44.1

This is the output from an argon laser. This laser is used for a scanning microscope. In most systems, the laser is deflected into a set of mirrors mounted on two perpendicular axes.

As the mirrors move, the laser beam is "rastered" over a specimen.

We have a single photon detection element, in the form of a photo-multiplier tube. For contrast, our CMOS or CCD cameras have many photon-sensitive elements crammed into a 2-D chip, placed at the back focal plane of the camera lens.

How do we take that single pixel from our scanning microscope to form an image? We form the image by registering the position of the mirrors with the light that is emitted from the sample. As the mirrors deflect the laser beam across the sample (row by row), we get a readout of the light coming for each "pixel" location. This is essentially the intensity information present on our digital cameras.

One additional note: we don't get RGB, nor do we get reflected light. This type of system generally requires the use of light emitting agents (termed fluorophor). The incoming laser actually excites (or stimulates) the fluorophor; the emitting light generally is red-shifted (so for a blue laser, we will generally get green, to orange, to red light).

We can place a bandpass filter to get only the peak of the emitted light; this allows us to use different fluorophors in the same sample, since different fluors have different excitation and emission wavelengths (i.e. color).

My profile picture is one example of a specimen "imaged" with a laser scanning microscope. It is a mouse brain structure called the olfactory bulb. The mouse had been modified to produce a green fluorescent protein. When scanned with a blue laser, the structure emits green light.

Since the structure is a 3-D sample, I took images at different focal planes and stacked them. The principle is much the same as focus stacking in photography; the laser scanning microscope has a "pinhole" (i.e. aperture). Closing it down allows us to reject out of focus light. We can alter the focus by simple moving our sample up and down, while the laser continues to scan in the x-y directions.