# Theory **URL:** https://forum.microlist.org/c/theory/7.md [Latest](https://forum.microlist.org/latest.md) · [Categories](https://forum.microlist.org/categories.md) · [Tags](https://forum.microlist.org/tags.md) --- ## [About the Theory category](https://forum.microlist.org/t/about-the-theory-category/16)
**Author:** [@talley](https://forum.microlist.org/u/talley)\ **Replies:** 0
A category for topics related to microscopy theory and procedural strategies. --- ## [Ripples through a focus stack (and other motion / digitisation artefacts)](https://forum.microlist.org/t/ripples-through-a-focus-stack-and-other-motion-digitisation-artefacts/3111)
**Author:** [@P\_Tadrous](https://forum.microlist.org/u/P_Tadrous)\ **Replies:** 0\ **Last updated:** [May 12, 2025, 6:00pm UTC](https://forum.microlist.org/t/ripples-through-a-focus-stack-and-other-motion-digitisation-artefacts/3111 "2025-05-12T18:00:07Z")
While testing the through focus mechanism on my new (under development) CNC microscope stage I found that I get this peculiar rippling effect while moving in Z with this silicon chip specimen - it looks like the sample i… --- ## [How am I obtaining higher energy photons from a fluorescein solution than the ones I'm exciting it with?](https://forum.microlist.org/t/how-am-i-obtaining-higher-energy-photons-from-a-fluorescein-solution-than-the-ones-im-exciting-it-with/3010)
**Author:** [@JelleP](https://forum.microlist.org/u/JelleP)\ **Replies:** 3\ **Last updated:** [February 28, 2025, 4:24pm UTC](https://forum.microlist.org/t/how-am-i-obtaining-higher-energy-photons-from-a-fluorescein-solution-than-the-ones-im-exciting-it-with/3010 "2025-02-28T16:24:27Z")
Hi all, I have a theory question that is probably obvious to some of you! I’m confocal scanning a FITC solution as follows: Imgur: The magic of the Internet with excitation at 520 nm and emission around 500 nm. I’m usi… --- ## [Guide to Microscopy about Cellular Ploidy Variation](https://forum.microlist.org/t/guide-to-microscopy-about-cellular-ploidy-variation/2301)
**Author:** [@Matheus\_C\_Casotti](https://forum.microlist.org/u/Matheus_C_Casotti)\ **Replies:** 2\ **Last updated:** [February 13, 2025, 8:54pm UTC](https://forum.microlist.org/t/guide-to-microscopy-about-cellular-ploidy-variation/2301 "2025-02-13T20:54:47Z")
Good morning My name is Matheus, I’m starting out in the area of image analysis and I would like to develop an analysis and identification of polyploid cells in histological slides stained in HE and also with fluorescen… --- ## [Airyscan imaging issues](https://forum.microlist.org/t/airyscan-imaging-issues/2940)
**Author:** [@bufobufo](https://forum.microlist.org/u/bufobufo)\ **Replies:** 3\ **Last updated:** [December 31, 2024, 4:46am UTC](https://forum.microlist.org/t/airyscan-imaging-issues/2940 "2024-12-31T04:46:40Z")
Hi, I am a PhD student, and my project heavily depends on imaging. I’ve been using LSM880 for my confocal images; however, recently, my PI suggested I start using airyscan for some of my images. I am trying to visualize… --- ## [Abbe's Diffraction Theory and Fourier Optics](https://forum.microlist.org/t/abbes-diffraction-theory-and-fourier-optics/2915)
**Author:** [@P\_Tadrous](https://forum.microlist.org/u/P_Tadrous)\ **Replies:** 0\ **Last updated:** [December 9, 2024, 12:18pm UTC](https://forum.microlist.org/t/abbes-diffraction-theory-and-fourier-optics/2915 "2024-12-09T12:18:18Z")
My latest video on YouTube covers how a microscope ‘works’ in terms of wave optics with an easy-to-understand explanation of Abbe’s diffraction theory backed up by practical demonstrations on the 3D printed PUMA microsco… --- ## [Diffraction - and modelling the focussing of light](https://forum.microlist.org/t/diffraction-and-modelling-the-focussing-of-light/2878)
