Is focus stacking with microscope lenses really necessary to reproduce small structures in detail in a photo? Or is a single shot with a good macro lens completely sufficient?
Occasionally you can read in social media that focus stacking with microscope lenses is actually completely redundant in order to produce micro photos with a high level of detail, because you can produce comparable images with macro lenses, reversing attachments or super macro lenses such as the Canon MP-E 65 mm, which then simply have to be scaled up accordingly. After all, these lenses have an aperture that can be used to create more depth of field so that focus stacking is not necessary. In this way, it is sometimes said, comparably detailed images can be produced with much less effort.
From my 18 years of experience with focus stacking, I am firmly convinced that this is not the case because special micro-optics simply have a much higher resolution and therefore better detail capture. But I was interested to see how big the difference between these two methods actually is. I therefore created a series of images to show what the individual lenses can get out of the same subject in terms of detail. I naturally chose my favourite butterfly Chrysiridia rhipheus as the subject, because its wing scales not only offer indescribably beautiful color combinations, but also filigree groove structures that make it easy to judge the richness of detail in a shot. And in a nutshell: the differences were even greater than I had expected.
The test candidates were the following lenses:
1. Canon macro lens RF 100 mm f/2.8 Macro L, up to 1.4:1
2. Canon MP-E 65 mm f/2.8 magnifying lens, 1:1 to 5:1
3. Mitutoyo microscope objective M Plan Apo 5 x with Raynox DCR 150 tube lens
4. Mitutoyo microscope objective M Plan Apo 10 x with Raynox DCR 250 tube lens
5. Mitutoyo microscope objective M Plan Apo 10 x with Raynox DCR 150 tube lens
6. HLB microscope objective Plan Apo 20 x with Raynox DCR 150 tube lens
Procedure for comparison
To ensure that blurring did not occur due to a poor or faulty stacking process, I created each series of images twice and also carried out the stacking process twice, because if there were any working errors, differences should be recognizable between the two resulting images. But this was not the case.
I also made sure that the part of the image to be enlarged was in the centre of the image in order to rule out the possibility that the error-prone edge zone of the lenses was being used with one of the lenses. Each lens delivers its best optical performance in the centre, and aberrations become more pronounced towards the periphery of the objective lenses. In such a comparison, it would therefore be fatal if different lens zones were used between the individual lenses. For this reason, only the central part of the images was enlarged for the comparison (content of the white frame).
I admit that I couldn't really do justice to the individual lenses with this approach, because in order to really represent their respective performance authentically, you would actually need to perform the upscaling of the image section (marked with a rectangular white frame) in an identical ratio for all lenses. However, as the image scales of the lenses differ radically, this made no sense in this comparison. I merely wanted to demonstrate that a high-quality microscope lens, which can only be used with complex focus stacking, produces considerably more detail than a normal 35 mm macro lens for an SLR or a mirrorless camera. The images of the two macro lenses therefore had to be greatly enlarged, those of the 5x and 10x microscope lenses only moderately, and those of the 20x and 50x should actually have been reduced in size. But I wanted to show how much more detailed information the microscope lenses can provide, in comparison to macro lenses, and I did not intend to demonstrate or evaluate the performance of the individual lenses.
Core of the article: 5x magnifying lens and 5x microscope lens
The exception here, however, were two lenses that entered the race with the same magnification: the Canon EF lens MP-E 65 at 5:1 and the Mitutoyo microscope lens M Plan Apo 5 x with Raynox DCR 150 tube lens at an extension length (distance from sensor to tube lens) of 210 mm. These two lenses produce a largely identical object magnification (the image scale of the Mitutoyo is a bit higher because the tube lens focal length of 208 mm was slightly longer than the required 200 mm). The detail reproduction of the images can therefore be directly compared here, which is why these two lenses are to a certain extent the core of this blog post, as they demonstrate quite authentically the resolution advantages that a microscope lens has over a highly specialized magnifying 35-mm-lens at the same magnification.
