Flat mirrorPhysics

And if you are exploring fluorescence in nature? Longpass is definitely the way to go. There is no way you could capture this image of an Ageratum flower with a bandpass filter.

** Zebrafish courtesy of Dr. Martha Marvin (Williams College), transgenic line bred by Dr. Lara Hutson (University of Buffalo).

Flat mirrordefinition

At point a, a wavefront is generated due to the secondary source on ray 2. At the same time, other wavefronts are generated at points c and b. Since wavefronts at points, a and b are generated at the same time ac = cb. Thus, the triangle acb is isosceles and the angles θ1 = θ2.

The purpose of any barrier filter in a fluorescence application is to increase the viewing contrast of what you want to see (the ‘signal’). The main job is to block reflected light from the excitation source and transmit the fluorescence emission. The next source of potential interference (‘noise’) is fluorescence from other substances in the viewing area that can mask the signal. This might be background fluorescence from things like growth medium (common when working with C. elegans) or parts of the subject itself (e.g., chlorophyll in plants or the yolk of a developing zebrafish).

Flat mirrorExamples

Have you ever thought about why we can see our image in a plane mirror? It’s because of the phenomenon known as reflection. Light waves, sound waves, and water waves can undergo reflection. In this session, let us learn about the reflection of light and the types of reflection in detail.

The bandpass filter can potentially cause confusion if the noise has spectral overlap with the signal. With the longpass filter you have two potential ways to distinguish the signal from the noise – intensity and color. With the bandpass filter you remove the color (spectral) dimension and leave only the intensity dimension. The images below of a fluorescent transgenic zebrafish illustrate this. This line of fish** expresses GFP in the heart and mCherry in the blood cells. The image below is a composite of two images – one taken using the longpass filter and the other with the green bandpass filter in place.

Since GFP (green fluorescent protein) and other green emitters (e.g. FITC) are the most widely used fluorochromes, our Royal Blue excitation/emission sets are the most popular option for the Stereo Microscope Fluorescence Adapter. We are often asked for advice in selecting between the yellow longpass and green bandpass barrier filter options. This article will address the pros and cons of each.

If the noise that you want to eliminate does not overlap (spectrally) with the signal then the bandpass filter is a good choice. This is the case, for example, with the red fluorescence from chlorophyll in Arabidopsis or other plants, as in the images below*. Chlorophyll emits in the far red, with its peak at about 685 nm. In the image on the left, made with the longpass filter in place, the red fluorescence makes it hard to distinguish the green GFP fluorescence. The green bandpass filter eliminates this, making it easy to see the GFP fluorescence in the leaves.

Flat mirroruses

Flat mirrorreflection

Consider the light rays 1, 2 and 3 shown by solid lines. The wavefronts which are perpendicular to these light rays are shown by the thin lines. The secondary wavefronts generated are the circular fronts described.

In the top image you can see the green-fluorescent heart, the red-fluorescent blood cells, and the natural yellow fluorescence of the yolk. These are easy to distinguish. In the lower image everything appears in varying intensities of green. The heart is bright but the yolk is not that much dimmer. It would be incorrect to say that all green is associated with GFP expression. In this case you would be better off viewing the subject with the longpass filter.

When a ray of light hits a polished surface, and the ray bounces back, it is known as the reflection of light. The incident light ray that falls on the surface is reflected off the polished surface. The light ray that bounces back is known as the reflected ray. A perpendicular drawn on a reflecting surface is called normal.

Flat mirrorimage

In practical terms, that means that the bandpass transmits only green wavelengths, while the longpass transmits greens, yellows, oranges and reds.

While you might think that it would be better to choose a filter that is closely matched to the expected emission, this is not always the case.

When the light rays get stroked on the flat mirror, they get reflected back. According to the laws of reflection, the angle of reflection is equal to the angle of incidence. The image is obtained behind the plane, which is present in the mirror. This process of obtaining a mirror image which is virtual and erect is known as a reflection on a plane mirror.

There are cases where the choice is less obvious. The growth medium for C. elegans has some background green fluorescence. The green bandpass filter does seem to add some enhanced clarity and contrast compared to the yellow longpass. In a number of tests we have had several observers look at the same specimens. Some have preferred the longpass because they feel it actually helps to see some of the background for context, while others preferred the bandpass for the same sample. In this case, and there may well be others, the choice is not completely obvious and may come down to a matter of personal preference.