66 MICROSCOPY
details), resolution is often indicated by the total number of pixels, measured in mega-pixels (MP).
Generic camera
– Image is noisy – Sample is not clearly visible
Dedicated fluorescence camera
– Image is clear – Sample structures are well defined
Fig. 2. Sensitivity is important for fluorescence applications. The benefits of using a dedicated monochrome camera are clear, with large pixels and active cooling for high sensitivity.
Fig. 3. Stereo microscopy image of a GFP-expressing Drosphila sample, overlaying brightfield and fluorescence images. DP80 pixel-precise centring mode and HDR processing of fluorescence image were used.
presentations and discussion sessions.
Te number one priority for low light and fluorescence applications is high sensitivity. Colour is not required for these techniques, meaning that superior sensitivity is achieved
by first removing the RGB colour filters covering the chip. Furthermore, a dedicated monochrome chip will have a higher sensitivity by simply having larger pixels, enabling the capture of a greater number of photons and thus producing a clear and noise-free image (Fig. 2). In highly sensitive chips, however, the image noise can be more evident, since
the measured signal is so low. Te most notorious contributor of noise is thermally derived, and so it is important to actively cool the camera chip, either with a Peltier- effect plate or with forced air or water flow.
In the past it seemed that a
choice must always be made between having a colour or a monochrome camera, but this is no longer the case with dual-chip cameras. For example, the Olympus DP80 camera houses both a dedicated colour and a monochrome chip, with automatic switching depending on the type of snapshot needed. Tis versatility is valuable and it also presents opportunities for overlaying colour and fluorescent images with single pixel precision (Fig. 3).
Digital technologies yield many advantages for documentation and archiving, making images highly accessible and easily shared across the globe. Te transition towards digital sample libraries is facilitated by high resolution imaging, allowing the sample to be analysed retrospectively with the digital zoom (Fig. 4). Defined by pixel size (smaller pixels capture finer
High resolution is, however, only effective when working at low magnifications. For example, when using a 100X objective a resolution of just 1 MP will be generally sufficient: any additional resolution will merely increase data volume without providing any extra information. An image instead captured at 4X magnification might require more than 10 MP to capture all the details from the large field of view. Currently, ‘pixel shifting’ technology allows the greatest number of pixels to be rendered by a camera.
Te finer pixel size necessary to obtain higher resolution will decrease the camera’s overall sensitivity. In this case, ‘pixel binning’ provides the option to group smaller pixels into bigger ‘virtual pixels’, increasing sensitivity when the application needs it, albeit at the expense of the overall resolution.
Which camera? Te camera sits at the very core of ocular-free microscopy, and each application requires specific features:
n Colour imaging – colour profiling technologies;
n On-screen viewing – progressive readout;
n Fluorescence imaging – monochrome chip with low noise;
n Documentation & digital archiving – high resolution.
For more information ✔ at
www.scientistlive.com/eurolab Flavio Giacobone is with the
Micro-Imaging Solutions Division, Olympus Europa SE & Co KG, Germany.
www.olympus-europa.com/microscopy
Low resolution
www.scientistlive.com High resolution
Fig. 4. High resolution is crucial for documentation purposes. At low magnification, retrospective analysis is only possible with high resolution.
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92