ChemoMetec NC-3000 - Manual (Page 5)

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Optimizing signal acquisition can easily be performed on the NC-3000 Nu-
cleoCounter by adjusting the exposure time in a manner similar to that used
in photography. If the image is under-exposed, it will be darker and much of
the finer detail may not be seen. Similarly, if it is over-exposed the pixels will
become saturated and information will also be lost. Optimizing of the exposure
time in the NC-3000 needs to be determined empirically for the initial experi-
ment but once determined, the settings can simply be applied to all the follow-
ing samples. That said, the default setting (200 milliseconds for LED(365) and
LED(405) and 1000 milliseconds for LED(475), LED(530) and LED(630)) will
fit most applications and optimization of exposure time may not be required.
As an example GFP coupled to a highly expressed protein have a significant
chance of being over-exposed with the default 1000 milliseconds, whereas
most fluorochrome coupled antibodies bound to an expressed protein will be
appropriately exposed with 1000 milliseconds.
Optimising Exposure Time
Figure 3 . A full list of possible LED/Emission filter combinations available.
The denotation of emission filter as e.g. EmS30/15 means a bandpass filter
that allows light of wavelength 530nm ± 15nm (515nm - 545nm) to pass.
Blue LED Green LED Red LED
Darkfield / Ex365 Light source
Violet LED UV LED
Ex365 Ex405 Ex475 Ex530 Ex630
Available emission filters Em430/20
Em470/55
Em475/15
Em560/35
Em675/75
Em630LP
Em740/60
- / Em470/55 Em475/15
Em530/15
Em560/35
Em560/35
Em580/25
Em675/75
Em675/75
Em630LP
Em740/60
Em740/60
UV
(Couterstain)
Darkfield
Insert a sample stained with a single fluorophore of interest and masking
stain (DAPI or Hoechst 33342) if flourescent masking is chosen into the
NC-3000.
Set the exposure time to be evaluated in ‘Protocol Adaption Wizard’ (See
section: Editing Image Capture and Analysis Parameters).
Run the sample using the protocol with the adjusted exposure time.
The data will automatically open in ‘Plot Manager’. Add a histogram to
data by clicking on the histogram icon, on the left-hand side of the
data row.
Double-click on the small histogram to open the large histogram in edit-
ing mode. Change the x-axis to the appropriate channel and the param-
eter to ‘Max Intensity’. This will display the signal intensity for the most
intense pixel/cell rather than average intensity for the area defined as a
cell when the scale is set to ‘Intensity’.