Practical Guide to Digital Imaging for Microscopy

Source: Optronics
The current microscope image acquisition marketplace is filled with new products, each one promising excellent images at a variety of price points
By William G. Hand, Ph.D., General Manager, Optronics

The current microscope image acquisition marketplace is filled with new products, each one promising excellent images at a variety of price points. What do you really need to know when you get ready to buy?

Your first priority should be to look at your application requirements. You should answer the following questions:

  • If fluorescence microscopy is used, can you see the latent fluorescent image through the eyepieces? If you can't see an image, you will need a camera that integrates the image past a few seconds (integration is the digital camera equivalent of extended or time lapse exposure in a film camera).
  • What field of view does the application require? This can be measured as a percent of the field of view as seen through the eyepieces. You can use a stage micrometer to determine this.
  • Do you feel comfortable with integrating image processing boards in to your computer? If you have not had experience doing this, you should work with a dealer who has an imaging department or consider purchasing a camera that uses an existing interface (Serial, Parallel, or if newer USB or IEEE 1394).
  • Do you really need a digital camera? There are still some applications where film is the best medium for image acquisition, especially if you require special lighting. Remember that you can post-process a photo image through a high quality scanner to get digital results.
  • What about the price/performance issue in digital cameras? As with any product, you get what you pay for. A lower price means some sacrifice in performance. Make sure that the performance hit does not effect your application.

Now that you have a idea of what your requirements are, make a features list and use it to select your ideal camera. Stay true to your list. The following description will provide you with some overview areas to consider when composing your camera "wish list".

When selecting a camera for microscope imaging, you have the following choices:

Traditional Film Cameras (Leica, Zeiss, Olympus, Nikon, Polaroid)

Whether standard emulsion, or Polaroid formats, film cameras provide the necessary high resolution and color fidelity required by microscopy.

There are several drawbacks to film cameras:

  1. You can not preview the image you are saving to film.
  2. Processing the film and providing prints or slides is cumbersome and expensive.
  3. The learning curve for successful application of film technology is extensive.
  4. No direct computer interface.

Films advantages are:

  1. A high latitude of film light sensitivity makes the medium ideal for some low light and high speed applications.
  2. Large emulsion formats provide unequaled resolution.

Video (Optronics, Sony, Hitachi, Javelin, Toshiba, Panasonic, Cohu, Dage)

Video has emerged over the past 10 years as a viable alternative to film. With CCTV high resolution imaging, video provides:

  1. WYSIWYG (What you see is what you get) capability.
  2. Ample resolution for high magnification, fine structure characterization microscopy.
  3. With 3 chip (RGB) technology, excellent color rendition.
  4. Easy digital conversion for computer input, storage and manipulation.
  5. Acceptable sensitivity for low light level applications, marginal sensitivity for high speed applications.

Video has some down sides too. These include:

  1. Complex microscope interface issues (image magnifiers, adapters, filters, etc.)
  2. Often complex computer interface issues (image processing board configurations, software compatibility issues).
  3. Video image display issues (RF interference, color balancing, display resolution, video tape storage resolution, video duplication image loss).

Digital Imagers (Optronics, Kodak, Diagnostic Instruments, Roper)

In the past 5 years, technology applied to video has been modified to provide high quality, high resolution digital images. By creating new imaging "chips", and ignoring standard video image standards (NTSC, PAL), it is possible to produce an electronic format camera that has high resolution, a simple direct computer interface, and excellent color rendition. The advantages of digital imagers are:

  1. High resolution with excellent field of view (where chip format is 1K x 1K pixels, or "megapixel" quality).
  2. Direct digital "mapping" of the image into a computer, eliminating the need for image processing boards (but you still may require dedicated image transfer boards).
  3. Relatively high sensitivity and image integration for low light applications such as fluorescence imaging.
  4. Some newer models have near "real time" image viewing.

Digital image cameras have some inherent disadvantages as well. These include:

  1. Slow image creation and transfer provide little or no WYSIWYG capability.
  2. Long integration times, often coupled with multiple image captures (for color imaging) make the use of these devices impractical for some high sensitivity fluorescence applications.
  3. Large physical size makes the mounting on some microscopes (especially tissue culture or other "inverted" optics microscopes) cumbersome or impossible.

Video Hybrid Imagers (Examples: Optronics, SONY, Cooke)
These cameras feature the ease of use of a video device with a method of taking a digital "snap shot". Most transfer the digital image over an existing computer interface (serial, parallel, or USB). These cameras provide the following benefits:

  1. Complete WYSIWYG functionality at standard video rates (NTSC, PAL).
  2. High Resolution 3-CCD imaging with real time image enhancement rivals the image quality of megapixel imagers.
  3. Direct digital input without image processing boards.
  4. TWAIN compliance for direct input into image manipulation and storage programs.
  5. Excellent sensitivity and cooling provide short duration integration times for low light (fluorescence) requirements.
  6. Small remote-head design provides a simple, low mass microscope mount.

The disadvantages of the hybrid system are few:

  1. Smaller field of view when compared to megapixel digital imagers .
  2. Some color mis-registration when compared to color wheel megapixel imagers.