Of course, the enlargements go far beyond what you would normally do to a photo, because viewed on this scale, the photo would cover an entire wall with a high-resolution camera. But here I wanted to make the differences visible, and that requires scaling up an image section far beyond the pain threshold.
When looking at the images, you also have to bear in mind that each individual wing scale of the butterfly photographed is practically a speck of dust about a tenth of a millimetre wide. If such a wing scale falls onto a white table top, it simply disappears to our eyes and can only be detected with a strong magnifying glass of at lest 10 x magnification. Reproducing such tiny structures with such great detail is a masterly achievement for each of the lenses compared here. In this respect, the performance of the two Canon 35 mm lenses in the extremely enlarged sections is also absolutely impressive, even if they appear blurred there.
When comparing the 100 mm macro lens with the MP-E 65, however, it must also be remembered that the latter is ultimately nothing more than a 65 mm 35 mm lens mounted in reverse and equipped with a telescopic tube to enlarge the extension similar to what a bellows system would do. In principle, this is similar to a standard focal length mounted on the camera with a reversing ring and extension rings. Although this makes the image subject appear larger in the photo because the shooting distance can be reduced, the detail capture is no greater in this way than with a comparable standard lens in the normal position, apart from the fact that the aforementioned magnifying lens has been optimized for the close-up range, i.e. it performs at its best here, while the standard fixed focal length has most probably been optimized for infinity and would possibly have imaging errors at the edges in the close-up range. For a considerably higher level of detail reproduction in the photo, you simply need different lenses.
1. Canon 35 mm macro lens 100 mm f2.8
With the 100 mm Canon RF 100 mm f/2.8 macro lens, the enlarged image section shows the lowest quality in terms of detail reproduction. This is of course due to the fact that it has to be scaled up considerably more than all other images at only 1.4x magnification*. This is just to show how much less detail there is in a photo taken with this excellent macro lens. It was designed for a different purpose, and it absolutely fulfills that purpose. However, the example shows that a post-magnified macro photo does not have anywhere near the same level of detail as one taken with a microscopic lens. (*In contrast to the older EF version, the Canon RF 100 mm f/2.8 macro does not achieve only 1:1, but 1.4:1)
2. Canon MP-E 65 35 mm lens
Set at 5:1, the Canon MP-E 65 mm shows a little more detail in the extreme magnification (white rectangle) than the 100 mm macro. However, this is probably not really due to a higher resolution, but rather contributed to the fact that here part of the magnification effect was achieved optically, i.e. without loss through the objective lenses, whereas with the 100 mm macro lens the entire magnification process was carried out with simple upscaling, i.e. with a loss. If this magnifying lens were in the 1.4:1 position, i.e. synchronised with the macro lens described above, its reproduction of detail in a post-magnification of a section would probably hardly be better than that of the macro lens.
It should also not be overlooked that the market launch of the MP-E 65 mm was many years ago, while the lenses of the relatively young (mid-2024) RF 100 mm f/2.8 macro have been intensively revised and represent the state of the art. One indication of this is, for example, the chromatic aberrations, the colored fringes, that the Canon MP-E 65 mm reveals in some magnified images when looking closely at critical subjects.
Mitutoyo microscope objective M Plan Apo 5 x
The Mitutoyo M Plan Apo 5 x microscope objective, which was used here with the Raynox DCR 150 tube lens (focal length 208 mm), is clearly in a different league. This image allows a direct comparison with the previous Canon MP-E 65 mm magnifying lens, and here it becomes clear that the detail reproduction is far superior to that of a conventional 35 mm macro lens. This can be seen particularly well in the light green-turquoise wing scale in the centre, whose groove structure is already clearly visible, whereas with the MP-E 65 mm these grooves can at best only be guessed at.
Mitutoyo microscope objective M Plan Apo 10 x + DCR 250
Even greater detail precision can be achieved by using a microscope objective with higher magnification and higher numerical aperture, but not giving it the tube lens focal length actually required, but a smaller one, combined with a smaller distance between the camera sensor and the tube lens. This sounds complicated, but is actually quite simple, and I will describe it in detail in a future blog post.
