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Digital Photography and the Microscope: How to take successful digital photographs through the microscope Enter your search terms Submit search form   Search This Website - InspectAPedia.com

• How to use a digital camera to take photographs through the microscope
• Recommendations for digital cameras, adapters, photograph organizing software for microscope digital photography
• Recommended camera adjustments and settings for use with a microscope

ABSTRACT: The purpose of this paper is to help microscopists photograph, store, and use digital microphotographs using modern digital cameras and transmitted-light microscopes. Discussion focuses on selection of cameras adapt well to microscope eyepieces or trinocular heads, and on camera settings and procedures to obtain best quality photographs. I also discuss image resolution and size settings and make recommendations. Cameras used as examples in this paper include the Nikon Coolpix series 990, 995, and 4500. [The photograph at page very top is one of our earliest attempts at photographing Aspergillus sp. using lacto phenol cotton blue stain. Even a beginner can obtain very good microphotographs with just a little care. The photograph shown here is of one of our stereoscopic microscopes in use for making digital photographs. The techniques discussed in this paper work well with digital cameras and any type of microscope, low power stereo zoom to high power forensic polarized light microscopes.] © Copyright 2008 Daniel Friedman, All Rights Reserved. Information Accuracy & Bias Pledge is at below-left. Use links at the left of each page to navigate this document or to view other topics at this website. Green links show where you are in our document or website. PAAA 2005 Symposium, University of Tulsa, Tulsa Oklahoma - June 2-5, 2005 Note: the latest version of this document can be found at www.inspect-ny.com/digipix.htm last update 20 August 2007 © Daniel Friedman All Rights Reserved

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 my 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 I 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 K�hler 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 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�hler 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.

Selection of Digital Cameras for Microphotography

There are many digital cameras on the market that might work to make acceptable photomicrographs. I've found that the Nikon Coolpix series has several advantages for through-eyepiece hand-held photomicrographs: the lens size fits nicely on most eyepieces without scratching anything; the auto focus and other automatic settings produce excellent photographs.

I've used the Nikon Coolpix models 990, 995, 4500 with quite satisfactory results in preparing about 250,000 digital photomicrographs and a large number of macro photos as well. The Nikon Coolpix 990, 995, and 4500 will work identically well though some of the menu settings are found in different places.

The Nikon Coolpix 850, and similar models from any manufacturer whose cameras employ external moving telescoping lens components are less successful for photomicrography. The sliding/telescoping barrel does not provide a rigid mount against the microscope eyepiece. Though it's official macro-lens close-up range is not as close as the other two models, this camera's auto-focus is able to adjust itself for successful photomicrographs. These Nikon models are available as new, factory reconditioned, and used from EBay and from several large camera dealers at prices typically in the $300-$400. range depending on accessories included.

Using other camera models made by Nikon, Canon, Kodak, or other manufacturers may be satisfactory, but I have found that the lens diameter and the internal rather than external telescoping parts of this Nikon 900/4500 series works particularly well for photomicrography. Some of the Canon digital cameras have superb lenses and outstanding macro photography ability, but the large lenses on these cameras do not so perfectly fit the microscope eyepiece and field size. Other smaller hand-held digital cameras I've used, such as the Canon digital elph can be used with the microscope but results were disappointing as the image formed is very small and telescoped. Microscopists may want to avoid some of the older small-pixel small-format earlier systems which produce only very small, low-resolution images.

Special systems which connect the camera output directly to a computer for on-computer image capture are particularly efficient for in-lab use. I have not (yet) included specifics on these features in this paper as herein I am focusing on image formation at the microscope and in the camera.

Camera mounts and trinocular heads for taking photographs through the microscope

Cameras with an appropriate front lens diameter can be hand-held against a microscope eyepiece to take successful photographs. Adapters which screw into the front of the camera lens and slide into a microscope eyepiece tube or into a trinocular head are a better approach for regular use in the laboratory. I use three such cameras permanently mounted on microscopes in my lab, for example.

My mount supplier also provided a mount stabilizing ring which provided a more secure position on my trinocular stereoscope.

In a comparison test using the same camera hand-held at eyepiece and mounted on the trinocular head with two different adapters we observed no detectable difference in the image produced with eyepiece mounts and hand-held shots.

