What is light microscopy?Parts of a microscope and how a light microscope worksSimple and compound microscopesTypes of light microscopy-     Bright field microscopy-     Dark field microscopy-     Phase contrast microscopy-     Differential interference contrast microscopy-     Polarized light microscopy-     Fluorescence microscopy-     Immunofluorescence microscopy-     Confocal microscopy-     Two-photon microscopy-     Light sheet microscopy-     Total internal reflection fluorescence microscopy-     Expansion microscopy

Depth of field vs depth of focusnikon

Jan 27, 2016 — How do polarized test image cards work? Polarized test image cards use polarizing filters to create two images, one for each eye. When viewed ...

Figure 16: Sample preparation for expansion microscopy. A cell is first stained and then linked to a polymer gel matrix. The cell structure itself is then dissolved (digested), allowing the stained parts to expand isotropically with the gel, allowing the stained structure to be imaged with more detail.

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.

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.

Figure 8: Differential interference contrast microscopy. Left: Schematic setup for DICM. Right: Live adult Caenorhabditis elegans (C. elegans) nematode imaged by DICM. Credit: Bob Goldstein, Cell Image Library. Reproduced under a Creative Commons Attribution 3.0 Unported license (CC BY 3.0).

Shallowdepth of field

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.

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.

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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.

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.

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 imaging system may also include elements such as apertures and filters that select certain portions of light from the sample, for example to see only light that has been scattered off the sample, or only light of a certain color or wavelength. As in the case of the illumination system, this type of filtering can be extremely useful to single out certain features of interest that would remain hidden when imaging all the light from the sample.Overall, both the illumination and the imaging system play a key role in how well a light microscope performs. To get the best out of light microscopy in your application, it is essential to have a good understanding of how a basic light microscope works, and what variations exist today.

Figure 9: Polarization microscopy. Photomicrograph of olivine adcumulate, formed by the accumulation of crystals with different birefringence. Variations of thickness and refractive index across the sample result in different colors. Credit: R. Hill, CSIRO.

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.

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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.

What is light microscopy? Light microscopy is used to make small structures and samples visible by providing a magnified image of how they interact with visible light, e.g., their absorption, reflection and scattering. This is useful to understand what the sample looks like and what it is made of, but also allows us to see processes of the microscopic world, such as how substances diffuse across a cell membrane.

