There are two major forms of laser scanning microscopy, namely confocal laser scanning microscopy (CLSM) and multiphoton laser scanning microscopy (MPLSM).
The two forms are very similar at the illumination side (as opposed to the detection side of the methodology).
A beam of laser light is focused into a small point at the focal plane of the specimen, for example inside a cell loaded with a probe.
A computer controlled scanning mirror can move or scan this beam in the X-Y direction at the focal plane.
Thus the name scanning microscopy.
The florescent emission created by the point as it scans in the focal plane is detected by a photon multiplier tube.
This detection input is reconstructed by computer hardware into an image.
There are two major modes of scanning when conducting dynamic imaging (where a series of images are taken in living cells or tissue).
In line-scanning the laser is "parked" such that it continually scans the same X line through the specimen.
Line scanning give a very high temporal resolution.
For the work horse of confocal microcopy, the Biorad MRC 600 or 1000, it takes approximately 6 ms to make one X scan.
Thus the microscope is generating new information every 6 ms, a very fast time resolution.
The spatial resolution however is very poor, consisting of one line through the cell or specimen.
Higher spatial resolution is obtained by conducting X-Y scans.
Here the laser scans the specimen in the X direction and then moves slightly in the Y direction before again scanning across the focal plane in the X direction.
The laser point goes back and forth at the focal plane until the entire X-Y plane has been scanned.
This gives an obvious gain in spatial resolution over line scanning.
Now the entire X-Y region of the specimen is examined.
This increased spatial resolution comes at a cost, however, namely in the temporal resolution.
It takes about 400 ms to complete an X-Y scan on the Biorad MRC 600 (using only 1/4 of the screen to speed things up).
The X-Y scans can be visualized as a stack, with each image giving new information every 400 ms.
Image processing software can be applied to analyze the behavior of the probe in different regions with time.
Finally, it should be mentioned that X-Y-Z scans are also possible.
Here the stage of the microscope is driven by a computer controlled motor to take the specimen to different Z positions following each X-Y scan.
In this way 3-dimensional reconstruction's can be made of the specimen.
The X-Y-Z mode of scanning is very common in immunocytochemistry and other staining methods but is rarely applied in dynamic imaging, where time is important, because it is too slow.
Confocal laser scanning microscopy uses gas lasers, the most common being an Ar/Kr laser.
Gas lasers give a steady stream of photons (represented by the blue circles in the figure to the right).
While the laser light is focused on the focal plane (f.p.) considerable fluorescence is created above and below the focal plane as well.
This fluorescence, if it makes its way to the photon multiplier tube, will cause blurring of the final image.
To get rid of this light a pin hole is introduced between the detector and the specimen.
Light outside of the focal plane (yellow in the figure to the left) is largely excluded from hitting the photon multiplier tube.
The geometry of the light in the focal plane (green in the figure) is such that it passes through the pin hole and is thus detected by the photon multiplier tube.
Thus, the focus plane of illumination is the same as the focal plane of detection.
In other words they are Confocal !!!
This combination creates a sharp image or optic section of the situation inside the specimen.
In multiphoton laser scanning microscopy solid state lasers are use (titanium sapphire lasers are very popular).
An intrinsic property of such lasers is that they give off photons in pulses, which, for a titanium sapphire laser are at 10 ns intervals.
Also, the solid state lasers give light off at a longer wave length that the gas lasers use in CLSM.
Thus the energy of the light is lower...in fact so low that when a probe absorbs a photon from the first pulse of light the electron is not elevated completely to an excited state.
Rather it is elevated to an intermediate or "pseudo" excited state.
From this state it will fall back to the ground state (S0) without giving off any fluorescence.
If, by chance, the probe is to absorb a second photon, coming from a subsequent pulse of the laser, then the electron can be elevated to the true excited state.
Consequently, florescent light will be given off as it returns to the ground state.
In this example 2 photons were required to get the electron to the excited state and thus we have a 2-photon event.
In practice three or four sequential events are often required, referred to as multiphoton events.
The region excited by MPLSM is much smaller than that excited by CLSM.
In fact, the region of probe activation is so restricted around the focal plane (f.p.) in MPLSM that no pin hole is required.
Essentially all the fluorescent light is coming from the f.p. and thus it can go directly to the photon multiplier tube.
The reason the fluorescence is so restricted in MPLSM is because the probability of a multiphoton event is extremely low and occurs only where the laser light is the most intense i.e. at the focal point.
To explain this, consider a probe being illuminated with laser light at the intensity of sunlight on earth.
In this case a 1 photon (1 P) event occurs about once every second.
This is the situation with short wave length lasers used in CLSM.
A 1P event is rather easy to induce and it occurs even outside the focal plane.
Now consider a 2 P event.
At sunlight intensity such an event would occur once every 10 million years!
So, in MPLSM where do we find the 2P events?...... the answer is where the laser light is most intense i.e. at the focal point of the laser beam.
The chance of having a 2P event outside the focal point is drastically reduced and thus we have the fluorescent light confined to a very small region, at the very point of focus of the laser beam.
A 3P event is even more rare, occurring only once in the life time of the universe at the intensity of sunlight.
High power laser beams, focused into a very small points, generate a light intensity considerably higher than that of sunlight and thus 2P and higher multiphoton events are useful in generating extremely sharp images, without the necessity of a pin hole.
MPLSM is more sensitive that CLSM because all the light generated to make an image is sent directly to the photon multiplier tube.
This contrasts with CLSM where a pin hole is required to select the light from the focal plane. In CLSM there is considerable loss of signal in the optics required to direct the light to the pin hole.
MPLSM gives a sharper image than CLSM because of the lack of extraneous light and improved geometry of detection. In MPLSM the photon multiplier tube can be placed very close to the specimen whereas CLSM has all the intervening optics and the pin hole.
MPLSM uses solid-state lasers which are more stable that the gas lasers used in CLSM.
The longer wave length used in MPLSM has an advantage because it is inherently less damaging to biological material than shorter wave lengths. Short-wave length laser light used in CLSM is known to produce free radicals which can damage biological material.
Also, the longer wave length light used in MPCLSM is more penetrating than the shorter wave length used in CLSM. It has been estimated that MPLSM penetrates 5 times deeper into biological material (up to 500 mm versus 100mm for CLSM).
This point is an important consideration when one wants to image in tissue.
NOTE: The above illustrations can also be viewed in an animated slide show.