Camera Settings for Digital Photomicrographs

The Nikon Coolpix 900-series and the 4500 camera can take wonderful photos on fully automatic, but to make photomicrographs you may have more success if you make use of some of the manual camera settings as follows (for the Coolpix 990. Find and make the comparable settings in the 850, 950 and 4500 menus). Most other digital cameras will have a similar set of camera setting options but they may be organized around a different menu system. Find and adjust the equivalent settings on your particular camera, using the guidelines listed below.

Automatic Settings for digital cameras with the microscope

In general the "automatic" settings of the camera work pretty well - it has general modes of "Automatic", Manual, Program, and Shutter - set by holding Mode (Func 1) button on camera top and turning the thumb wheel - your camera should start in "Automatic".

Image print size, resolution, and disk space

The camera has a number of image size (3:2, XGA, VGA, FINE) settings and within XGA and VGA you also specify the resolution (Basic, Normal, Fine, and "HI" the last of which makes a huge TIFF file you don't normally want). Image size refers to the intended final display or print size. Resolution refers to the level of data compression in the stored image file. Larger size and higher resolution mean larger image files (and more disk space consumption).

I usually shoot at XGA-Fine which will let me print a high-resolution 8x10 photo if I want a printout, but keeps the disk file sizes down to a manageable 200-300K bytes. You could shoot at XGA-Fine with good result but if you ever want to crop and zoom in on a particle in your photo you may not like the quality. If you want to save disk space and if you never plan to zoom in or enlarge your images, shoot at VGA-Fine which will produce excellent but smaller photos. Remember however that often you may want to enlarge a portion of a photomicrograph to see detail or for publication. You can't do much enlarging of a "VGA" image.

To email photos to someone you'll want to use a photo editing program to make a smaller, lower-resolution image to get the file size down to about 30K. (Folks who have the CD-ROM from the Laurentides Spore Camp received down-sized images which you'll see don't stand up to close scrutiny nor to any zooming.)

Some experts argue that one need not bother to keep images of higher resolution than the resolution capacity of the printer to be used for final images. Because I use images for reference and identification (email-exchange) purposes, often with images cropped and enlarged, and because I rarely print large images, the choice of image size is not reduced to printer resolution.

In sum: for image library purposes and printing up to 8x10 you do not need to store high resolution, large image size files - which wastes disk space. However if you need to crop and enlarge a portion of a photographic image you will want to shoot at higher resolutions and perhaps sizes. If in the microscope I know in advance I'm going to want to crop and enlarge I shoot at fine or high resolution image sizes. I have not required use of uncompressed TIFF type images for these purposes.

Manual setting mode for Nikon Coolpix series Cameras with the microscope

Turn the camera on to "Manual" mode" Not to confuse with the "Automatic" overall MODE setting above, to use the Coolpix through the microscope eyepiece I shoot with the ON-OFF DIAL in "Manual" mode (as opposed to fully Automatic) - set the dial that turns the camera on to "M" rather than "A".

1. Access the camera settings menu for basic photo settings:

Press the "Menu" button on the back of the camera (above the display screen) and then using the round thumb button to scroll and select, and the "picture taking" button to select each setting (there are shortcuts which I'm leaving out for simplicity), choose the following settings:

• White Balance
  For digital photos through the microscope - A (auto) will work, but better, mount and focus on one of your typical slides on the microscope stage. Move the slide so that there are few particles in the field of view - mostly white space, or all white space. Then use the camera's menu, at "White Balance" select "White Bal Preset,"then select "measure," and the camera will set a reasonable white balance for your microscope and slide set-up. This should produce images with color very close to what you see through the eyepiece. If this is not the case, you may have changed some microscope settings and you'll need to repeat this process.
• Metering - Matrix or Center Weighted
• Continuous - S (single shot not continuous shooting)
• Best Shot Selector - Off
• Lens - Normal
• *** Image Adjustment: either Auto or I prefer "More Contrast" but you should try "Auto" or "Normal" alternatives as well.
• *** Image Sharpening: either Auto or I prefer "High" but you should try "Auto" or "Normal" alternatives as well.

The *** items are helpful for better photomicrographs but are not essential.

