Macro Image Techniques By W.P. Armstrong    © W.P. Armstrong 3 November 2017 Oversimplified Definitions: A macrophotographic image is a close-up image taken through a macro lens with a magnification ratio of at least 1:1. In other words the subject appears life size on the film or sensor. The Canon 5x Macro Lens produces extreme close-up images from a 1:1 magnification ratio to 5:1 ratio. The subject will appear up to 5 times its original size on the sensor. Under low magnifications, an image taken through a dissecting microscope could be considered an extreme macro image. Generally a true microphotographic image is taken through a compound microscope with a magnification ratio of 20:1 or greater (with magnifications up to 1000x). During My 40+ year career as a professor of botany & biology I have taken literally thousands of macro images with a variety of equipment, including film and digital cameras. For very small subjects I have used macro lenses with extension rings and low magnification dissecting microscopes. With cropped images I have also used uprezing programs such as the excellent PhotoShop plugins: Genuine Fractals and Perfect Resize. With these programs I can back away from subject a little to maximize depth-of-field and lighting, while still mantaining a desired image size. I am referring here to macrophotography, not to be confused with my numerous microphotographic images taken with an Olympus Compound microscope at magnifications up to 1000x. The following images show some of my different techniques for macrophotography. Note: I strive to show sufficient detail in my images so that  an authority could verify my identifications at least to genus. Links     Table Of Contents For Macro Images      1. Nikon     Images With Nikon Digital SLRs     2. Dissecting Microscopes     Nikon B & L Adapters B & L Stereomicroscpes 3. Sony Cameras Sony T-9 & T-10 Mini Cameras 4. Compound Microscope A True Microphotographic Image 1. Nikon Digital SLRs Photographing Live Ants Ants are one of the most difficult subjects for macrophotography. They are very small and are constantly moving. When placed in a refrigerator to slow them down, they assume unnatural positions until they "wake up." Pinned or preserved specimens in alcohol may be suitable for strictly morphological or anatomical images, but generally do not make natural photogenic subjects. In my opinion live ants make the best subjects, but they are quite challenging. I have fairly good results placing the live ant in a white Corelle dish or in a transparent bowl on a light box. When the ant settles down (pauses) I simply photograph it on the sides or rim of the dish. Of course, I must constantly encourage it to stay in the dish with my free hand. This method doesn't always work with very fast ants that can easily climb up the sides of the dish. I use a Nikon D-90 SLR with a good macro lens and ring flash or SB-400 flash with diffuser. I actually prefer an inexpensive Opteka Diffuser slipped over my SB-400 flash. With F-22 or F-32 and at least 1/200 shutter speed I get good depth of field and stop action results. This is particularly true when the ant pauses. This requires patience and careful ant tracking, and numerous shots. FP mode with higher shutter speeds can also be used, depending on the camera model (D-90 and above). Reflections on the glass dish (see left image) can be removed with Photoshop. In the following images there is very little reflection on the glass bowl. The circular ring flash reflections can be removed with photoshop, or a different flash with diffuser can be used (preferably Opteka Diffuser). The bottom line here is obtaining sharp images with good depth of field. In the lower image, the three minute simple eyes (ocelli) on the top of head plus the antennal and tarsal segments are all in sharp focus. [Ocelli pronunciation: oh-CELL-eye.] There are more elaborate macro-systems for ants; however, some are rather cumbersome, especially when carried into the field. There is a faint reflection of ring flash on the above image. Pseudomyrmex apache typically nests inside cavities of dead branches and abandoned galleries hollowed-out by beetles. According to Antweb.com: Pseudomyrmex apache is found throughout most of California except the mountains and extreme north. It occurs in chaparral, oak woodland, mixed (oak-pine-douglas fir) forest, coastal sage scrub, and desert riparian sites. Nests have been collected in dead branches of Arctostaphylos, Baccharis, Quercus and Umbellularia. There are also records from a Fraxinus gall and a Pinus attenuata cone. Workers appear to be generalist scavengers. My specimen was discovered in the coastal sage scrub of northern San Diego County (north side of Owens Peak) on a flowering Baccharis pilularis that resembles B. sarothroides. Dead branches of this common shrub often have hollow stems; however, I have yet to find an active nest of this remarkable ant species. Ring Flash May Not Be The Best Choice For Shiny Convex