Condenser: Two achromatic doublets (50 mm FL and 19 mm FL) to create a condenser with an approximate NA of .57 - This is hugely overkill for the objective I’m using but I plan to reuse this later since it appears to work well. In the future I think an Abbe style condenser would be a better and cheaper choice but it’s not easy to find large ball lenses like they tend to use. Anyway, using achromats here seems like overkill and I could definitely have gotten away with singlet lenses. There’s also a bayonet mount on the bottom of the condenser that I can mount aperture stops to. I haven’t tested that yet so all these images are with the condenser “wide open”

However, to achieve an identical proper exposure, the shutter speed is probably closer to 1/1000th to compensate for the increased amount of light entering the lens at f/2.8.

DIN standard ------------------------------------ Yes Magnification ------------------------------------ 40x Numerical Aperture ------------------------------ 0.65 Working Distance -------------------------------- 0.6 Field of View...

Objective: I tested a used Olympus 10X Plan achromat from Ebay. These have field numbers of 22 with their native tube lens focal length (180 mm) and are pretty easy to find used for cheap. I use similar objectives on commerical scopes and the quality is fine for a low-power objective. Most importantly, the field is quite flat across the whole field of view.

As for the condenser, it looks good but with such a low-NA objective it’s hard to tell if it’s actually doing a good job. Once I get a higher NA objective to test it with, we’ll see how it performs.

May I ask if you have tried imaging at low light, to get an idea for pixel-to-pixel heterogeneity, “hot pixels” etc? I wonder if these artefacts, if present, can be handled by libcamera which Raspi comes with. My application is 3D real-time tracking of microparticles, with each particle image occupying 10-20 pixels. I’ve done it with high-end cameras, NA1.40+ objectives and nano-stages on “proper” microscopes, and I wonder if the setup can be miniaturized w/o losing too much performance.

The last element affecting depth of field is the distance of the subject from the lens – you can adjust the DOF by changing that distance.

Well, the colors looking better has nothing to do with the optics; that’s down to the camera sensor and its bayer filter/microlens array. Using the HQ camera alleviates this but it’s not compatible with the OFM software yet.

Just wanted to chime in here to say that people in the macro photography world swear by the raynox DCR 150. Not sure the 200mm focal length is what you want for a rpi camera though.

Field curvature is definitely something that can be compensated for by focus stacking. It’s actually an ideal use-case for it! You mentioned that you’ve experienced this; are you using infinite conjugate objectives? I am curious if the current tube lenses works better with finite conjugate objectives.

The aperture is the opening at the rear of the lens that determines how much light travels through the lens and falls on the image sensor.

It is really interesting to see in this and other threads people really looking into the detail of the performance of their microscopes. The overall system works well enough that the optical quality of different objective lenses becomes apparent. With the motorized focus and scanning the microscope has access to all of the information that is necessary to characterise its own performance. A self-check routine is a goal of current developments of the software. I don’t think that evaluation of field curvature is planned initially, but it should be possible to quantify field curvature and tilt, and even identify the presence of aberration at the edges that is not just a different focus but blurring at any focus.

Each movement up the range (say f/2 to f.2.8) reduces the amount of light by one-half, and each movement down the range (say f/11 to f/8) doubles the amount of light passing through the lens.

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Basically, when you change the aperture size one stop, you have to shift the shutter speed one stop in the opposite direction to maintain a consistent exposure… and this change in aperture alters the depth of field (DOF) accordingly.

Stage: Just a (reasonably) flat surface that goes up and down. I’m using a bolt and captive nut to raise and lower it, and I built a simple double-paralellogram flexure to hold it in place. I also put some captive nuts in the stage that hold screws to adjust a platform above the stage, if I need a really flat field. This only sort of worked and probably isn’t worth the trouble.

For the tube lenses, I would have expected the Thorlabs achromats to be best when operated in their design configuration with the image at the focal distance from the lens - which you get with an infinity corrected objective. But that is moving in the opposite direction from your observation - a larger distance of 75mm for your lens.

