Getting started with Photomicrography

Macro photography challenges photographers, particularly when capturing objects at magnifications beyond 3:1 using standard lenses. The shallow depth of field and the need for equipment stability make achieving sharp focus difficult. Furthermore, limited lens options exist with magnifications beyond 2:1, with few offerings from Venus Laowa in the 1:1 to 5:1 magnification bands and virtually no offerings from Canon and Nikon beyond 1:1. To achieve extreme magnifications (greater than 4X), photographers turn to microscope objectives for high magnification photography, entering the fascinating field of photomicrography or Extreme Macro. This technique opens up a hidden world of microscopic wonders, involving the capture of images of minuscule subjects, including small wildlife and plant life, typically imperceptible to the human eye.

When selecting a microscope objective, consider the following important factors:

Optical Systems

Microscope objectives fall into two categories: finite conjugate and infinite corrected designs, offered by companies such as Nikon, Olympus, and Mitutoyo. Finite conjugate objectives focus light from the objective onto the focal plane of the eyepiece. In contrast, infinite corrected objectives collect light from the object and form a parallel beam, with the image distance set to infinity. An additional lens then refocuses the beam onto the eyepiece. In recent years, major microscope manufacturers have favored the infinitely corrected optical system because it allows for the inclusion of different optical elements, such as polarizers and filters, in the tube without interfering with the beam’s focusing.

When coupling a microscope objective with a camera, use a combination of extension tubes, coupling rings, and bellows. The recommended optimal tube length is approximately 200mm, which constrains the focal length of the tube lens to be around 200mm as well. Many macro photographers choose the Raynox 150 clip-on lens as a tube lens for additional magnification. The Raynox 150 has a focal length of 208mm, low spherical and chromatic aberration, making it well-suited for this purpose. Note that finite conjugate objectives do not require a tube lens, and the objective’s focal length determines the tube length.

Optical qualities

Select objectives with low spherical and chromatic aberrations, specifically Plan Achromat objectives with optically corrected distortions and flat fields, as they make excellent candidates for photomicrography. Additionally, choose an objective that produces a reasonably large image circle to cover the entire camera sensor and provides consistent image quality across the entire frame. Nikon Plan Achromat and Mitutoyo M Plan Apo series of objectives are good choices that provide excellent image quality.

Working distance and Numerical Aperture

Working distance (WD) and numerical aperture (NA) are essential factors in photomicrography. The working distance determines the farthest focusing distance from the objective, while the numerical aperture measures the objective’s ability to gather light and resolve fine specimen detail at a fixed object distance. Smaller working distances can make compositions and optimal lighting harder to achieve. Higher values of numerical aperture allow for more highly resolved images and better clarity in visualizing smaller structures. A NA of 0.25 at 10X is reasonably good and practical for photomicrography.

Lighting techniques

Lighting techniques play a crucial role in photomicrography, where you can use either reflected or transmitted light for creating photomicrographs. Use remote flashes or even a set of LED lights for reflected light, which works particularly well for opaque subjects such as insects and plant life. It’s also essential to diffuse the light to prevent harsh shadows and highlights.

Use transmitted light to photograph transparent/translucent subjects with the light source directly behind the subject. This technique works well for photographing crystals exhibiting birefringence under polarized transmitted light.

Other Challenges:

At extreme magnifications, the depth of field becomes shallow, and you need to move the entire camera setup by tiny distances to capture images at different focal planes for image stacking. Use electronic macro rails with capabilities to move the rig by a few micrometers for every step. Additionally, employ stacking software and techniques that can handle large stacks for artifact and noise control, as stacks for frames at extreme magnifications can be as high as a few hundred shots. Specimen preparation and staging also play important roles.

Single shot vs Stacked Image

From RAW to Final Image: A Photomicrography Workflow Example

To efficiently process the large number of images required to create a final image in photomicrography, design a well-planned workflow. Here’s an exemplary workflow used to create an extreme macro frame using a 10x microscope objective of a Morning Glory flower’s pollen-laden stamen and an early-stage Hibiscus bud.

1. The entire scene was imaged using a panoramic stack and stitch method, dividing the scene into four sections and imaging each separately. The specimen was moved using a precision XYZ linear stage to capture each section separately.
2. Each section consists of a separate stack of approximately 170 and 200 shots, covering the entire scene of the stamen and bud, which are about 5mm in length.
3. The focus shift was made at 14um distances using an electronically controlled macro rail to move the camera forward precisely for the focus stack.
4. Shooting in RAW format allows for subtle adjustments to highlights, shadows, and colors during raw image processing.
5. Assembling the stack in a lossless format such as TIFF is ideal, but is not always practical, as this requires 100’s of GB’s of hard disk space. The practical alternative will be to convert Raw files to Jpegs and stack in Jpeg format. Using Jpeg as a format allows a medium sized stack such as this one, to be assembled with as low as 50-100GB of disk space. While this introduces slightly more noise due to the very nature of the lossy compression of Jpeg, this can be controlled in the image post-processing stage.
6. The sections are stitched together seamlessly in software after stacking each section separately.
7. Final assembled image has the following post processing sequence to correct the background colours (in this case to true blacks), noise management, a tighter crop, healing to remove sensor dust trails and stack halo correction.

Here are a few other example frames created using the above-described techniques.

In summary, photomicrography requires attention to detail, patience, and expertise to capture images of minuscule subjects. By considering the factors mentioned above, you can create stunning and detailed images of subjects that are otherwise unseen to the human eye.