Subjects! Desert harvester ant (Messor pergandei). Photographed with a Nikon D-90, SB-400 Flash and Opteka Diffuser. A winged Myrmecocystus queen and three workers. Photographed in a field collection container with a Nikon D3200, SB-400 Flash and Opteka Diffuser. In our southwest desert, big-headed ants (Pheidole) have major workers to defend their nest against army ant raids. The major worker has an enormous head in proportion to its body. Some species actually block the nest entrance with their heads! Nocturnal Carpenter Ant (Camponotus fragilis) near Tucson, Arizona. This carpenter ant was found in the Poleta Limestone outcrops along the road to the Schulman Grove of bristlecone pines in the White Mountains of eastern California. It resembles on-line images of Camponotus sansabeanus which is reported from this area. Live Cyphomyrmex wheeleri caught in pitfall trap. This small ant is about 2 - 2.5 mm in length. It belongs to the subfamily Myrmicinae (tribe Attini) and includes the larger Arizona leafcutters Acromyrmex & Trachymyrmex, and the tropical leafcutters Atta. Like the larger leafcutters, Cyphomyrmex is a fungus-gardening ant. It carries organic material such as insect droppings and pieces of plants back to its nest, using the material as a food for their fungus gardens. The ants then eat the fungus that they cultivate on the debris.Their garden consists of a particular fungal species that these ants have been cultivating for millions of years!   See Images Of Other Leaf-Cutter Ants On Ant Introduction Page   Army ant worker (Neivamyrmex) from under a rock on Superstition Mountain. These minute ants are without eyes. According to Neivamyrmex authority Gordon Snelling (Personal Communication, 26 Jan. 2017), it is N. leonardi! Live Pogonomyrmex rugosus workers taken with Nikon D-90, 60mm MicroNikkor A-F-2 F/2.8G Macro Lens, SB-400 Flash and Opteka Diffuser. The image was uprezzed 300 percent with Genuine Fractals. The detail is sufficient to separate this species using the Key To North American Pogonomyrmex in AntWiki. Pogonomyrmex sp. using another technique for extreme close-ups: I placed my Nikon D-90 with 60mm MicroNikkor A-F-2 F/2.8G Macro Lens and one Kenko Extension ring on a Bausch & Lomb stereomicroscope stand. Lighting was from two studio lamps on either side without electronic flash. The microscope stand made it easier to do critical focusing on the subject. This effect produced a sharp image of the ant specimens (red arrow) without hot spots or the annoying rings of a ring flash. In my humble opinion, there are 3 important factors for good macro images: (1) A good camera with macro lens, (2) A good lighting system, and (3) Experience and/or knowledge of macrophotography. I would rate each of these at about 30 percent. For good on-line Internet images, you don't need the latest or most expensive camera. Even with 12 megapixels you can uprez to 24 MP without noticeable deteriation to the image. 16 Ant Species From Owens Peak On Ant Introduction Page Many of the ants from Owens Peak were photographed alive on the white Corelle dish discussed above using Nikon D-90 with 60mm MicroNikkor A-F-2 F/2.8G Macro Lens, SB-400 Flash and Opteka Diffuser.   Table Of 16 Ant Species On Owen's Peak   A live yellow jacket (cf. Vespula pensylvanica) portrait taken on New Year's day 2018! Three ocelli are clearly visible between the 2 multifaceted compound eyes. Nikon D-90 with 60mm MicroNikkor A-F-2 F/2.8G Macro Lens, SB-400 Flash and Opteka Diffuser. Image enlarged 300 percent with Genuine Fractals. The basket of hairs (psammophore) around & below mandibles helps this queen carry mouthfuls of dry, loose sand without spilling out. Nikon D-90 with 60mm MicroNikkor A-F-2 F/2.8G Macro Lens and three Kenko Extension rings. Image taken hand-held with Nikon SB-400 Flash (without Opteka Diffuser). To verify the ID of Pogonomyrmex magnacanthus in Borrego Valley you must count the number of teeth. It has 7 teeth per mandible. The rare P. anzensis is similar in size to P. magnacanthus but has only 6 teeth per mandible. A species of Apsena in the Tenebrionidae: My latest beetle discovery on Owens Peak. It is always a thrill to figure out the name of a new beetle (new to me), especially considering that there are at least 350,000 described species. Nikon D-90 with 60mm MicroNikkor A-F-2 F/2.8G Macro Lens. Image taken hand-held with Nikon SB-400 Flash and Opteka Diffuser. I am reminded by a number of on-line photography pages that f-32 is not the best camera setting for optimal image quality. In my humble opinion, APS digital cameras & higher f-stop settings gives me slightly more depth-of field which is more important to me. This is especially true in ant ID where you sometimes need to count antennal segments, number of ommatida (facets in compound eyes) and shape of their anus; however, in the latter cases, a stacking program or dissecting microscope would probably produce better images. 