Image Acquisition
In any microscopy set-up – fluorescence or brightfield - the optical detector being utilized must be carefully selected to deliver the highest quality image possible for the application. This selection is not often trivial, as the researcher must take into account a myriad of variables, most of which are mutually exclusive. Often times, one variable is optimized while others are negatively impacted. Therefore, in most live cell microscopy situations, the primary task becomes balancing the health of the cells with the quality of the image; taking into consideration factors like cell viability, signal to noise ratio, and required resolution or speed of acquisition. The aims and goals of the particular investigation should determine the order in which these factors are prioritized.
Detector choices include color CCD cameras to capture brightfield images or samples containing multiple fluorochromes, infrared sensitive cameras often utilized in DIC mode imaging, electron-multiplying CCD cameras to capture low-level fluorescence signals, high-resolution monochrome sensors for TIRF and other specific fluorescence techniques, and many more.
Pixel Shift and Multiband Filter Sets
When making a multiple-exposure photograph or multiple electronic images of specimens stained with several fluorochromes, using separate filter cubes in a standard microscope, there will be unavoidable registration shifts between exposures. Varying amounts of wedge in the emission filter and beamsplitter, variations in thickness and alignment of the beamsplitter, and mechanical vibration that occurs when the cubes are switched all contribute to this shift. Although for some applications these effects can be reduced to acceptable levels, many others require more sophisticated filter designs and optical apparatus. Table 1 lists some available methods for applications involving multiple probes. These include the multi-band filter sets introduced earlier on the multiband filter page, and various configurations that utilize both multi-band and single-band filter components.
Methods for applications using multiple probes
All of these methods are designed to eliminate the aforementioned registration errors. Method 2 offers the ability to visually observe up to three colors simultaneously. (Methods 3 through 6 can also do this if multi-band filters are added to the filter sets.) It should be noted that this list is only a guide, not an exhaustive list of all possible methods and configurations.
METHOD | COMPONENTS | ADVANTAGES | DISADVANTAGES |
1. Separate single-band filter sets | Standard microscope | No extra equipment is necessary Brightest images | Simultaneous imaging is not possible Imprecise imaging of combined images |
2. Multi-band filter sets | Standard microscope | Simultaneous visual observation with no registration errors No extra equipment required (other than special filter set | Reduced brightness Not recommended with xenon arc illumination Color balance is fixed |
3. Multi-band beamsplitter and emitter, single-band exciters | Microscope with a filter wheel or slider in the illumination path Photo camera, or electronic camera with image processing | Sequential visual observation with no registration errors Precise registration of combined images; adjustable color balance Optimized exciters offer brighter image than method 2 | Extra equipment (a filter wheel or slider) is necessary |
4. Multi-band beamsplitter, single-band emitters and exciters, single camera | Microscope with filter wheel or sliders in both the illumination path and imaging path Photo camera, or electronic camera with image processing | Exciters and emitters can be designed to have brightness similar to method 1. Reduction in registration error (by eliminating movement of the beamsplitter) | Extra equipment (two filter wheels or sliders) is necessary
Registration errors between emitters still might be present. |
5. Multi-band beamsplitter, single-band emitters and exciters, multiple cameras | Microscope with filter wheel or slider in the illumination path. Beamsplitter assembly for separating channels, each channel having a separate emission filter | In addition to the advantages in method 4: Registration errors from emitters can be eliminated. Additional applications are supported (e.g. ratiometry) | More complicated apparatus requiring additional beamsplitters and cameras |
6. Neutral beamsplitter, replacing multi-band beamsplitters in methods 4 and 5 |
| Any valid combination of emitter and exciter can be used | Brightness may be reduced by as much as 80%, so special light sources are recommended (e.g. laser illumination) |