References1. Rochow TG, Tucker PA. A Brief History of Microscopy. In: Introduction to Microscopy by Means of Light, Electrons, X Rays, or Acoustics. Springer US; 1994:1-21. doi:10.1007/978-1-4899-1513-9_12. Smith WJ. Modern Optical Engineering: The Design of Optical Systems. McGraw-Hill; 1990. ISBN: 00705917413. Shribak M, Inoué S. Orientation-independent differential interference contrast microscopy. Collected Works of Shinya Inoue: Microscopes, Living Cells, and Dynamic Molecules. 2008;(Dic):953-962. doi:10.1142/9789812790866_00744. Gao G, Jiang YW, Sun W, Wu FG. Fluorescent quantum dots for microbial imaging. Chinese Chem Lett. 2018;29(10):1475-1485. doi:10.1016/j.cclet.2018.07.0045. Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D. Green fluorescent protein as a marker for gene expression. Science. 1994;263(5148):802-805. doi:10.1126/science.83032956. Baranov M V., Olea RA, van den Bogaart G. Chasing Uptake: Super-Resolution Microscopy in Endocytosis and Phagocytosis. Trends Cell Biol. 2019;29(9):727-739. doi:10.1016/j.tcb.2019.05.0067. Miller DM, Shakes DC. Chapter 16 Immunofluorescence Microscopy. In: Current Protocols Essential Laboratory Techniques. Vol 10.; 1995:365-394. doi:10.1016/S0091-679X(08)61396-58. Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer EHK. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science. 2004;305(5686):1007-1009. doi:10.1126/science.11000359. Huisken J. Slicing embryos gently with laser light sheets. BioEssays. 2012;34(5):406-411. doi:10.1002/bies.20110012010. Fish KN. Total Internal Reflection Fluorescence (TIRF) Microscopy. Curr Protoc Cytom. 2009;50(1):273-275. doi:10.1002/0471142956.cy1218s5011. Wassie AT, Zhao Y, Boyden ES. Expansion microscopy: principles and uses in biological research. Nat Methods. 2019;16(1):33-41. doi:10.1038/s41592-018-0219-412. Lam F, Cladière D, Guillaume C, Wassmann K, Bolte S. Super-resolution for everybody: An image processing workflow to obtain high-resolution images with a standard confocal microscope. Methods. 2017;115:17-27. doi: 10.1016/j.ymeth.2016.11.00313. Hedvat C V. Digital microscopy: past, present, and future. Arch Pathol Lab Med. 2010;134(11):1666-1670. doi: 10.5858/2009-0579-RAR1.114. Fatermans J, den Dekker AJ, Müller-Caspary K, et al. Single Atom Detection from Low Contrast-to-Noise Ratio Electron Microscopy Images. Phys Rev Lett. 2018;121(5):56101. doi:10.1103/PhysRevLett.121.05610115. Zhang C, Huber F, Knop M, Hamprecht FA. Yeast cell detection and segmentation in bright field microscopy. In: 2014 IEEE 11th International Symposium on Biomedical Imaging (ISBI); 2014:1267-1270. doi:10.1109/ISBI.2014.686810716. Nair RR, Blake P, Grigorenko AN, et al. Fine Structure Constant Defines Visual Transparency of Graphene. Science. 2008;320(5881):1308-1308. doi:10.1126/science.115696517. Xu D, He Y, Yeung ES. Direct Imaging of Transmembrane Dynamics of Single Nanoparticles with Darkfield Microscopy: Improved Orientation Tracking at Cell Sidewall. Anal Chem. 2014;86(7):3397-3404. doi:10.1021/ac403700u18. Neu-Baker NM, Dozier AK, Eastlake AC, Brenner SA. Evaluation of enhanced darkfield microscopy and hyperspectral imaging for rapid screening of TiO2 and SiO2 nanoscale particles captured on filter media. Microsc Res Tech. doi:10.1002/jemt.2385619. Li K, Miller ED, Weiss LE, Campbell PG, Kanade T. Online Tracking of Migrating and Proliferating Cells Imaged with Phase-Contrast Microscopy. In: 2006 Conference on Computer Vision and Pattern Recognition Workshop (CVPRW’06); 2006:65. doi:10.1109/CVPRW.2006.15020. McFadzean JA, Smiles J. Studies of Litomosoides carinii by Phase-contrast microscopy: the Development of the Larvae. J Helminthol. 1956;30(1):25-32. doi:10.1017/S0022149X0003294621. Sun W, Wang G, Fang N, Yeung ES. Wavelength-dependent differential interference contrast microscopy: selectively imaging nanoparticle probes in live cells. Anal Chem. 2009;81(22):9203-9208. doi: 10.1021/ac901623b22. Xiao L, Ha JW, Wei L, Wang G, Fang N. Determining the full three-dimensional orientation of single anisotropic nanoparticles by differential interference contrast microscopy. Angew Chemie Int Ed. 2012;51(31):7734-7738. doi: 10.1002/anie.20120234023. Wolman M, Kasten FH. Polarized light microscopy in the study of the molecular structure of collagen and reticulin. Histochemistry. 1986;85(1):41-49. doi:10.1007/BF0050865224. Slámová M, Očenášek V, Vander Voort G. Polarized light microscopy: utilization in the investigation of the recrystallization of aluminum alloys. Mater Charact. 2004;52(3):165-177. doi:10.1016/j.matchar.2003.10.01025. Lichtman JW, Conchello J-A. Fluorescence microscopy. Nat Methods. 2005;2(12):910-919. doi:10.1038/nmeth81726. Franke W, Appelhans B, Schmid E, Freudenstein C, Osborn M, Weber K. Identification and characterization of epithelial cells in mammalian tissues by immunofluorescence microscopy using antibodies to prekeratin. Differentiation. 1979;15(1-3):7-25. doi:10.1111/j.1432-0436.1979.tb01030.x27. Seto S, Layh-Schmitt G, Kenri T, Miyata M. Visualization of the attachment organelle and cytadherence proteins of Mycoplasma pneumoniae by immunofluorescence microscopy. J Bacteriol. 2001;183(5):1621-1630. doi:10.1128/JB.183.5.1621-1630.200128. Pawley J, Pawley JB. Handbook of Biological Confocal Microscopy. 2006;(August 2010). doi:10.1007/978-0-387-45524-229. Ellis-Davies GCR. Two-Photon Microscopy for Chemical Neuroscience. ACS Chem Neurosci. 2011;2(4):185-197. doi:10.1021/cn100111a30. Helmchen F, Denk W. Deep tissue two-photon microscopy. Nat methods. 2005;2(12):932-940. doi:10.1038/nmeth81831. Sako Y, Uyemura T. Total internal reflection fluorescence microscopy for single-molecule imaging in living cells. Cell Struct Funct. 2002;27(5):357-365. doi:10.1247/csf.27.35732. Axelrod D. Total internal reflection fluorescence microscopy in cell biology. Traffic. 2001;2(11):764-774. doi:10.1034/j.1600-0854.2001.21104.x

