Etaluma Solid State Optics
With No Pixel Shift
Etaluma Solid State Optics Provide Composite Images With No Pixel Shift
Multicolor fluorescence microscopy is traditionally done by introducing an excitation filter, a dichroic filter, and an emission filter for each channel and sequentially changing them to acquire images in multiple channels of excitation/emission. Each of these filters is generally located on a filter wheel or linear positioner. The resulting individual images are then overlaid to achieve a color composite. Slight variations in each optical path as these filter wheels are physically repositioned cause each channel to be slightly displaced relative to the others. The end result is that co-localized fluorescence of more than one color appear adjacent to each other and not superimposed (see figure to right), or fluorescence colors that are separate from each other can overlap. This is referred to as pixel shift.
In an attempt to mitigate this problem in typical digital microscopy, a variable “expected pixel shift” can be entered into certain software so that some level of compensation can take place. For exact microscopists desiring the most accurate localization by immunofluorescence, having to rely on an approximate compensation that can be dialed in at any level is far from ideal. Furthermore, determining the level of compensation needed is time consuming and requires calibration under the same experimental conditions.
Etaluma’s LS Microscopes do not show pixel shift because our optics modules are solid state, i.e., there are no moving parts. The LEDs and filters are all stationary and the LEDs turn on in series. In contrast, when using a filter wheel or linear positioner, different filters are moved in and out of the optical path, causing pixel shift. Etaluma realized that “Pinkel” filter sets in which the same multiband beam splitter (dichroic) filter and multiband emission filter are used to image the different colors could be capitalized on to develop solid state optics.
To illustrate, the schematic to the right shows Etaluma’s solid state 3-color optics module used in the LS620 and automated LS720. The 405 nm LED is turned on first. The violet light goes through its excitation filter, is reflected by the dichroic filter up to the sample, returned as a narrow band of excited blue light that goes through the multiband dichroic filter and multiband emission filter, and then reaches the camera sensor. Second, the blue 488 nm LED is turned on resulting in a relatively narrow band of green fluorescence reaching the sensor, followed by the amber 590 nm LED turning on for capture of red fluorescence. Because of no movement within the optics, the resulting composited images show no pixel shift.
The light path in the LS microscopes is very efficient; there are relatively few lenses and filters as light makes its way from LED to sample to camera. This, combined with a highly sensitive CMOS sensor, means that LS microscopes achieve extremely high sensitivity compared to traditional microscopes.