2. Macrophotography With Bausch & Lomb Dissecting Microscopes Sony DSC W-300 Mounted On B & L Stereomicroscope The Sony W-300 can be mounted on the ocular tube of a stereozoom (dissecting) microscope using a Sony adapter ring and a 37mm (M) to T step ring. When attached to a Scopetronix Maxview Plus adapter, the camera slips into the eyepiece housing. You simply focus through the camera's high resolution LCD screen. In order to avoid camera movement at slow shutter speeds, it is advisable to use the 2 second self timer. It is also advisable to use manual (M) settings on the camera. From the left: Sony DSC W-300 Cybershot camera; Sony VAD-WF adapter ring; 37mm (M) to T step ring (37 mm to 42 mm); Scopetronix Maxview Plus adapter; Note: the Maxview Plus shown above slips into an ocular tube 24 mm in diameter. [24 mm extension can be replaced with a 30 mm extension for other microsopes.] Advantages of Sony W-300 over a digital SLR: Smaller and lighter than SLR with extension rings. Brighter LCD display. Extension rings greatly reduce viewing light. Digital SLR requires 3 extension rings in order to eliminate vignetting.    The 13.6 megapixel sensor has plenty of room for cropping. Magnified view of Denver mint date on U.S. Penny Rose bud photographed with Sony W-300 through Bausch & Lomb dissecting microscope. The dew droplet is one millimeter (1/25th of an inch) in diameter. One gland-tipped hair is trapped within the tiny water droplet. The individual red gland at the tip of stalk is about 250 micrometers in diameter. Magnification 30x Using Nikon SLR Adapters With B & L Stereomicroscope Comparison Of Nikon Adapters With Sony W-300 Attached To B & L Summary & Conclusions In my humble opinion, images #1 and #2 are slightly better in terms of sharpness. In addition, image #3 was more difficult to view through the Nikon camera body. However, ant images taken with above Nikon adapters and Bausch & Lomb stereomicroscope (B & L) were disappointing. Undoubtedly, better lighting would have improved these images. I used a high intensity incandescent illuminator that worked fine for other subjects, but not for these minute, shiny ants. In fact, I have had good results using this illuminator with other subjects using B & L with Sony W-300, and especially photographing duckweeds many years ago with Kodachrome 64 and my Nikon FM-2. With the Sony W-300 attached to B & L (eyepiece removed), I tried two 5000K daylight fluorescent lamps, one on either side of microscope stage. In my opinion the results were quite remarkable compared with the 2 Nikon adapters. In fact, there was insufficient light using these daylight lamps with the Nikon adapters. One of the advantages with the W-300 is that the camera menu has different white balance settings: Daylight, fluorescent, incandescent, cloudy, etc. The daylight setting had the best color. Since the camera only has a maximum resolution of 14 megapixels, I simply uprezzed my image with the Photoshop plugin "Genuine Fractals." You can easily double an image resolution without any noticeable degradation of the image, at least for the Internet! The following W-300/B&L image shows a considerable improvement over images taken with 2 Nikon adapters: Image of the minute, big-headed ant Pheidole navigans (shown above) taken through Bausch & Lomb stereomicroscope. The right eyepiece was replaced with Sony W-300 digital camera. The light source was two 5000K daylight, fluorescent lamps instead of the high intensity B & L all purpose, incandescent illuminator typically used with this dissecting microscope (see left image). The image also shows a fluorescent illuminator for substage lighting. Along with good lenses, lighting is an extremely important factor in highly magnified subjects. My original hypothesis is true: "Images of subjects such as minute ants (1.5 - 2 mm in length) taken with this B & L stereomicroscope do not show as much detail as a high quality macro lens with electronic flash and high f-stop." This is largely due to the lack of depth of field compared with a good macro camera setup (see following image). Another important factor is the electronic flash used with the macro lens. Of course, multiple images using a stacking program would greatly enhance these images. Subjects such as minute ants are very sharp and appear in 3d looking through the B & L; however, our greatly evolved, remarkable brain & stereoscopic vision are superior to images transferred directly to the Nikon sensor from the microscope. In a way, our eyes work like an instantaneous, incredibly sophisticated method of stacking!   Excellent Article By David Molnar: Human Eye Compared With Camera   Image of Pheidole navigans taken with Nikon macro lens, extension rings and electronic flash. The following image shows the equipment I used to photograph this minute tropical ant species introduced into Twin Oaks Valley, San Diego County via local nurseries. My modest, relatively inexpensive photographic equipment is borderline for the identification of ants of this size using scholarly taxonomic keys.

