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Digital microphotography guide: The purpose of this paper is to help microscopists and other photographers take high quality digital photographs, store them for easy editing and retrieval, and use digital microphotographs as an information or forensic database. We discuss using modern digital cameras and transmitted-light microscopes together.

Introduction to Digital Photography and Microscopy

Before specifying a camera choice and recommending camera settings let's review the purposes of digital microphotography of particular interest to aerobiologists and other microscopists:

1. To develop a large reference library of photo images of known particles - so images will be mostly stored on a computer, in some data base form.[1] Images need to sharp, often with multiple exposures at different focuses.
2. For research: to exchange email images with other professionals to obtain assistance in identification - similar to #1 above, but images by email should be reduced in file size for convenience and transmission speed.
3. To print with professional reports - usually these are limited in physical size. This is a distant third in importance in our practice.

Some Factors Involved in Obtaining and Using Good Photomicrographs

Start with a well prepared slide, choosing a mountant with good optics for the particles being examined. Fungal spores or pollen grains may benefit from hydration but not excessive hydration. Heavy use of colored stains risks obscures natural colors and features needed for identification, though we may succumb and use fuchsin, lacto phenol, or even India ink for occasional development of certain features.

After a well prepared and clean slide, be sure the microscope is properly set up for Koehler illumination, and be sure the objective is clean.

Select a camera which has a very good lens - among the current crop of 3+megapixel cameras, this is perhaps more important than a higher number of pixels. Lens resolution in macro mode and with the camera focused at infinity are both important.

Post processing of images may improve the image color balance, sharpness, or contrast, but no amount of processing can produce accurate image data if that information was not captured in the first place.

Even excellent photomicrographs are not useful if they cannot be located later for reference purposes. A good photographic data base system is important.

The Digital Image Formation Chain determines image quality in digital photos through the microscope

1. Microscope resolution, including the quality, cleanliness, and adjustment of every lens in the light path. Before starting the process be sure the microscope is clean and that K�Koehler illumination has been established.
2. Light source (color temperature, focus, etc.)
3. Adjustment of the condenser (N>1 oil-rated condenser lens for high magnifications such as the 100x oil-immersion objective - don't forget to oil the top of an oil-rated condenser lens when using the oil objective)
4. Adjustment of the field diaphragm, condenser aperture, and where provided, objective aperture and eyepiece aperture)
5. Placement of the well-prepared and clean slide on the microscope stage
6. Choice of objective lens and use of oil-immersion (higher magnifications reduce the demands on the camera's photo array of pixels)
7. Eyepiece and camera mount adjustment (and where provided,
8. calibration of the camera lens focal distance to match the eyepiece distance - or infinity)
9. Camera resolution
• Camera lens resolution
• Pixel resolution of the camera used
• Camera settings used for image capture
• Camera's focusing or macro-focusing algorithm if it is not being used with lens set at infinity
• Image data compression selection, ranging from zero (tiff) to low-loss, to high-loss compression
10. Post capture image processing
• enhancement and cleanup, such as using the "digital camera" or "jpeg" cleanup functions of image management software to remove noise, sharpen edges, adjust color balance, adjust contrast
• dithering algorithm on compressed-image expansion
• Level of image compression, e.g. JPEG, used to store final image - determines file size, maximum print resolution, and limits of ability to crop and enlarge or "zoom" into the image.
11. Observation Resolution
• Image display or printing capability, including effects of printer capability, dpi, dot size, dot spacing, ink types, and choice of paper used to prepare the print - but remember, that contrary to the argument of one expert microscopist who teaches that there is no reason to store large images, if you want to be able to crop and enlarge or zoom into an image you need to have recorded the original with sufficient megapixels and resolution.
• Visual resolution ability of the human eye
• The limits of light to resolve particles are pertinent throughout these steps, at the microscope during observation and making of the photograph in particular.

What this list means is that the quality of the end result of photomicrography is limited by the weakest link in the image-formation chain. For example, no amount of camera pixels will provide a sharp image of an object which is not resolved sharply in the microscope nor will a high-pixel camera produce a sharp image if the camera's lens is of limited ability. Beware: not all high pixel cameras have equally sharp lenses, so the "resolution" in megapixels can be misleading. High megapixels defines how many data points of image are being recorded. But if the lens and other steps in the image formation chain are not producing a sharp image, high megapixels means you're recording a lot of fuzzy data.

With these preliminaries, let's look at choosing a camera, selecting camera settings, using mounts, etc.

Questions & Answers regarding this article

Questions & answers about digital microphotography: how to take succcessful, sharp digital photographs through the microscope

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Digital Photography & the Microscope
Introduction to Digital Photography and Microscopy
Some Factors Involved in Obtaining and Using Good Photomicrographs
The Digital Image Formation Chain
Selection of Digital Cameras
Camera mounts
Camera Settings
  Automatic Settings
  Image print size
  Manual setting mode
Focusing tips for Digital Cameras
Take some test photos

Getting your photos from camera to the computer
  Simple method - flash card
  Manufacturer's cables
  Video bus

Building and Organizing a Digital Photo Library on a computer
  Photo library management
  Photo Library design
  First Pass Screening
  Saccardo Method

Image Library Backup Suggestions
References for Digital Photography

References for Digital Photography Through the Microscope - Micro Photography

A Short Course in Nikon Coolpix 990 Photography, Dennis P. Curtain, http://www.shortcourses.com - RECOMMENDED

Resolution of Digital Photomicrographs from Scanned Film, Theodore M. Clark, Microscopy Today, Feb/Mar 2001 http://www.couger.com/microscope/Ted-Clarke/papers/FilmScanner/

Light, Michael I. Sobel, University of Chicago Press, 1987, ISBN 0-226-76751-5

ThumbsPlus® image software, provides highly functional image database including basic editing, cropping, enlarging, enhancement of photos, image organization, keywords and comments attached to each image, and search capability - HIGHLY RECOMMENDED for PC platforms. Typically less than $100. Network support available.