Focus enables you to isolate a subject and specifically draw the viewer’s eye to exactly where you want it.The first thing to understand about focus is depth of field.1Depth of FieldThe depth of field (DOF) is the front-to-back zone of a photograph in which the image is razor sharp.As soon as an object (person, thing) falls out of this range, it begins to lose focus at an accelerating degree the farther out of the zone it falls; e.g., closer to the lens or deeper into the background. With any DOF zone, there is a Point of Optimum focus in which the object is most sharp.There are two ways to describe the qualities of depth of field – shallow DOF or deep DOF. Shallow is when the included focus range is very narrow, a few inches to several feet. Deep is when the included range is a couple of yards to infinity. In both cases DOF is measured in front of the focus point and behind the focus point.DOF is determined by three factors – aperture size, distance from the lens, and the focal length of the lens.Let’s look at how each one works.2ApertureThe aperture is the opening at the rear of the lens that determines how much light travels through the lens and falls on the image sensor.The size of the aperture’s opening is measured in f-stops – one of two sets of numbers on the lens barrel (the other being the focusing distance).The f-stops work as inverse values, such that a small f/number (say f/2.8) corresponds to a larger or wider aperture size, which results in a shallow depth of field; conversely a large f/number (say f/16) results in a smaller or narrower aperture size and therefore a deeper depth of field.3Small vs Large ApertureManipulating the aperture is the easiest and most often utilized means to adjust Depth of Field.To achieve a deep, rich and expansive DOF, you’ll want to set the f-stop to around f/11 or higher. You may have seen this principle demonstrated when you look at photos taken outside during the brightest time of the day. In such a case, the camera is typically set at f/16 or higher (that Sunny 16 Rule) and the Depth of Field is quite deep – perhaps several yards in front of and nearly to infinity beyond the exact focus point.Let’s take a look at these two photos as examples. The left side of the photo has an expansive DOF, most likely shot around noon (notice the short, but strong shadows), with an f/22 aperture. The right side of the photo has an extremely shallow DOF; probably an f/2.8 aperture setting.However, to achieve an identical proper exposure, the shutter speed is probably closer to 1/1000th to compensate for the increased amount of light entering the lens at f/2.8.4Aperture RangeThe aperture range identifies the widest to smallest range of lens openings, i.e., f/1.4 (on a super-fast lens) to f/32, with incremental “stops” in between (f/2, f/2.8, f/4, f/5.6, f/8, f/11, f/16, and f/22).Each f-number is represents one “stop” of light, a stop is a mathematical equation (which is the focal length of the lens divided by the diameter of the aperture opening) that determines how much light that enters the lens regardless of the length of the lens. Such that an f/4 on a 50mm has smaller opening than an f/4 on a 200mm, but an equivalent amount of light travels through both lenses to reach the image sensor thus providing the same exposure.Each movement up the range (say f/2 to f.2.8) reduces the amount of light by one-half, and each movement down the range (say f/11 to f/8) doubles the amount of light passing through the lens.It’s important to understand this concept and how it affects exposure because it works in tandem with the shutter speed (we’ll discuss this in another section) to establish a given exposure value.Basically, when you change the aperture size one stop, you have to shift the shutter speed one stop in the opposite direction to maintain a consistent exposure… and this change in aperture alters the depth of field (DOF) accordingly.5Distance from the LensThe last element affecting depth of field is the distance of the subject from the lens – you can adjust the DOF by changing that distance.For example, the closer an object is to the lens (and the focus is set on that object) the shallower the DOF. Conversely, the reverse is true – the farther away an object is and focused on, the deeper the DOF. Changing the distance to subject is the least practical way to manipulate the depth of field, and by changing the distance from a subject to the lens, you immediately change your image’s composition. To maintain the compositional integrity of the shot, but still have the change in DOF from a distance, you can change the focal length (either by changing lenses or zooming in).Why does changing the focal length negate the effects on DOF? This is because the visual properties of a given lens either provide either greater DOF (shorter lenses) or shallower DOF (longer lenses). The physical properties of a lens at a given focal length also affect the depth of field. A shorter focal length lens (say 27mm) focused at 5 meters, set at f/4 has a deeper DOF (perhaps from 3 meters in front and 20 meters behind) than a longer focal length (say 300mm), also set at f/4 focused at 5 meters. The 300mm lens has a remarkably shallow depth of field.Incidentally, to help you with this, every lens has a manual with a DOF chart for each f/stop and the major focusing distances. DOF is just a matter of physics, and it’s important to grasp this concept.CConclusionManipulation of depth of field is a good way to modify the characteristics of your photo, and manipulating the aperture is the ideal way to do this because it has little or no effect on composition.You simply need to change the shutter speed (or change the light sensitivity – ISO) to compensate for the changes in the exposure from the adjustments to the f-number. Changes in distance and focal length also affect DOF, but these changes have trade-offs in terms of composition.Therefore, changes to aperture are the best way to manipulate DOF without affecting a photo’s composition.