The table below summarizes the features of each camera type with respect to the nature of the microscope application:

Camera Application Table I: General Issues
Camera Type Resolution Sensitivity How to make digital image Input to computer
Film Excellent Excellent Scanner Scanner
Video Marginal Very Good Image Processor Image Processor
Digital Very Good Very Good Direct or board Direct or board
Hybrid Very Good Very Good Direct Parallel Direct Parallel
Excellent Able to reproduce image at or beyond the resolution of the microscope.
Very Good Matches the resolution of the microscope without image enhancement.
Marginal Does not match the microscope image in one or more ways.
Scanner A separate hardware device which copies (digitizes) the printed film image to the computer.
Image processor A board-level addition to the computer that converts analog video images to digital images in real or delayed time.
Direct Permits a digital image to be stored in a computer without a scanner or image processor. This method may require a proprietary interface board and/or a parallel or serial cable, or proprietary cable.

Camera Application Table II: Low Light Applications
Camera Type Fluorescence WYSIWYG Color Generation
Film Seconds to minutes No Various emulsions
Video Seconds to minutes Yes NTSC, PAL
Digital Seconds to minutes No Matrix CCD or Filter
Hybrid Seconds to minutes Yes NTSC, PAL

Microscope imaging is often plagued by uneven and/or low illumination levels. Some hybrid cameras (Optronics, Sony) provide the easiest-to-use solution for low light imaging. With high speed image integration and Peltier cooling, low light level fluorescence images may be produced in a short time (seconds), with a single exposure, and viewed live before they are stored. With some models (Optronics) 3-CCD true RGB imaging, color correction and digital image enhancement allow the user to control color appearance and image detail in real time.

Digital cameras use color wheels or color matrix CCDs to capture color information in the image. What are the pros and cons of these image acquisition solutions?

Mosaic CCD Digital Imaging Cameras (Roper, Apogee, Kodak)

Most imaging "chip" manufacturers (primarily SONY and Kodak) provide color versions of their megapixel imagers. All of the lower cost digital imaging cameras use this form of sensor. Color is provided by segregating the color information into a mosaic on the chip surface. This is accomplished by taking a group of four pixels and assigning a color frequency to each in a specific pattern. There are usually single red, single blue and two green pixels in the array. This array is replicated over the chip surface to create a color mosaic on to which the incoming color image is mapped. The chip is "read", and the color image is reconstructed using software or dedicated firmware.

What is gained and what is lost in this process? There is an obvious gain in speed. A color image is possible with a single exposure. Single pass color imaging has the further advantage of permitting the frame and focus functions of the camera to be in color as well (often at 12 images/second or more). What is lost is resolution. The color and image definition produced by this method will not tolerate the same degree of enlargement as will an image produced by a multi-pass imaging camera. Color fidelity and registration will not be as good, since each pixel in the array is not exposed to each color of the image. What you get is an "averaged" image, with the resultant compromises.

Color Wheel and Tunable Filter Digital Imaging Cameras (Optronics, Diagnostic Instruments)

Color wheel and "tunable" filter imaging cameras require multiple subject exposure (at least three; red, green and blue). Often, a fourth exposure is taken, a "dark" exposure, which may be used to subtract image noise from the result. The four exposure method, although longer, generally provides superior image results, with superior color rendition and minimal noise artifact. The down side is that the additive exposure and subsequent image transfer and computer color reconstruction can create a total image acquisition time of a minute or more in cameras with slow data transfer rates and/or long integration times.

In addition, there are distinct differences between color wheel and tunable filter technologies that are significant to long exposure (fluorescent) applications:

  1. Color wheels add a mechanical component to the camera design that increases size and adds an additional service component. The good news is that you maintain the high sensitivity of the imager. The fact that all pixels read the RGB component of the image results in better color fidelity when compared to mosaic imagers.
  2. Tunable filters greatly reduce camera sensitivity (by as much as 60% when compared to color wheel cameras). Reduced sensitivity extends exposure time (more integration: often to critical exposure levels with highly light sensitive fluorescent preparations). The good news is that the camera package can be made smaller and less expensive (there are fewer mechanical and electronic components).
Camera Application Table III: Ease of Use
Camera Type Time to Acquire Image Time to Store Image Learning Curve
Film Depends on scanner speed and resolution, generally minutes. Depends on storage program used, generally 5-15 seconds. Long, requiring knowledge of emulsions, developers, and printing papers.
Video 1/30 second Up to 1/30 second Depends on image processing components
Digital Seconds to minutes depending on need for color and light level Seconds to minutes depending on camera model Shorter than film, but similar in nature. Trial and error exposure
Hybrid 1/30 second Up to 15 seconds Very short , easiest of all to use


In summarizing this article, I would recommend the following (assuming that your choice meets your application requirements):

  1. If you want the ultimate in ease of use, excellent image quality, "real-time" image display and you are not concerned with a large field of view, I would recommend a hybrid camera.
  2. If you want the ultimate in image quality and format size, go with the multi-pass digital camera.
  3. If you want a large format image, with digital image quality and cost is a factor, go with a mosaic digital imaging camera.
  4. If you need real time, and the ability to record strings of images (motion studies, etc.), go with a high quality hybrid or video camera and an appropriate video digitizing board.
  5. If you have lots of time, and you enjoy darkroom photography, film is still an option

This article Copyright, ©1999 Optronics All rights reserved.
WGH 11/11/99 rev.4


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