Update on two-color imaging

November 5, 2019

by Abhijit Marar

The parts for the Light-sheet illumination scheme have arrived and we have begun assembling it.

Two-color imaging:

A common concern when performing two-color imaging is the lateral chromatic shift caused by optical elements in the emission path of the optical system. For example, in the emission path of the optical system being used to perform two-color imaging of the NMJ, we employ a multiband emission filter (Semrock, FF01-446/523/600/677-25, USA) to separate out the emitted wavelength of interest (600 nm and 677 nm in our cases). In addition to the multiband emission filter, we also use single-band emission filters centered at 600 nm and 677 nm to avoid cross-talk between the two channels. Doubling up on the filters has the added advantage of suppressing light from other wavelengths. Since the two single-band filters are different optical elements, one notices a slight lateral shift in the image when acquiring an image of the two channels. This can be seen in Fig.1(C) and Fig.1(F).

Fig 1: Chromatic shift seen when imaging 200 nm tetrapeck beads using laser light of 647 nm and 561 nm. (A) Image of 200 nm tetraspeck beads seeing using 647 nm laser (B) Image of 200 nm tetraspeck beads seeing using 561 nm laser
(C) Composite of (A) and (B) showing two-color data (D, E, F) Magnified images of the central 128 X 128 pixels of (A), (B) and (C)

One can see from Fig.1(F), that the images of the same beads acquired under the two channels are slightly shifted from each other in the lateral dimensions. In order to have an accurate representation of two-color super-resolution images, it is imperative that this shift is corrected for. There are several algorithms that perform the registration of two-color SMLM data. In order to correct for the chromatic shift in our data, we used the NanoJ plugin in Fiji. Using the plugin, a translational mask was estimated as shown in Fig.2. The translational mask shows the translational shift in both the x-dimension (Fig. 2(A)) and y-dimension (Fig. 2(B)) across the entire field of view.

Fig.2: Translational mask estimated from data shown in Fig. 1. (A) Estimated chromatic shift in x across the 512x512 field of view (B) Estimated chromatic shift in y across the 512x512 field of view

Using the translational mask shown in Fig.2, any data acquired from the optical system can now be corrected for the chromatic shift. Fig. 3 shows the results of using the translational mask on the data acquired in Fig.1.

Fig 3: Results of chromatic shift correction on data shown in Fig 1. (A) Two-color image of beads acquired using the two channels pre-registration (B) Two-color image of beads acquired using the two channels post-registration

The translational mask shown in Fig.2 has also been used to correct the chromatic shift in data collected when imaging the growth cone tagged with hrp-AF647 (red) and synapsin-CF568 (green) (Fig.4).

Fig 4: SMLM image of the growth cone tagging HRP (red) and synapsin (green) (A) before chromatic shift correction (B) after chromatic shift correction.