2. Set the camera to Macro Mode
   On the back of the camera the bottom left button "M focus" press repeatedly until you see the little Tulip Flower in the upper right display. Now on the upper right of the camera back adjust the zoom (Ranges between "wide angle" and "telephoto") until you see the tulip flower turn yellow - which means you are in "Macro Lens" mode. (On the 850 just turn the dial to "CSM" mode.)
3. Infinity as an alternative to Macro Mode
   Macro mode and auto focus can often focus the camera to produce a better image than a human can do manually. However if the object of interest lacks strong lines and contrast, the camera may have trouble focusing. Alternatively, you may wish to be sure the focus concentrates on specific features or ornamentation. For these purposes, set the camera to infinity mode, and then observing the image in the camera LCD, you can fine-tune the camera focus by adjusting the microscope stage height slightly up or down as needed.
4. Turn off auto flash:
   On the back of the camera bottom center button, press until you see a circle with a jagged arrow - which means flash is turned "OFF"

Take some test photos through the microscope

Focus your microscope on your slide for best image Try shooting first through the eyepiece that has a reticule or other scale - you'll see the camera sometimes focuses on the scale rather than the image but at times, if the particle is close to the scale in depth this gives sharp results; Try now shooting the same image through the other eyepiece - one with no scale mounted in it - forces camera to focus on the particle - which I put in the center of the field. Also I set this eyepiece on my microscope to "0" image adjustment since I think the camera would not prefer to have to focus through the correction I otherwise set for my own eye.

Test Images for evaluating image resolution: you may already have images on file suitable for this purpose. I use slides of diatoms or radiolarians - particles which have very fine detail and sharp edges. One can also purchase slides specially prepared for this purpose (but they are expensive.) I tried using my stage micrometer but other images have finer detail.

Focusing tips for digital photography at the microscope

Hold the camera dead still against the eyepiece while shooting; the rim of the camera lens should press against your eyepiece sides, so you're not scratching anyone's lens. Or use a standard lens mount that replaces the eyepiece, or use a microscope with a trinocular head and mount your camera there.

• On my forensic microscope I mount the camera on the trinocular head and I add a Nikon MC-EU1 remote control which connects to the camera by a cable and permits me to take photos using a button on the desktop, avoiding touching the camera at all for least disturbance. However this device is not really necessary. This may be the only instance where the shutter delay on older digital cameras is an advantage. Simply press the shutter button and then remove your hands from the camera, giving it time to settle down before the shutter activates.
• If you're not getting a sharp photo the camera may be auto-focusing on a different part of the image.
• Be sure the object of interest is located in the center of the viewing area or use the camera's optional "manual focus" mode to select which area of the field is to be focused-upon.
• If you find the camera just can't get a clear photo no matter what, it might be that the image of interest lacks sufficient points of contrast (lines work best) for the camera to "see" what to focus upon.
• Try turning off the camera's auto-focus by setting it to focus at infinity. Then with the camera turned on, adjust the microscope stage up or down for the sharpest image shown on the camera LCD screen. You can improve your performance doing this by zooming in on the camera during focusing, then zooming out to get the image you want.
• For example shooting Periconia if you focus the microscope on the warty surface the edges of the spore will be blurred and it may take several tries at 100x to get a good shot. If you re-focus the microscope to show the sharp edges of the spore you'll see the camera hesitates much less before deciding it's got the image in focus.
• Try several shots, review on the camera screen, then load into your PC for really better examination.

Getting your photos from camera into the computer

Fast, simple photo transfer method - flash card readers for photo transfer

Remove the camera's flash card, plug it into

• a flash card slot on your computer,
• into a flash card reader which connects to your desktop computer's USB port
• into a flash card holder which connects to your laptop computer's PCMCIA card slot

open the image processing software (ThumbsPlus from Cerious Software [recommended--DF]for example)

open a Windows Explorer (PC-computers) or Finder (Apple Mac computers) window, and drag/drop the image files seen on the flash card hard drive into the desired image management software library window

I carry a flash card reader on long trips, along with my laptop, as this is a compact and very fast means of image upload. This is the most-general means of image upload as the flash card simply appears as an additional hard drive on the computer. Use of this method should not require the installation of any special software on your PC or Mac.

Photo Transfer by Manufacturers USB cables

Use the USB connector, connecting the camera to the computer's USB port.Turn on the camera, use manufacturer's driver (on the computer) or plug-and-play to see the camera appear as a hard drive; move (drag and drop) images to the desired library location - this works fine though I find that I might need the camera manufacturer's software installed on the computer in order to recognize the camera as a USB device. With this special software installed, usually a unique program will run automatically when the camera is detected, offering to show, copy, or otherwise handle your photos. This method of data transfer is often slower than the flash-card approach because software in the camera is acting as a data transfer device.