Depth of field vs depth of focusmicroscope

What's the difference between light microscopy vs electron microscopy? Light microscopy typically uses wavelengths of light in the visible spectrum, which inherently limits it spatial resolution due to the Rayleigh criterion to approximately half the wavelength used (approximately 200 nm at best). However, even when using objectives with high NA and advanced image processing, this fundamental limit cannot be overcome. Instead, observing smaller structures requires the use of electromagnetic radiation of shorter wavelength. This is the underlying principle of electron microscopy, where electrons are used to illuminate the sample instead of visible light. Electrons have an associated wavelength which is much shorter than visible light, which allows magnifications of up to 10,000,000 x to be achieved, such that even single atoms can be resolved.

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).

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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.

Depth of field vs depth of focusphotography

Depth of focusin photography

Figure 7: Phase contrast microscopy of a human embryonic stem cell colony. Credit Sabrina Lin, Prue Talbot, Stem Cell Center University of California, Riverside.

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Some of the most fundamental processes in nature occur at the microscopic scale, far beyond the limits of what we can see by eye, which motivates the development of technology that allows us to see beyond this limit. As early as the 4th century AD, people had discovered the basic concept of an optical lens, and by the 13th century, they were already using glass lenses to improve their eyesight and to magnify objects such as plants and insects to better understand them.1 With time, these simple magnifying glasses developed into advanced optical systems, known as light microscopes, which allow us to see and understand the microscopic world beyond the limits of our perception. Today, light microscopy is a core technique in many areas of science and technology, including life sciences, biology, materials sciences, nanotechnology, industrial inspection, forensics and many more. In this article, we will first explore the basic working principle of light microscopy. Building on this, we will discuss some more advanced forms of light microscopy that are commonly used today and compare their strengths and weaknesses for different applications.

Depth of fieldanddepth of focusPDF

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.

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).

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.

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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.

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Strong background suppression from non-birefringent areas of a sample, allows measurement of sample thickness and birefringence

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).

Depth of field vs depth of focusreddit

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.

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Depth of field vs depth of focuscamera

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.

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.

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.

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.

Figure 17: Image deconvolution. Left: Original fluorescence image. Right: Image after deconvolution, showing increased detail. Credit: Author.