Your 40x has less severe softness but it’s still noticeable. It’s supposed to be a semi-plan objective which means that 80% of the field of view is supposed to be in focus at the same time. This doesn’t look like it’s performing as designed but where the issue is isn’t clear to me.

Depth of field vs depth of focusreddit

Absolutely. I think I might explore C-mount photo/video lenses with 50-60mm focal lengths eventually - they might work well, are often inexpensive, and would be a lot more compact than my current system

As you say, the filter/microlens array of the PiCamera 2 gives problems that are hard to correct completely (discussed in other threads about colour saturation) the High-Q camera does not have that issue and so should be a lot better. Unfortunately until now the Pi Camera V2 has been the only one that could be integrated into the software to give the live illumination and flat field correction and fast autofocus, which are necessary for scanning and tiling. The upcoming Openflexure server V3 should allow us to use the other Pi cameras.

DIN standard ------------------------------------ Yes Magnification ------------------------------------ 40x Numerical Aperture ------------------------------ 0.65 Working Distance -------------------------------- 0.6 Field of View...

Results Raspberry Pi Camera v2 with two-lens tube lens (I manually flat-fielded these images with varying degrees of success) reticle_flatfield1920×1439 135 KB flat_field smear1920×1439 315 KB flat_field pinworm1920×1439 267 KB

You simply need to change the shutter speed (or change the light sensitivity – ISO) to compensate for the changes in the exposure from the adjustments to the f-number. Changes in distance and focal length also affect DOF, but these changes have trade-offs in terms of composition.

The aperture range identifies the widest to smallest range of lens openings, i.e., f/1.4 (on a super-fast lens) to f/32, with incremental “stops” in between (f/2, f/2.8, f/4, f/5.6, f/8, f/11, f/16, and f/22).

These last images look amazing!. No out of focus areas and the colors are perfect. Please tell me your secret! I am use finite conjugate objectives: 160/0.17 1) Parco Scientific 58A-0445 20X and 40x Semi-Plan Achromatic DIN Objective Lens

Manipulation of depth of field is a good way to modify the characteristics of your photo, and manipulating the aperture is the ideal way to do this because it has little or no effect on composition.

Depth of field vs depth of focusphotography

Because I was using the HQ camera, I didn’t use the OFM software and couldn’t leverage its automatic exposure, white balance, and flat fielding. I used RaspiCam, which worked quite well otherwise.

There are two ways to describe the qualities of depth of field – shallow DOF or deep DOF. Shallow is when the included focus range is very narrow, a few inches to several feet. Deep is when the included range is a couple of yards to infinity. In both cases DOF is measured in front of the focus point and behind the focus point.