**Author:** [@P\_Tadrous](https://forum.microlist.org/u/P_Tadrous)\ **Replies:** 0\ **Last updated:** [November 4, 2024, 10:45am UTC](https://forum.microlist.org/t/diffraction-and-modelling-the-focussing-of-light/2878 "2024-11-04T10:45:34Z")
My latest Photology video discusses diffraction and how it models the focussing of light by a lens. This video features results from the seminal 2017 JCB paper by @talley and @jennifer as well as explaining the modern co… --- ## [High NA objectives](https://forum.microlist.org/t/high-na-objectives/1013)
**Author:** [@Edalat](https://forum.microlist.org/u/Edalat)\ **Replies:** 10\ **Last updated:** [October 10, 2024, 10:00am UTC](https://forum.microlist.org/t/high-na-objectives/1013 "2024-10-10T10:00:00Z")
Dear all I have a very basic question about objective lenses with NA larger than ~1.4: can for instance a 1.49 NA lens gather more light than a 1.4 (1.33 to 1.38) NA lens from a biological sample? To me it seems that th… --- ## [Fourier 3: The outputs and symmetries of the Fourier Transform](https://forum.microlist.org/t/fourier-3-the-outputs-and-symmetries-of-the-fourier-transform/2723)
**Author:** [@P\_Tadrous](https://forum.microlist.org/u/P_Tadrous)\ **Replies:** 0\ **Last updated:** [September 6, 2024, 1:27pm UTC](https://forum.microlist.org/t/fourier-3-the-outputs-and-symmetries-of-the-fourier-transform/2723 "2024-09-06T13:27:25Z")
Continue your education of the Fourier transform with my latest video that explains how the numbers output by a DFT relate to all those waves (harmonics) you see in graphical demos. I also introduce you to another Frenc… --- ## [How to decide the number of iterations used in iterative deconvolution algorithms?](https://forum.microlist.org/t/how-to-decide-the-number-of-iterations-used-in-iterative-deconvolution-algorithms/2463)
**Author:** [@Qiqi-Lu](https://forum.microlist.org/u/Qiqi-Lu)\ **Replies:** 2\ **Last updated:** [April 11, 2024, 7:12pm UTC](https://forum.microlist.org/t/how-to-decide-the-number-of-iterations-used-in-iterative-deconvolution-algorithms/2463 "2024-04-11T19:12:16Z")
Hi, I wonder how many iterations used when do deconvolution using iterative deconvolution algorithms (for example, the Richardson Lucy Deconvolution). For some situations, the deconvolution method will converge to a fix… --- ## [How to generate PSF of iSIM using PSF generator](https://forum.microlist.org/t/how-to-generate-psf-of-isim-using-psf-generator/2381)
**Author:** [@Qiqi-Lu](https://forum.microlist.org/u/Qiqi-Lu)\ **Replies:** 6\ **Last updated:** [January 27, 2024, 8:34am UTC](https://forum.microlist.org/t/how-to-generate-psf-of-isim-using-psf-generator/2381 "2024-01-27T08:34:11Z")
Hello, I have troubles to generate a theorical PSF for instant SIM (iSIM) using PSF generator. For example, the iSIM mentioned in the paper ’ Guo, M. et al. Rapid image deconvolution and multiview fusion for optical mi… --- ## [Imaging live culture neurons on a Plastic Plate](https://forum.microlist.org/t/imaging-live-culture-neurons-on-a-plastic-plate/2346)
**Author:** [@tristangliu](https://forum.microlist.org/u/tristangliu)\ **Replies:** 2\ **Last updated:** [December 13, 2023, 10:14pm UTC](https://forum.microlist.org/t/imaging-live-culture-neurons-on-a-plastic-plate/2346 "2023-12-13T22:14:49Z")
Hi, I want to image live culture neurons using plastic plate instead of recommended glass plate, because they grow better on the plastic plate. We will collect Gcamp signals from mouse neurons and aim to take those imag… --- ## [Zoom factor in confocal microscopy](https://forum.microlist.org/t/zoom-factor-in-confocal-microscopy/2287)
