Digital Imaging – New Opportunities for Microscopy
The explosive growth in digital imaging technologies is taking the imaging market by storm. The latest digital cameras combined with powerful computer software now offer image quality that is comparable with traditional silver halide film photography. Moreover, digital cameras are also easier to use and offer greater flexibility for image manipulation and storage.
Digital imaging is increasingly applied to image capture for microscopy - an area that demands high resolution, color fidelity and careful management of, often, limited light conditions. These are challenging requirements, even for traditional film based photography, so why consider digital imaging? This article addresses how digital imaging performs under these conditions and what the advantages are of digital imaging for the optical microscopist.
Illustrated in Figure 1 is a digital image of a thick section of mouse intestine stained with three fluorophores and recorded with a Nikon DXM1200 digital camera system coupled to the SMZ1500 stereomicroscope. The image was captured utilizing a combination of fluorescence and Nikon's proprietary oblique coherent contrast (OCC) illumination techniques. A total of four integrations were performed and the image was composed in Photoshop to yield the final version presented in the figure.
Why consider digital imaging?
It is important to emphasize that the quality of the final image, whether digital or film, is dependent on the quality of the original microscope image. No matter how good the digital or conventional camera is, it cannot produce outstanding images from a poorly configured microscope. In addition, both film and digital imaging systems can reveal imperfections that are not immediately visible when looking through the microscope eyepiece.
With the general growth in electronic communications, there is a real requirement for digital images, which can be easily transmitted to a wide number of users. Digital images, for example, may be simply emailed for consultation and discussion, incorporated into other digital documents, exported to image analysis systems or posted on a website since they are easy to copy, store and archive. They can also be easily annotated with appropriate software for inclusion in presentations or archives. While photographic images can be scanned into a computer to produce digital images - digital image capture from the start saves time and effort.
Most digital cameras are of the "point and click" principle - little or no photographic expertise is needed. Traditional photomicrography, in contrast, requires some knowledge of photographic techniques. Users need to understand the benefits and drawbacks of different kinds of photographic film, should pay careful attention to the filters they use and have some understanding of the relationship between lens apertures, shutter speeds, depth of field and, for color photomicrography, color temperature. Results can be variable, especially for novices, hence the important practice of "bracketing" exposures for critical images, i.e. taking at least three separate pictures to ensure that at least one of the pictures is successful. This however increases film and processing costs. Digital imaging does not incur ongoing costs - there are no film or processing fees.
Digital imaging is almost instantaneous. Most cameras have an LCD screen that enables viewing of the image and pictures can be quickly transferred to a PC. A decision can be immediately taken on whether the picture is satisfactory. Film, on the other hand, needs developing and processing before it can be seen. By this time, the subject may no longer exist especially in cases where microscopists are recording dynamic events in living cells.
What to look for in a digital camera
Cameras are available with either a digital or analog output. For analog output there are several different standards like PAL, NTSC, or RS-170 and for transmitting the data there are also different formats such as RGB, S-VHS or composite. Analog signals must be transformed into digital signals using a frame grabber before they can be sent to a computer whereas in a digital camera the signal to the computer is already in digital format. This reduces noise and obviously omits the need for a frame grabber. Digital signals can be transferred to the computer using serial or parallel ports (both slow), USB (faster but available on almost all-modern computers), Fire Wire (faster than USB but not so widespread) or through boards for the PCI bus (fastest, widely available but requires board installation).
Digital imaging systems vary in the speed that images can be transferred to the computer and this is an important consideration for laboratories taking large numbers of pictures. For the low volume user a wait of a minute for downloading a picture to the PC may be acceptable but for the busy lab it can be severely rate limiting.
Presented in Figure 2 is the Windows interface for Nikon's DXM1200 digital camera system. The camera requires a proprietary card be inserted into the computer motherboard and input/output is controlled through the accompanying Automatic Camera Tamer (ACT) software.
The live image stream is important for focusing and positioning - a camera would either have an on-board LCD viewing monitor or the user should be able to view the image to be taken live on a PC screen. The LCD monitor needs to be large enough to see the image well, especially if there is no live stream to a PC. Some LCD screens can be tilted in the direction of the user for easier viewing. This can be important, especially for microscopists.
For users that need to use their camera for a variety tasks around the laboratory, a camera that can be easily attached and removed form the microscope will be an advantage.
Resolution is perhaps the most important parameter for the selection of the camera. Images must be able to record the fine detail revealed by the microscope magnification. Digital images are made up of millions of tiny squares called picture elements or pixels. These tiny pixels are used to display or print images and the more pixels in a given area the higher the resolution of the image. If a digital image is enlarged there will come a point when the individual elements can be seen as separate dots - similar to graining in a silver halide photograph and the more pixels an image contains the more it can be enlarged before the separate pixels start to show. The size of the image can be described by its dimensions, for example, 1500 x 1700 pixels or by the total number of pixels present, in this case, 2.55 million. Resolution is also often quoted as the size of charged coupled device (CCD), which is effectively the number of pixels on the chip. It should be noted, however, that the size of the individual pixels varies amongst different types of CCDs. For microscopy, a pixel size (square) of 6.7 micrometers is thought to be ideal.
As light enters the camera it passes a filter that divides the pixels into red, green and blue tone pixels - the colors used create the overall color image. The light rays are then directed to the CCD, which is specialized semiconductor that transforms the light rays into electrical charges. The intensity of the electrical charges is proportional to the intensity of the light coming from the subject. Values stored in the digital image specify the brightness and color of each pixel.
Some cameras achieve their resolution by an additional extrapolation step using software that "guesses" the values between two pixels. This value is used in the final "extrapolated" image. Other cameras acquire three separate images - one each for red, green and blue, which are then combined into a full resolution (non extrapolated) image. The disadvantage of this method is that exposure times are tripled.
High resolution can also be achieved through a novel technology introduced recently by Nikon for its DXM1200 digital camera (Figures 2 and 3). The technology known as IPS (Inter Pixel Stepping) uses a piezo electric mechanism to increase the resolution of the chip by moving it back and forwards, for example, in a total of nine steps, by around 1/3 of a pixel. This increases, in this case, both the resolution and the size of the image by a factor of 9. Using this method, several images are averaged out to produce a sharper image with less noise. The DXM1200 produces high quality images with approximately 12 million output pixels. This is roughly equivalent to the number of silver halide grains in a conventional 35mm film. Performance, therefore, rivals conventional film based silver halide images or even surpasses them for enlargement purposes. Its low noise design is especially suitable for capturing low light images, for example, in fluorescence studies. Three levels of sensitivity are available and a long exposure time of up to 170 seconds ensures the capture of dim specimens.
Software is obviously an important feature of a digital camera. More powerful software confers greater flexibility to the user but again the software capability should be matched to the laboratories' needs. For professional users, ACT-1 image acquisition software supplied with the DXM1200 provides sophisticated functions with ease of use.
Digital imaging offers a world of opportunity for the microscopist, providing an easy-to-use image acquisition system that enables easy image storage, manipulation and management.
When selecting a digital camera, resolution, image transfer speed and color fidelity are good starting points but software and ease of operation are also important consideration. Because of the simplicity of digital imaging, high quality images are now within everyone's reach.
Peter Drent - Production Manager, Microscopy Division, Nikon Europe, Schipholweg 321, 1171PL Badhoevedorp, The Netherlands.