In term of alignment, I used a reasonably stable laser and the two-pinhole “alignment” tool from Thorlabs. Lens tubes from Thorlabs were OK, to make sure the obj and the tube lens were “mostly OK”.

I guess the “rolls Royce” of optics would be a purpose-built microscope tube lens like the Thor labs ttl200, but they are real expensive (much more than the raynox). You can also use any “long” camera lens like a telephoto lens. I’m not suggesting any of this would be appropriate for the OFM since these optics are big and chunky too.

In this section we’re going to discuss several crucial elements for exercising greater creative control over your final photographic image.Other than lighting, composition and focus (which includes depth of field) are the main elements that you can exercise complete command over.Focus enables you to isolate a subject and specifically draw the viewer’s eye to exactly where you want it.The first thing to understand about focus is depth of field.1Depth of FieldThe depth of field (DOF) is the front-to-back zone of a photograph in which the image is razor sharp.As soon as an object (person, thing) falls out of this range, it begins to lose focus at an accelerating degree the farther out of the zone it falls; e.g., closer to the lens or deeper into the background. With any DOF zone, there is a Point of Optimum focus in which the object is most sharp.There are two ways to describe the qualities of depth of field – shallow DOF or deep DOF. Shallow is when the included focus range is very narrow, a few inches to several feet. Deep is when the included range is a couple of yards to infinity. In both cases DOF is measured in front of the focus point and behind the focus point.DOF is determined by three factors – aperture size, distance from the lens, and the focal length of the lens.Let’s look at how each one works.2ApertureThe aperture is the opening at the rear of the lens that determines how much light travels through the lens and falls on the image sensor.The size of the aperture’s opening is measured in f-stops – one of two sets of numbers on the lens barrel (the other being the focusing distance).The f-stops work as inverse values, such that a small f/number (say f/2.8) corresponds to a larger or wider aperture size, which results in a shallow depth of field; conversely a large f/number (say f/16) results in a smaller or narrower aperture size and therefore a deeper depth of field.3Small vs Large ApertureManipulating the aperture is the easiest and most often utilized means to adjust Depth of Field.To achieve a deep, rich and expansive DOF, you’ll want to set the f-stop to around f/11 or higher. You may have seen this principle demonstrated when you look at photos taken outside during the brightest time of the day. In such a case, the camera is typically set at f/16 or higher (that Sunny 16 Rule) and the Depth of Field is quite deep – perhaps several yards in front of and nearly to infinity beyond the exact focus point.Let’s take a look at these two photos as examples. The left side of the photo has an expansive DOF, most likely shot around noon (notice the short, but strong shadows), with an f/22 aperture. The right side of the photo has an extremely shallow DOF; probably an f/2.8 aperture setting.However, to achieve an identical proper exposure, the shutter speed is probably closer to 1/1000th to compensate for the increased amount of light entering the lens at f/2.8.4Aperture RangeThe aperture range identifies the widest to smallest range of lens openings, i.e., f/1.4 (on a super-fast lens) to f/32, with incremental “stops” in between (f/2, f/2.8, f/4, f/5.6, f/8, f/11, f/16, and f/22).Each f-number is represents one “stop” of light, a stop is a mathematical equation (which is the focal length of the lens divided by the diameter of the aperture opening) that determines how much light that enters the lens regardless of the length of the lens. Such that an f/4 on a 50mm has smaller opening than an f/4 on a 200mm, but an equivalent amount of light travels through both lenses to reach the image sensor thus providing the same exposure.Each movement up the range (say f/2 to f.2.8) reduces the amount of light by one-half, and each movement down the range (say f/11 to f/8) doubles the amount of light passing through the lens.It’s important to understand this concept and how it affects exposure because it works in tandem with the shutter speed (we’ll discuss this in another section) to establish a given exposure value.Basically, when you change the aperture size one stop, you have to shift the shutter speed one stop in the opposite direction to maintain a consistent exposure… and this change in aperture alters the depth of field (DOF) accordingly.5Distance from the LensThe last element affecting depth of field is the distance of the subject from the lens – you can adjust the DOF by changing that distance.For example, the closer an object is to the lens (and the focus is set on that object) the shallower the DOF. Conversely, the reverse is true – the farther away an object is and focused on, the deeper the DOF. Changing the distance to subject is the least practical way to manipulate the depth of field, and by changing the distance from a subject to the lens, you immediately change your image’s composition. To maintain the compositional integrity of the shot, but still have the change in DOF from a distance, you can change the focal length (either by changing lenses or zooming in).Why does changing the focal length negate the effects on DOF? This is because the visual properties of a given lens either provide either greater DOF (shorter lenses) or shallower DOF (longer lenses). The physical properties of a lens at a given focal length also affect the depth of field. A shorter focal length lens (say 27mm) focused at 5 meters, set at f/4 has a deeper DOF (perhaps from 3 meters in front and 20 meters behind) than a longer focal length (say 300mm), also set at f/4 focused at 5 meters. The 300mm lens has a remarkably shallow depth of field.Incidentally, to help you with this, every lens has a manual with a DOF chart for each f/stop and the major focusing distances. DOF is just a matter of physics, and it’s important to grasp this concept.CConclusionManipulation of depth of field is a good way to modify the characteristics of your photo, and manipulating the aperture is the ideal way to do this because it has little or no effect on composition.You simply need to change the shutter speed (or change the light sensitivity – ISO) to compensate for the changes in the exposure from the adjustments to the f-number. Changes in distance and focal length also affect DOF, but these changes have trade-offs in terms of composition.Therefore, changes to aperture are the best way to manipulate DOF without affecting a photo’s composition.

