the entire field of view is blocked from reaching the detector, except for the fluorescence emission coming from exactly the center of the field of view.

One way out of this problem would be to install a second set of scan optics in the detection path, before the pinhole. This second scan optics (descan optics) will have to be precisely synchronized with the first one in such a way that it cancels the deflection of the emitted light. This way the emitted light will remain stationary at the pinhole while the excitation light is being scanned across the sample plane. So the scan optics will scan the excitation light on the sample plane and the descan optics will descan and  keep the emitted light beam stationary at the pinhole.

It turns out that we donot need to two sets of scan optics. A single set of scan optics can do both scanning and descanning if it is placed in the path that is common for both excitation and emitted light (between the primary dichroic mirror and the objective). This is what modern confocal microscopes do. 

Lets us assume that the X scanner is deflecting the excitation light by +10 degrees. When the emitted light returns it retraces the path and is traveling in the opposite direction. So as it passes through the scan mirrors it is deflected -10 degrees. The net deflection would be +10 + -10 = 0 degrees. This is true for any deflection angle for both the X and Y axis scan mirrors. The net deflection of the emitted light in the descanned configuration is 0 degrees. The emitted light remains stationary at the pinhole.

A single set of scan optics hence performs both scanning and descanning.

It must be noted that the scan mirrors are continuously moving. There is a time delay between the excitation light leaving the primary dichroic mirror and the emission light arriving at the dichroic mirror. The mirrors would have moved slightly during this time. Note that the fluorescence lifetime is in nanoseconds and light travels at the speed of light. So compared to the speed of motion of the mirrors (even for the high-speed scanners) the fluorescence emission is arriving almost instantaneously after the excitation has left the primary dichroic mirror. The mirrors are practically at the same position.