This does not work to the same extent with all microscope lenses, because the flexibility of the optics varies greatly here, and you simply have to work it out with trial and error. However, the two Mitutoyo objectives used here are excellent.
In this way, the Mitutoyo M Plan Apo 10 x with the Raynox DCR 250 tube lens (focal length 125 mm) was brought to a magnification of about 6 x. You can see that the overall image is slightly larger than that of the two aforementioned lenses at 5x, so that a little less digital magnification was required by upscaling. The detail rendition is again significantly better than with the previous Mitutoyo M Plan Apo 5x lens, which can be explained by the higher numerical aperture (details in article "Microscope objectives – what is the numerical aperture?"). And it is precisely this that opens up the little-known possibility of creating higher-resolution images than is possible with a microscope objective with a specific magnification. In a way, it's like using the standard version of an objective (e.g. Mitutoyo M Plan Apo 5x), but creating images that are comparable to the detail of the much more expensive HR version (high resolution). As I said, I would like to present details about this in a separate article in the future. The gain in detail can be seen very clearly in the turquoise wing scales in the centre of the image section.
Mitutoyo microscope objective M Plan Apo 10 x + DCR 150
Once again, the image section becomes noticeably more detailed if the Mitutoyo M Plan Apo 10x microscope objective used is given the tube lens focal length that it actually requires in order to achieve the nominal magnification (the value printed on the outside of the objective body). In this case, this is 200 mm, which the Raynox DCR 150 almost equalled with its 208 mm. This means that even greater detail precision can be achieved with this lens than with the smaller tube lens focal length mentioned above, which can be seen in the comparison of the turquoise-coloured wing scales in the centre of the image section: the groove structure becomes clearer once again. This shows that although the 10x objective loses resolution when the tube focal length deviates downwards (125 mm instead of the required 200 mm), the fine detail it achieves in the photo is still better than that of the lower magnification 5x objective with the actual matching tube focal length of 200 mm (or 208 mm).
HLB microscope objective Plan Apo 20 x + DCR 150
To top it all off, the same section of the butterfly's wing was then to be photographed again with the HLB Planapo 20x, using the matching Raynox DCR 150 tube lens. There was no need to enlarge the image by upscaling because the image display was larger than in the sections of the previous experiments anyway. The turquoise-coloured wing scale shows that the resolution is incomparably higher, and even upscaling (image in the white frame) cannot overtax the resolution. A look at the high NA value of the objective of 0.42 explains this fine detail, as the 10x objective is only 0.28.
Conclusion
This brings me back to the question I posed at the beginning: „Is focus stacking with microscope lenses necessary to produce photomicrographs with a high level of detail, or can comparable images be produced with macro lenses, reversing rings, close-up lenses or super macro lenses such as the Canon MP-E 65 mm?“
If you compare the detail reproduction of the wing scales of all three microscope lenses with that of both 35 mm objectives, it is obvious that optics from completely different application areas are competing here. Standard focal lengths with reversing rings and intermediate rings, as well as lenses fitted with a close-up lens, are able to get closer to the object compared to a standard lens and thus to show it larger. However, this alone does not improve the capture of the tiniest details.
In macro and magnifying objectives, the lenses are optimized for close-ups and their line reproduction is considerably higher than that of a standard objective. But their detail capture simply does not go beyond a certain level.
Microscope lenses, on the other hand, are specially designed for maximum detail reproduction, virtually regardless of losses, because you pay dearly for this with a decreasing depth of field. The depth of field of microscope lenses is so adventurously shallow that it is impossible to obtain a usable photo in this way. The situation is of course different when looking through the eyepieces of a microscope, because here you can move the focal plane forwards and backwards by turning the fine adjustment knob and thus make all the details visible. A photo, however, only shows a wafer-thin plane of sharply reproduced details, and here the focus stacking technique is actually indispensable in order to obtain a usable photo that shows far more details than a conventional 35 mm lens would ever be able to.
Whether you work with a macro lens or a microscope lens simply depends on the level of detail you expect from the photos.
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