The OFM software is really doing a tremendous job improving the camera’s output as I found it nearly impossible to work with the unprocessed output from the standard raspberry pi camera. That said, the photos from the HQ camera looked really good without post processing beyond a simple white balance.

Depth of fieldanddepth of focusPDF

In my digital photography classes I learned that optical magnification is always superior to digital magnification. But the former is way more expensive than the latter. Adding $70 USD is a significant amount when you look at the total cost of the current microscope. Could this image aberration be programmatically fixed? Can z stacking be used to obtain a sharper image? If money was not an issue, what would it take to build the OFM optics “Rolls Royce” ?

I went a bit off the deep end after building my first OFM, trying to see how I could modify the optics a bit. I wanted to answer these questions:

The HQ cam is a very substantial increase in quality. The issue isn’t the detector size, resolution, or the mounting hardware (which is nice to have), but the CRA mismatch between the bayer filter/microlens array designed for webcam/cellphone lenses and the lenses used in microscopy really compromises the quality achievable from the standard camera. Some of this can be post-processed out, but the images I took today were saturated in one or more channels in part of the image, which makes correction not worth it.

Raspberry Pi HQ camera with 75 mm achromatic doublet tube lens (no postprocessing) 2024-02-07T14-20-27Z1920×1439 72.9 KB 2024-02-07T14-21-39Z1920×1439 364 KB 2024-02-07T14-22-30Z1920×1439 393 KB

I tested a new Plan 40x/0.65 objective. It looks better and the out of focus area on the left is less evident but still there. Besides the color problems, it looks overall better, sharper, and more magnified . OFM_2024-02-22T16_41_26.212Z1920×1442 227 KB

Raspberry Pi HQ camera with two-lens tube lens (no postprocessing) 2024-01-28T04-23-12Z1920×1439 75.3 KB 2024-02-07T14-05-14Z1920×1439 410 KB 2024-02-07T13-59-08Z1920×1439 480 KB

The softness is worse to one side (you say it’s consistently the left) which does suggest either sensor tilt, objective tilt, or another other misalignment somewhere between objective and sensor. I had a similar problem before and the problem was that the objective was not seated exactly straight on it’s mount.

