The lab that I work in recently set up a microscope suite for high quality imaging of cell and tissue samples prepared mainly for immuno-staining and morphologic analysis. The suite is composed of a
Leica DM4000 B Microscope, an
EXFO X-Cite illumination system and a SPOT RT KE 3-shot color camera with accompanying SPOT software from Diagnostic Instruments (www.diaginc.com). This set-up has proven to be extremely popular and easy to use for both fluorescence and light microscopy. Although image quality from the system is undoubtedly a function of the three components together, it is the integrated camera and software package that ultimately produces the high quality images – the role of this suite.
Diagnostic instruments market a variety of color and monochromatic cameras for different imaging requirements, their SPOT RT cameras are a mid-line range. Like many companies, Diagnostic Instruments uses Sony (SE range) and Kodak (KE range) CCDs – the Charge Coupled Device that registers the spatial distribution of light intensities from a sample – in their cameras. The light capturing efficiency of the SPOT RT cameras may not be the highest on the market but these cameras do give excellent results and in conjunction with a good optical system (microscope, objectives and light source) should prove suitable for most immuno-fluorescence, GFP-expression and organic fluorophore (BCECF etc) applications. CCDs are inherently monochromatic and only register light intensity, however different formats of colored filters can be used to generate colored images. The method employed by the RT 3-shot cameras retains optical resolution, a key requirement for our applications, by using separate red, blue and green filters in front of the CCD.
The SPOT software that accompanies the CCD camera is a key element of this system and comes in both Basic and Advanced versions for Macs (OS X) and PCs (using at least Windows 95). Which software version you use is determined by your requirements, your imaging experience and by how qualitative your downstream image analysis will be. To date most of my imaging has used the Basic software run on a Mac. The basic package is easy to navigate and will definitely get you up and running rapidly. It is also worth noting that many third party software packages have compatibility with SPOT cameras. Communication between the camera and computer is by a high speed PCI interface.
SPOT software differs from older programs that I have experienced in that exposures can be automatically determined. This feature is available, however, with several newer imaging packages. An Image Type (fluorescence, bright-field, phase contrast, etc.) is selected within the Image Settings tool and this determines how the SPOT program captures and processes the image. The user can influence the final image though by biasing certain parameters displayed as sliders. For instance, quality/gain of an image can be varied and an algorithm can be evoked to balance intensities in samples with high light-to-dark contrast. Users can also store recallable parameter settings within the Image Settings tool for samples that are repeatedly prepared. These features certainly allow a user to ‘point-and-shot’ with excellent results but additional flexibility is available. Images can be acquired with manually selected exposures (which can be useful for dull fluorescent sample) and samples can be viewed in a live/real time format and images captured whilst the camera works in this mode.
The Basic SPOT software offers a plethora of further features: regions of interests can easily be defined; time-lapse sequences can be acquired and played back; final Reports for publication or presentations can be generated, annotated and formatted. Finally, extensive software tools exist to manipulate images, in much the same manner as ImageJ or Adobe Photoshop, such as adjustment of brightness and contrast, merging and subtracting of images, smoothing, sharpening and so on.
So what does the Advanced SPOT program offer? The user is afforded much greater control in how images are captured, edited and Reports generated through extended options in the software. The Advanced program also allows macros to be written which can control and automate camera operations and image processing. Finally, the PC version of the advanced program has an extensive database facility. This feature could be especially useful for a clinical histology or pathology lab or a very ‘image intensive’ project such as a developmental study.
I really like the SPOT RT KE color 3-Shot CCD, indeed there are many more features that I have not mentioned. It is very easy to use, due to the great software interface, and the system generates excellent images. The trial-and-error element that used to exist with imaging is now gone, a real bonus especially with fluorophores that are prone to bleaching. As I said earlier, the efficiency of the CCDs is not the highest on the market but this camera has proven to be more than sufficient for all the fluorophores I have used so far. The SPOT RT really performs like the Diagnostic Instruments literature says: ‘nothing exotic, just solid, reliable performance for all your typical brightfield and fluorescence microscopy imaging’.
Peter Haggie, Ph.D.
Post-Doctoral Fellow
University of California, San Francisco