Linearity with Exposure Time

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When 5.27 Mbq of 18f-FDG is placed in IVIS spectrum, it is noted that the intensity of the amount of luminescence and the total count for all pixel increase with exposure time. Exposure time affects two factors that determine intensity of luminescence observed;

The number of luminescence molecules that get excited and

The amount of energy absorbed from incident radiation

Luminescence attributed to fluorescence which determines the intensity of the color observed. The principles of fluorescence require the fluorescent molecules of the fluorophore be excited from ground state by exposure to photons of light. As the molecules return from excited from an excited state to ground state or any other lower energy level they dissipate the absorbed energy in various forms; heat and light (luminescence observed) being the major forms. When the fluorophore is exposed to incident radiation for a long period of time, its molecules gets more excited increasing the activity of the molecule. This is because they get more time to absorb more radiation. Considering that the energy absorbed by the molecules from the incident radiation determines the energy of the emitted radiation, then increasing the energy absorbed by the molecules results in an increasing in the energy of the emitted light which relates to the intensity of the observed luminescence. Therefore, particles that have been exposed to IVIS spectrum for a short time produce less luminescence compared to those that have been exposed for a shorter time.

The intensity of luminescence observed also depends on the number of molecules exhibiting fluorescent properties. Increasing the time of exposure enables more of the fluorescent molecules of the 18F-FDG to absorb photons energy from the incident radiation. As a result, more molecules are excited and as they return to a lower energy level most of them release the absorbed energy in the form of luminescence; hence increased the intensity of luminescence. Low time of exposure results in fewer fluorescent molecules getting excited hence lower intensity of luminescence emitted.

Linearity with dilution

Tonic water and tap water fluoresce with bright blue lamination due to the presence of quinine (a fluorophore) in tonic water. Quinine absorbs light and gets excited. It then releases blue light as it returns to the less excited state. Dilution affects the concentration of the fluorophores. At high concentrations the quinidine particles can interact resulting in overlapping of their electronic orbitals. This results in changes in their (orbitals) energetic levels seen as peak fluorescence shift. The greater the dilution, the less the fluorescent luminescence emitted. This explains why as the drops of water were added quinine was unable to absorb light and hence could not emit blue light. Moreover dilution does not only affect fluorescent radiation but also affects Cerenkov radiation. The principle of Cerenkov luminescence is the traveling of charged particles at a speed more than that of light in a specific medium which they are moving in. Dilution alters the intensity of Cerenkov luminescence in a linear manner. As dilution increases it is, Cerenkov luminescence diminishes.

Linearity with Field of View

It was noted that the ROI measurements in count mode decreased with increase in the field of view. The sensitivity of the measurement is dependent on several factors; the field of view being one of them. Smaller FOV give higher sensitivity measurement while large FOV gives lesser sensitivity measurements. As a result as the FOV increases less count are registered indicating an inversely proportional relationship between FOV and ROI measurement in count mode. This is also supported by the fact that as the count mode of FOV increase the number of spots visible also increase. The number of spots observed depends on the sensitivity of the instruments which is dependent on the FOV used. Smaller FOV results in higher sensitivity which results in a higher total count of ROI measurements and observation of more spots. On the other hand, as the FOV increases the sensitivity decreases leading to a less total count of ROI measurements and observation of fewer spots. Ultimately we end up with a linear relationship between the total count and the number of spots of the field of view.

Linearity of drops

Cerenkov radiation was taken from several numbers of drops approximately 50uL of 18F-FDG from one to seven that were placed separately in a plastic container and combined them together with additionally three drops of Tonic water, tap water and sample of 18F at the same exposure time which was 60 s. It is clear that when the activities increase the luminescence increases too. The activity of the dots is dependent on the number of fluorescent molecules that are available to absorb incident light and emit it as fluorescent luminescence. The different dot samples had different concentrations of fluorescent molecules meaning that they had different activity. Those with higher luminescence had greater activity resulting from possession of more fluorescent molecules. This conclusion becomes valid because the fluorophore was the same for all the spots (quinidine from tonic water) and only its concentration differed among the spots resulting to a different activity.

While the first spot over images and as more spots added and background increased was changed

A background is caused by direct interactions of photons produced by positrons emitted from 18F in the CCD chip. This leads to the production of a high signal within a single pixel. These events occur across the field of view, even when the activity source is localized. This may result from lack of focusing the gamma rays of the IVIS spectrum by the optics of the imaging system. Practically, this becomes problematic when high amounts of activity are present in the field of view. Even then, most CCD cameras have a cosmic ray rejection mode that can be effective in removing these events

When the dots are placed at different background both of them, emit light. The ROI measured for both the background were different; the air background was less than that of the plastic background. Utilizing of different backgrounds is essential of for obtaining a background-free signal of a given ROI measurement. This is because background can be a source of errors and differs from place to place hence making it essential to change the background.

How the image for dot 4 was affected by image 1, 2 and 3 because all place in one container.

The images of the fourth dot were affected by that of the first, second and third dots. The fourth dot had more luminescence compared to the first three dots due to higher activity. The first three dots contributed to this higher activity in the dots. The quinidine in the fourth dot absorbed both fluorescent and Cerenkov radiation from dot 1, 2 and 3 together with light from the IVIS spectrum resulting to more excitation leading to more activity and ultimately brighter luminescence. This made its image more conspicuous than those of dot 1, 2 and 3

How the amount of each spot affected the outcomes over images

Volume is also a key factor in determining the image to be observed. However, it is essential to note that volume and concentration are two factors that are interlinked. The greater the volume of the spot the more the Cerenkov luminescence generated. This is because charged particles travel faster than air for a longer distance in a medium of larger volume generating more Cerenkov luminescence. The effect of volume on fluorescent luminescence is limited however concentration of the drops is an essential determinant of the intensity of fluorescent luminescence in the drops.

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