As for the focus, I’m not 100% sure what the source is - but it could be the objectives themselves. Your 20X doesn’t purport to be a plan objective which could account for the very soft edges.

I wouldn’t discourage this type of exercise. In fact, I learned a lot by exploring how different objectives affect the quality of the image. I realized that I was mistakenly using a semi-plan when I should have been using a plan objective.

Let’s take a look at these two photos as examples. The left side of the photo has an expansive DOF, most likely shot around noon (notice the short, but strong shadows), with an f/22 aperture. The right side of the photo has an extremely shallow DOF; probably an f/2.8 aperture setting.

Depth of field vs depth of focuscamera

Very nice work and thanks for sharing. This explains the reason of the out of focus areas in all of my builds. I noticed the same artifact mostly in the upper left corner of the field. I was under the impression that could be a problem with an uneven stage due to a problem with my printer, or something with the doublet lens position inside the tube. However, neither tilting the slide, sanding the stage, nor repositioning the lens improved the image.

Shallowdepth of field

Incidentally, to help you with this, every lens has a manual with a DOF chart for each f/stop and the major focusing distances. DOF is just a matter of physics, and it’s important to grasp this concept.

As soon as an object (person, thing) falls out of this range, it begins to lose focus at an accelerating degree the farther out of the zone it falls; e.g., closer to the lens or deeper into the background. With any DOF zone, there is a Point of Optimum focus in which the object is most sharp.

As to your question about magnification, the first set of 3 images use the Raspberry Pi v2 Camera, which has a smaller sensor than the HQ camera. The smaller sensor produces larger apparent magnification (crop factor).

To achieve a deep, rich and expansive DOF, you’ll want to set the f-stop to around f/11 or higher. You may have seen this principle demonstrated when you look at photos taken outside during the brightest time of the day. In such a case, the camera is typically set at f/16 or higher (that Sunny 16 Rule) and the Depth of Field is quite deep – perhaps several yards in front of and nearly to infinity beyond the exact focus point.

The Thor labs achromatic doublets don’t seem to have flat fields and the lens I used produced a pretty noticable field curvature when used alone. I think this is actual field curvature and not a tilted slide based on focusing up and down and seeing a pronouced circumferential difference in focus. It’s probably not an aberration, per se, since I could focus the edges of the image nicely, just not the center and the edges at the same time. I guess this is expected as these lenses aren’t really meant for imaging. Interestingly, when combined with the Raynox (which is intended for photography) the issue was not really noticable. I think this is due to the distance between the achromatic doublet and the detector. Longer distance = more noticeable curvature.

The f-stops work as inverse values, such that a small f/number (say f/2.8) corresponds to a larger or wider aperture size, which results in a shallow depth of field; conversely a large f/number (say f/16) results in a smaller or narrower aperture size and therefore a deeper depth of field.

Depth of field vs depth of focusnikon

Illuminator: A “high CRI” white LED and an aspheric collector lens to roughly collimate the beam. I also made a little bayonet mount where I can fit field stops.

Depth of focusin photography

Each f-number is represents one “stop” of light, a stop is a mathematical equation (which is the focal length of the lens divided by the diameter of the aperture opening) that determines how much light that enters the lens regardless of the length of the lens. Such that an f/4 on a 50mm has smaller opening than an f/4 on a 200mm, but an equivalent amount of light travels through both lenses to reach the image sensor thus providing the same exposure.

The size of the aperture’s opening is measured in f-stops – one of two sets of numbers on the lens barrel (the other being the focusing distance).

These aren’t high sensitivity monochrome cameras though. I don’t know how the Bayer filter affects resolution especially with regard to repeatability of a stage

Why does changing the focal length negate the effects on DOF? This is because the visual properties of a given lens either provide either greater DOF (shorter lenses) or shallower DOF (longer lenses). The physical properties of a lens at a given focal length also affect the depth of field. A shorter focal length lens (say 27mm) focused at 5 meters, set at f/4 has a deeper DOF (perhaps from 3 meters in front and 20 meters behind) than a longer focal length (say 300mm), also set at f/4 focused at 5 meters. The 300mm lens has a remarkably shallow depth of field.