® image editing software, provides sophisticated image editing, available for PC and Macintosh computer platforms. Not recommended for reference library use - this is overkill for image editing and lacks good database functions. Typically more than $400.

iPhoto® image software, provides easy user interface and limited function to organize and print photos for MAC computer platforms - not recommended, limited function, very wasteful of disk space, lacks good database functions. Free included with new Apple computers or operating systems.

Photo Explosion® image software - free from Microsoft with other OS purchases. Untried.

Picture Easy software from Kodak - free with some Kodak cameras, simple organizing and photo manipulation.

Nikon View® software - free from Nikon with purchase of Coolpix cameras, easy image import and simple image manipulation and printing. Great for printing contact sheets of photos (which themselves are pretty useless).

[1] Thumbs Plus image data base software from Cerious software, www.cerious.com is a top choice low-cost option), and will be searched-through for reference purposes

Books & Articles on Building & Environmental Inspection, Testing, Diagnosis, & Repair

• Our recommended books about building & mechanical systems design, inspection, problem diagnosis, and repair, and about indoor environment and IAQ testing, diagnosis, and cleanup are at the InspectAPedia Bookstore. Also see our Book Reviews - InspectAPedia.
• Aerobiology, Building Science, Microscopy, & Laboratory References, an extensive technical bibliography
• Atlas of Clinical Fungi, 2nd Ed., GS deHoog, J Guarro, J Gene, & MJ Figueras, Centraalbureau voor Schimmelcultures, Universitat Rovira I Virgili, 2000, ISBN 90-70351-43-9
• Atlas of Mold Related Illness: Index of Symptoms and health, physical, neurological, psychological, and other complaint which people suspect may be mold or building-related.
• Atlas of Indoor Mold, Online Clinical Mold Atlas, Toxins, Pathogens, Allergens and Other Indoor Particles - Medical Health Effects of Mold (separate online document)
• Building Floods: quick steps after a building flood or plumbing leak can prevent costly mold contamination
• Classes of Mold: what types of cosmetic, allergenic, or toxic mold are a problem? Can mold be cleaned-up successfully?
• Clinical Mold References - Detailed bibliography of mold reference texts
• "A Brief Guide to Mold, Moisture, and Your Home", U.S. Environmental Protection Agency US EPA - includes basic advice for building owners, occupants, and mold cleanup operations. See http://www.epa.gov/mold/moldguide.htm
• "Disease Prevention Program for Certain Vegetable Crops," David B. Langston, Jr., Extension Plant Pathologist - Vegetables, University of Georgia (PDF document) original source: www.reeis.usda.gov/web/crisprojectpages/209797.html
• "Disease Prevention in Home Vegetable Gardens," Patricia Donald, Department of Plant Microbiology and Pathology, Lewis Jett
  Department of Horticulture, University of Missouri Extension - extension.missouri.edu/publications/DisplayPub.aspx?P=G6202
• "Management of Powdery Mildew, Leveillula taurica, in Greenhouse Peppers," Ministry of Agriculture and Lands, British Columbia - Original source: www.agf.gov.bc.ca/cropprot/peppermildew.htm
• Environmental Health & Investigation Bibliography - our own technical library on indoor air quality inspection, testing, laboratory procedures, forensic microscopy, etc.
• Fifth Kingdom, Bryce Kendrick, ISBN13: 9781585100224, is available from the InspectAPedia online bookstore - we recommend the CD-ROM version of this book. This 3rd/edition is a compact but comprehensive encyclopedia of all things mycological. Every aspect of the fungi, from aflatoxin to zppspores, with an accessible blend of verve and wit. The 24 chapters are filled with up-to-date information of classification, yeast, lichens, spore dispersal, allergies, ecology, genetics, plant pathology, predatory fungi, biological control, mutualistic symbioses with animals and plants, fungi as food, food spoilage and mycotoxins.
• Fungi, Identifying Filamentous, A Clinical Laboratory Handbook, Guy St-Germain, Richard Summerbell, Star Publishing, 1996, ISBN 0-89863-177-7 (English)
• Mold Action Guide: Step-by-Step Instructions, What to do about mold, mildew, and other indoor allergens
• US EPA: Mold Remediation in Schools and Commercial Building US EPA
• US EPA: Una Breva Guia a Moho - Hongo - en Espanol
• Most Common Indoor Molds Found in buildings, A Table of
• Mycology, Fundamentals of Diagnostic, Fran Fisher, Norma B. Cook, W.B. Saunders Co. 1998, ISBN 0-7216-5006-6
• ...