I have definitely taken some very good images with this B & L setup. This is particularly true of extremely small subjects, such as head shots of minute ants in the genus Strumigenys that were only about 0.5 mm long. And I have never surpassed my wolffia images with my old Nikkormat FTN or Nikon FM-2 film camera body attached to the B & L. In addition, I was able to get above lighting and substage illumination with the B & L which brought out more detail of the microscopic subject matter; however, I must add that most of the macro images on Wayne's Word were taken with Nikon digital cameras with a macro lens and extension rings.

L eft image was taken during the early 1980s with film camera body attached to B & L microscope. I did a lot of duckweed photography in those years. My film & processing bill one year was over $3,000. This would equate to more than $7,000 at today's prices! The following minute wolffia plant is seldom seen and it is rarely observed in flower. In fact, some botanists have stated in the literature that it only reproduces asexually by budding and no longer produces flowers! Floating on the water surface, it is a difficult subject to photograph.

Wolffia also produces the world's smallest fruit. Each one-seeded fruit is about the size of a cuboidal grain of ordinary table salt (0.3 mm on a side) and weighs about 70 micrograms (1/400,000 of an ounce). It is roughly five billion times lighter than a 900 pound squash. The wolffia fruits were photographed in an alcohol (ethanol) solution and the salt grains have dissolved slightly resulting in rounded corners and the appearance of ice cubes.

During my teaching career of 40 years I have taken countless thousands of images using various cameras with close-up (macro) lenses, from 35mm film cameras using Kodachrome and Ektachrome to modern digital SLRs. I have shared these images in my lecture presentations at Palomar College and elsewhere, using carousel color transparency projectors and modern digital projectors. In fact, many of these copyrighted images are on my Wayne's Word website. Although my on-line images are only 72 dpi, they project very well on large classroom screens with high quality LCD projectors. Many of my images have also been published in magazines, peer reviewed journals and textbooks.

Although my first 35mm cameras were Canon and Tower (Sears), I have used Nikon & Sony cameras during most of my career. I have pushed my introductory Nikon SLRs and macro lenses to the limit, and acheived some fairly good images of ants. However, in my opinion, I have never quite acheived the quality of Alex Wild's ant images using Canon SLRs and his fabulous Canon MP-E 65mm 1-5x macro lens. Unfortunately, this lens will not work with a Nikon body. Another major factor in macrophotography is diffuse lighting with an effective external flash system. Of course, years of experience with ant photography is invaluable! In conclusion, my Nikon setup is relatively inexpensive and very portable for field work, especially long, arduous hikes over rough terrain.

When it comes to duckweeds in flower (family Araceae subfamily Lemnoideae), the macro images with Bausch & Lomb dissecting microscope and substage illumination are hard to beat, particularly with Kodachrome 64 and my old Nikkormat and Nikon film cameras.

Although probably not in the same league as digital SLRs, I have actually taken some pretty good macro images with small Sony digital cameras. The built-in flash allows shutter speed of 1/1000th, and the close focusing and small sensor produce good depth of field. The cameras can be held in one hand with the arm outstretched.

Magnification Ratio With Camera Attached To Microscope: E.g. sensor width = 36 mm. If the projected image on camera sensor is life size (i.e. fills entire frame when viewed through camera), then the magnification ratio is 1:1. In other words the object is the same width as sensor. If the projected image on sensor (when viewed through camera) is 2 mm across, then the magnification ratio is 18:1 (36/2 = 18). I.e. the subject is 18 times larger. When magnification ratios approach 20:1 you are in the realm of microphotography rather than macrophotography.