Other than lighting, composition and focus (which includes depth of field) are the main elements that you can exercise complete command over.Focus enables you to isolate a subject and specifically draw the viewer’s eye to exactly where you want it.The first thing to understand about focus is depth of field.1Depth of FieldThe depth of field (DOF) is the front-to-back zone of a photograph in which the image is razor sharp.As soon as an object (person, thing) falls out of this range, it begins to lose focus at an accelerating degree the farther out of the zone it falls; e.g., closer to the lens or deeper into the background. With any DOF zone, there is a Point of Optimum focus in which the object is most sharp.There are two ways to describe the qualities of depth of field – shallow DOF or deep DOF. Shallow is when the included focus range is very narrow, a few inches to several feet. Deep is when the included range is a couple of yards to infinity. In both cases DOF is measured in front of the focus point and behind the focus point.DOF is determined by three factors – aperture size, distance from the lens, and the focal length of the lens.Let’s look at how each one works.2ApertureThe aperture is the opening at the rear of the lens that determines how much light travels through the lens and falls on the image sensor.The size of the aperture’s opening is measured in f-stops – one of two sets of numbers on the lens barrel (the other being the focusing distance).The f-stops work as inverse values, such that a small f/number (say f/2.8) corresponds to a larger or wider aperture size, which results in a shallow depth of field; conversely a large f/number (say f/16) results in a smaller or narrower aperture size and therefore a deeper depth of field.3Small vs Large ApertureManipulating the aperture is the easiest and most often utilized means to adjust Depth of Field.To achieve a deep, rich and expansive DOF, you’ll want to set the f-stop to around f/11 or higher. You may have seen this principle demonstrated when you look at photos taken outside during the brightest time of the day. In such a case, the camera is typically set at f/16 or higher (that Sunny 16 Rule) and the Depth of Field is quite deep – perhaps several yards in front of and nearly to infinity beyond the exact focus point.Let’s take a look at these two photos as examples. The left side of the photo has an expansive DOF, most likely shot around noon (notice the short, but strong shadows), with an f/22 aperture. The right side of the photo has an extremely shallow DOF; probably an f/2.8 aperture setting.However, to achieve an identical proper exposure, the shutter speed is probably closer to 1/1000th to compensate for the increased amount of light entering the lens at f/2.8.4Aperture RangeThe aperture range identifies the widest to smallest range of lens openings, i.e., f/1.4 (on a super-fast lens) to f/32, with incremental “stops” in between (f/2, f/2.8, f/4, f/5.6, f/8, f/11, f/16, and f/22).Each f-number is represents one “stop” of light, a stop is a mathematical equation (which is the focal length of the lens divided by the diameter of the aperture opening) that determines how much light that enters the lens regardless of the length of the lens. Such that an f/4 on a 50mm has smaller opening than an f/4 on a 200mm, but an equivalent amount of light travels through both lenses to reach the image sensor thus providing the same exposure.Each movement up the range (say f/2 to f.2.8) reduces the amount of light by one-half, and each movement down the range (say f/11 to f/8) doubles the amount of light passing through the lens.It’s important to understand this concept and how it affects exposure because it works in tandem with the shutter speed (we’ll discuss this in another section) to establish a given exposure value.Basically, when you change the aperture size one stop, you have to shift the shutter speed one stop in the opposite direction to maintain a consistent exposure… and this change in aperture alters the depth of field (DOF) accordingly.5Distance from the LensThe last element affecting depth of field is the distance of the subject from the lens – you can adjust the DOF by changing that distance.For example, the closer an object is to the lens (and the focus is set on that object) the shallower the DOF. Conversely, the reverse is true – the farther away an object is and focused on, the deeper the DOF. Changing the distance to subject is the least practical way to manipulate the depth of field, and by changing the distance from a subject to the lens, you immediately change your image’s composition. To maintain the compositional integrity of the shot, but still have the change in DOF from a distance, you can change the focal length (either by changing lenses or zooming in).Why does changing the focal length negate the effects on DOF? This is because the visual properties of a given lens either provide either greater DOF (shorter lenses) or shallower DOF (longer lenses). The physical properties of a lens at a given focal length also affect the depth of field. A shorter focal length lens (say 27mm) focused at 5 meters, set at f/4 has a deeper DOF (perhaps from 3 meters in front and 20 meters behind) than a longer focal length (say 300mm), also set at f/4 focused at 5 meters. The 300mm lens has a remarkably shallow depth of field.Incidentally, to help you with this, every lens has a manual with a DOF chart for each f/stop and the major focusing distances. DOF is just a matter of physics, and it’s important to grasp this concept.CConclusionManipulation of depth of field is a good way to modify the characteristics of your photo, and manipulating the aperture is the ideal way to do this because it has little or no effect on composition.You simply need to change the shutter speed (or change the light sensitivity – ISO) to compensate for the changes in the exposure from the adjustments to the f-number. Changes in distance and focal length also affect DOF, but these changes have trade-offs in terms of composition.Therefore, changes to aperture are the best way to manipulate DOF without affecting a photo’s composition.

