 | Acta Crystallographica Section F Acta Crystallographica Section F STRUCTURAL BIOLOGY COMMUNICATIONS |
journal menu
![[Figure 3]](wd5124fig3mag.jpg)
|
|
Figure 3
A difficult test to compare the abilities of UV microscopes to detect a protein crystal. Two examples of a protein crystal are shown in hanging-drop experiments. (a) A crystal of the cytoplasmic domain of the sodium bicarbonate cotransporter (NtNBCe1) is shown. (b) Microcrystals of inositol 1,4,5-trisphosphate receptor-binding protein (IRBIT) are shown. Fluorescence (FL): the partial attenuation of UV light by a 0.96 mm glass cover slip distinguished the microscopes based on the quality of their optical hardware to detect emitted light by epi-illumination with 1 s exposures. The ability of microscope 1 to detect emitted light from the crystal is lower than the others, if the camera is indeed detecting any light. Microscope 2 is the most sensitive as is apparent from the bright and sharp images for both crystal examples. Interestingly, there seemingly appears to be a hint of external or reflected light, i.e. light that does not originate strictly from the crystal(s) and/or perhaps an enhanced sensitivity of the camera. The FL images for microscope 3 (zero gain) and microscope 4 are similar in intensity but not in clarity. Brightfield (BF): BF images are useful for identifying individual protein and salt crystals when compared with FL images. However, a few of the BF images shown have anomalies: the images from microscope 1 were actually inverted and reverted; here they have been correctly oriented using third-party software. Also, note that the BF image from microscope 2 is significantly dark or black around the rim, masking crystals (see white arrow) and prohibiting usable side-by-side images of BF and UV. Furthermore, note that both microscopes 1 and 4 do not give a one-to-one positional correspondence or register between the BF and FL images owing to the fact that there are two light sources on the camera that are mounted at different locations. Absorbance (AB): microscope 4 can also detect protein crystals by transilluminating UV light from the bottom of the tray and detecting from the top of the microscope. Note that microscope 4 clearly identifies the NtNBCe1 crystal as protein by AB, thereby suggesting that AB is a more robust method than FL. However, microscope 4 is not able to clearly identify IRBIT as protein by AB, perhaps owing to the location of microcrystals at the edge of the drop, thereby showing some limitation under these conditions (see Fig. 4 ![[link]](../../../../../../logos/arrows/f_arr.gif) ). |
![[Figure 3]](wd5124fig3.jpg)
| STRUCTURAL BIOLOGY COMMUNICATIONS |
ISSN: 2053-230X