Photo Transfer by Video bus connection

Use the camera video-out connector and cable to connect to a video input on your computer. - this approach also works fine though I find that I might need the camera manufacturer's software installed on the computer in order to recognize the camera as a USB device and as with the USB approach above, this special software installed, usually a unique program will run automatically when the camera is detected, offering to show, copy, or otherwise handle your photos. This method will be slower than the flash-card approach, as mentioned above.

Photo Transfer by Infra Red or Blue Tooth

Some cameras also permit photo transfer from the camera directly to a computer by infra-red (IR) or Blue Tooth technology. In my (limited) experience these methods are convenient but slow compared with a flash card.

Organizing and finding digital micro-photographs on a computer

Selection of a photo library management program for Digital Photography through the Microscope

Library design is critical if a large number of photographs are to be stored and ever found again. I have found that general purpose PC-type software such as the Thumbs Plus photo management software by Cerious (www.cerious.com) is particularly suited for this purpose for these reasons which also will describe what I consider important in photo library software:

• The program permits ready directory creation, image movement, copying, and deletion without hogging disk space by keeping multiple copies of each image every time one is moved or changed. [The Mac iPhoto program is the world's worst offender in this regard.]
• Basic image manipulation such as rotation, cropping, enlarging, jpeg cleanup, image enhancement, color balance, saturation, contrast, brightness, are readily at hand.
• Image file naming and image clean up or manipulation functions can be applied either to individual photo images or can be applied to groups of pictures. This makes the assignment of meaningful names to images a snap as it can be done en masse rather than having to type in individual photo names.
• Directories can be scanned for duplicate or similar images
• A watermark can be assigned to images, e.g. for © protection
• Annotations and keywords can be assigned to each image, recording technical or descriptive data for each photo.
• Data base functions are provided, permitting searching for images based on name, assigned keywords, or annotations.

Library design for digital photographs through the microscope

Build a file or disk directory library of folders which organize your photos according to your reference library schema. Example, if you're an aerobiologist, mold, or pollen specialist, create folders for:

Microscopy Pictures - a sample library organization

• to be sorted - this is a general import photo directory
• Bacteria
• Ascomycota
• Yeasts
• Amphisphaeria
• Apiosporina
• etc
• Basidiomycota
• Uredinales RUST
• Ustilaginales SMUT MILDEWS
• Acryia
• Agaricales
• Agaricosa
• etc
• Myxomycota
• Mitospores
• Algae
• Chrytidomata
• Oomycota
• Zygomycota
• Pollen
• Animals, Fibers, Insects, Other
• Unknown Particles

First Pass Screening Microscopic Photo Collection by Family or Type

Using a schema similar to the above, I place one or two representative photos of each spore of a general class (Ascomycetes) in the root directory Ascomata and then place all of the additional reference photos of each Genera/Species within its own subdirectory. On recognizing a spore as an Ascomycete, I can quickly scan through my Ascomycete library of representative photos, pick candidates, and look at more photos in their detailed subdirectories where there will be photos of more spore variations and details.

Saccardo organization for Mold Spore Photographs

I have experimented with forcing my higher level reference directory photos into a Saccardo schema, particularly under the Mitospore category, but it's been tough to maintain this order. Scrolling through the library is so fast that it's easy enough to spot and scan, for example, hyaline allantospores that I stopped forcing those photos into a false order and just order them by their individual names.

Using annotations, keywords, and data base search. I include keywords, annotations, comments, with each photo to assist in identification, such as remarks from reference sources about distinguishing characteristics, habitat, etc. I find the annotations extremely helpful, the keywords and search functions I use very little in my lab.

Typical photo annotations include:

• Photo source
• Unique identifying features to look for
• Other similar spores or pollen grains to cross-check against
• Habitat information
• Reference text citation
• Pathogenicity

Let me know how you succeed, or if you're having trouble obtaining successful, sharp photos with good exposure, email me a photo or two. Sometimes by looking at a bad photo we can figure out what camera or microscope changes might improve results.

I'm working on a visual spore and pollen key, and currently have about 250,000 images on hand, but still I'm lacking many of the common spores and pollen grains. Anyone willing to collaborate should send me a note.

Image file backups on trips

In the lab I make daily backups of my image library onto a second or even third device. One of these is portable and is carried home each night so that I have off-site bacup data. Once the volume of images becomes substantial, use of a portable hard drive is more practical than writing CD's or DVD's.

I also carry an external hard drive on critical excursions, so that I can back up my photos in at least two different media/physical devices. With a laptop along one can also write a CD or DVD for non-volatile storage as well.

Daniel Friedman

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.

PhotoShop 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

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