My application is transmitted light for which those issues wouldn’t crop up. You might want to check out people disussing the pi cameras for astrophotography as they have detailed knowledge of how the processing affects low light and long exposure captures. I know they disable some of the dead pixel correction as it interferes with some captures.

In terms of stage Z adjustment etc: for a limited budget one can find used spring-loaded translation stages on ebay (Standa, Thorlans, Newport etc). Also, ebay has “aliexpress-grade” stages which cost 5x+ less than Thorlabs ones. I ordered a few and I used them for laser alignment; they seemed OK, but I haven’t characterized drift systematically. Rack-and-pinion stages are very drifty.

For example, the closer an object is to the lens (and the focus is set on that object) the shallower the DOF. Conversely, the reverse is true – the farther away an object is and focused on, the deeper the DOF. Changing the distance to subject is the least practical way to manipulate the depth of field, and by changing the distance from a subject to the lens, you immediately change your image’s composition. To maintain the compositional integrity of the shot, but still have the change in DOF from a distance, you can change the focal length (either by changing lenses or zooming in).

The setup: I wanted a basic upright brightfield microscope that would use infinite-conjugate objectives to evaluate different tube lenses, sensors, and illumination conditions. I ended up building a simple setup that slides along a vertical “2020” aluminum extrusion. Alignment and vibration are problems with this “minimal viable microscope” consisting of illuminator, condenser, stage, objective, tube lens, and sensor, but it works and makes some nice images when I can get everything lined up and not shaking. I didn’t use OpenSCAD for modeling this but I can share models as STL or STEP if any are interesting. image1920×2957 408 KB

Depth of field vs depth of focusmicroscope

It’s important to understand this concept and how it affects exposure because it works in tandem with the shutter speed (we’ll discuss this in another section) to establish a given exposure value.

3) # 185 Objective Lens 60X https://www.amazon.com/gp/product/B0C55VKJYN/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&psc=1

This thread also brings up the quality of the tube lens as a weak point when using the best objectives. Working out the best price/performance for doing something about that is a substantial investigation.

I was not intending to discourage it at all. It is very useful to understand all the things that affect overall performance and I have learnt a lot from what you and others are doing.

I’m not sure what the best way to ensure alignment is, but one way I spotted the issue was removing the camera from the optics module and pointing the objective directly at a bright, small light source like a surface mount LED. I put a peice of paper on the back of the optics module so I could see the projected image when the room lights were dimmed. This showed me that despite centering the LED relative to the objective the image was not centered where the sensor should be. That clued me in that the objective was misaligned.