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Creation and Processing of Digital Photographs

Hardware and Software: (organized around a single workstation on a central table in our lab)

Workstation on lab table  Workstation on lab table
  1. A relatively fast PC computer with at least 4 GB of memory to easily process data using image-editing software.
  2. Many of the older photos present on this website were taken with an analog single-chip video camera—high quality digital cameras for microscope applications were prohibitively expensive at that time. Digital cameras have come down in price and are more affordable (now in the $6000-7000 range) and they are much smaller in size and weight. We use a Nikon DS-Ri1 high resolution camera. Uncropped images are 4.25×3.4 inches at 300 dpi.
  3. Adaptors which allow the video camera to be attached to either a compound microscope (to capture photos of mounted spores/mycorrhizae, etc.) or a stereomicroscope (to capture images of intact spores, roots, mycelium, etc.).
  4. A high-quality stereomicroscope. We use Nikon SMZ-U for routine photos because it uses the same adapter as the compound microscope and the camera can be readily interchanged between scopes.
  5. A high-quality compound microscope, preferably equipped with differential contrast optics to better resolve thin colorless layers and other difficult-to-see subcellular structures. We use a Nikon E600 Eclipse Microscope with DIC prisms matched to each objective (10, 20, 40, 100X).
  6. A dye-sublimation printer capable of printing from standard size photograph paper (8.5×11 inches). We use a Hewlett Packard Photosmart Pro B9180 printer that produces remarkably crisp images with good color rendition. The nicest feature of a dye-sublimation printer is that high quality photographs can be obtained with a minimum resolution of 150 dpi. This is important for printing page-size images.
  7. Image-editing software. Many different applications are available, but we use the workhorse that comes with the Nikon camera (at a high cost, of course) and that program is called NIS-Elements. White balance sometimes is hard to achieve, and so that is edited as needed in Adobe Photoshop.

Steps in creating a digital image using a digital camera:

  1. Load application that accompanies the digital camera (NIS Elements). A window appears that contains the “live” image from a microscope (see far left photo below).
  2. Arrange the specimen in the window to clearly show all the features desired, adjusting the fiber optic lighting to provide the best contrast.
  3. “Grab” the image using menu commands in the window and save the image as a TIF file (NOT jpeg, because jpeg uses a compression algorithm and some pixel data are lost).
  4. Once saved, the image then is opened in Photoshop and cropped to a size that best frames the object. The image is edited to match color under the microscope and the background darkened to optimize contrast. To play it safe, a duplicate image (go to “image” in the menu bar, and then “duplicate” in the menu—see photo below). SAVE the edited image in Photoshop’s native PSD format, thereby retaining all the layers so they can be edited or deleted later if necessary. Flatten the image and save it a JPG version. You now have three files: (i) the original TIF image to archive, (ii) the layered file containing the maximum information that also is editable individually and can be altered at any time to change resolution (e.g. highest for printed photograph, 72 dpi for presentation on computer monitors), and (iii) a compressed file of the edited image whose resolution also can be changed as needed. It is this multiple filing system which provides maximum flexibility for any future use, whether anticipated or not.
(i) i  (ii) ii
  1. For photos archived as information only (not for general viewing), notes or observations can be included either as separate layers on the PSD file or as metadata (the “file info” selection under “File” in the main menu). When the file eventually goes public, these layers can be deleted and the file resaved in another format (thus preserving the original).
  2. All photos should be stored in folders organized hierarchically, after which they can be backed up or archived on compact disc.

How and When to Change Image Resolution:

  1. Web-based images should not exceed 72 dots-per-inch (dpi). At higher resolution, the image will appear larger on a monitor because it has a fixed number of pixels. Larger monitors, especially newer flat-screen models, have more pixels and can be set at higher resolutions (1024×768, 1260×1600, etc.), and so these are ideal for viewing high resolution images.
  2. Images for Microsoft Powerpoint presentations should not exceed 100 dpi if the goal is to keep the file as small as possible. Extra pixels above this resolution are not used, even though the entire file is loaded.
  3. Images for printing should have a minimum resolution of 150 dpi for dye-sublimation printers and 300 dpi for other color printers.
  4. There are times when resolution may need to be increased to obtain a good print image (usually when resolution was low when first shooting the photo with an analog camera or from a low-end scanner). To preserve fine detail (such as inner wall structure of spores, etc.), then the image should not be resampled (pixels added) when increasing resolution. To do this, uncheck the “resample image” option. The result will be a smaller printed image (the same number of pixels are present, but with more per inch with no change in file size (see example below). The size of the image on the monitor will not change, since no pixels were added or subtracted.
  5. Resolution of images from a digital camera usually is greater than that which is optimal for monitor-based displays (web pages, image catalogs, etc.). Here, the image is resampled (“resample image” is checked) when the resolution is changed from 150 to 72 in the example below. Notice that the file size is greatly reduced (3.75Mb to 885 Kb), but the image size remains unchanged. It is amazing how much detail is preserved when so much pixel data is discarded. To experiment, duplicate the original image, perform these modifications on the copy, and compare quality with the original.

Data on the original image

Data on the original image

Increasing resolution (no resampling of pixels)

Increasing resolution (no resampling of pixels)

Decreasing resolution (resampling of pixels)

Decreasing resolution (resampling of pixels)