One aspect of our business, that we work very hard on at Leeds, is our ability to listen to our customers and see where we can help out. Here are some useful tips pertaining to the LSV’s operation regarding distance and diopters:
As with any ALS system, the sensitivity of the LSV for screening is largely affected by distance between the LSV imaging head and the sample. The sensitive design of the LSV’s camera allows the LSV to be able to capture stains even at relativity great distances for overviews, however, if a screener is trying maximize the systems sensitivity for intensive screening of dilute stains or faint samples, the imaging head will generally need to work relatively close (between ~7-13 inches of working distance). The rule of thumb here for sensitivity is the closer, the better.
The reason distance plays such an important role in screening is that as light travels from a source it spreads, losing energy concentration as a squared function of distance, known as the inverse square law.
This distance effect is felt in two areas when working with the LSV, one from the light being projected by the LEDs to the sample (the excitation light), and the other from the fluorescently emitted light from the sample traveling to the camera (the emission light). This causes the impact of distance to be compounded as a squared function – with an end result of small decreases in working distance between the LSV head and the sample dramatically increase the amount of light that can reach the LSV camera – and therefore dramatically increasing sensitivity of the system to faint stains.
In order to allow the LSV head to optically get closer to the sample for this increase in sensitivity, the LSV utilizes diopters, which are also known as close up lenses.
The LSV is equipped with 4 diopter positions, a +3.5, +2, +1 and an open position with no diopters equipped.
Rule of thumb – the larger the numbered diopter, the closer the LSV head can get to the sample and focus. The closest working distance is obtained at the +3.5 setting and the longest working distance (for the largest overviews) employs the open position with no diopters equipped.
Generally a good balance of working distance, sensitivity and field of view, can be obtained at the lower working distance of the +2 diopter position, however, if working on dilute or low fluorescence samples you may need to bring the head even closer and utilize the +3.5 diopter.
Each diopter has a working distance range, to see approximately what this range is in the visible light (as oppose the infrared setting), simply mouse over the diopter icon. Please note that when you switch from the visible light to infrared, you may need to go up one diopter number to get optimal focus.
Visible Light to Infrared Example: A user is imaging with the +1 diopter with the blue light and orange filter (in the visible spectrum), and they decide that they would like to inspect in infrared. To have optimum focus in the infrared at the same working distance as in the blue light orange filter, they may need to switch to the +2 diopter.
If the pictures from the LSV appear to be fuzzy, or out of focus; try looking at the diopter and verifying that the working distance of the LSV head matches the working distance range of diopter you are using.
A way to verify if the correct diopter is being used is to optically zoom in through use of the zoom slider in the software and attempt to focus. If the image cannot focus then the the LSV’s working distance is not matching up with the diopter equipped and you can either change the LSV’s working distance, or switch to a different diopter.