4
Optimizing signal acquisition can easily be performed on the NucleoCounter®
NC-3000
TM
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 the exposure time in the NC-3000
TM
needs to be determined empirically
for the initial experiment but, once determined, the settings can be applied to
all the following 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, has a significant
chance of being over-exposed with the default of 1000 milliseconds. However
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.
The denotation of emission filter as e.g. Em530/15 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
editing mode. Change the x-axis to the appropriate channel and the
parameter 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’.