**Author:** [@Xerise](https://forum.microlist.org/u/Xerise)\ **Replies:** 2\ **Last updated:** [November 23, 2023, 1:28pm UTC](https://forum.microlist.org/t/zoom-factor-in-confocal-microscopy/2287 "2023-11-23T13:28:09Z")
Hello, I need help to understand how to calculate the zoom factor of a confocal microscopy image. Despite a lot of research and asking other people, I can’t find the answer. Let’s imagine that an image (with a scale bar … --- ## [Theoretical excitation illumination area in fluorescence microscopy](https://forum.microlist.org/t/theoretical-excitation-illumination-area-in-fluorescence-microscopy/2310)
**Author:** [@szh141](https://forum.microlist.org/u/szh141)\ **Replies:** 0\ **Last updated:** [November 6, 2023, 3:57pm UTC](https://forum.microlist.org/t/theoretical-excitation-illumination-area-in-fluorescence-microscopy/2310 "2023-11-06T15:57:23Z")
Hello all. I was trying to calculate the illumination area in fluorescence microscopy to estimate the irradiance. Can you help me confirm if the following is correct? In point scanning confocal, we overfill the objectiv… --- ## [How to achieve (and measure) high NA illumination for microscopy - new paper](https://forum.microlist.org/t/how-to-achieve-and-measure-high-na-illumination-for-microscopy-new-paper/2291)
**Author:** [@P\_Tadrous](https://forum.microlist.org/u/P_Tadrous)\ **Replies:** 0\ **Last updated:** [October 17, 2023, 3:19pm UTC](https://forum.microlist.org/t/how-to-achieve-and-measure-high-na-illumination-for-microscopy-new-paper/2291 "2023-10-17T15:19:38Z")
You may be interested in my latest publication on the subject, just out - here: https://doi.org/10.1111/jmi.13237 Basically, I explain new ways to generate high NA illumination (and new ways to measure the NA of illumi… --- ## [Deconvolution for 2D brightfield microscopy](https://forum.microlist.org/t/deconvolution-for-2d-brightfield-microscopy/2267)
**Author:** [@billo](https://forum.microlist.org/u/billo)\ **Replies:** 3\ **Last updated:** [September 6, 2023, 9:04am UTC](https://forum.microlist.org/t/deconvolution-for-2d-brightfield-microscopy/2267 "2023-09-06T09:04:31Z")
I am interested in playing with deconvolution/deblurring on diagnostic 2D brightfield microscopy images, with H&E staining. Frankly, it’s more aesthetics and curiosity than real investigation. I have a little experienc… --- ## [Deconvolution vs. AI 'Deblurring' for microscopy](https://forum.microlist.org/t/deconvolution-vs-ai-deblurring-for-microscopy/2237)
**Author:** [@P\_Tadrous](https://forum.microlist.org/u/P_Tadrous)\ **Replies:** 1\ **Last updated:** [August 12, 2023, 4:59pm UTC](https://forum.microlist.org/t/deconvolution-vs-ai-deblurring-for-microscopy/2237 "2023-08-12T16:59:29Z")
Does anyone here have experience using or comparing general deconvolution methods with ‘AI’-based ‘de-blurring’ methods for microscopy data? I have seen some papers in the literature touching on this. I use the term ‘ge… --- ## [Objective back focal plane diameter](https://forum.microlist.org/t/objective-back-focal-plane-diameter/2235)
**Author:** [@szh141](https://forum.microlist.org/u/szh141)\ **Replies:** 15\ **Last updated:** [August 8, 2023, 9:19pm UTC](https://forum.microlist.org/t/objective-back-focal-plane-diameter/2235 "2023-08-08T21:19:00Z")
Hello. I’m wondering if someone could shed some light on how to calculate the objective back focal plane diameter. I’m using an Olympus IX81, 60x NA 1.2 water lens. I assume the tube lens focal length is 180mm. If mag =… --- ## [Fourier transform of 2D point spread function using Fiji](https://forum.microlist.org/t/fourier-transform-of-2d-point-spread-function-using-fiji/2038)
**Author:** [@szh141](https://forum.microlist.org/u/szh141)\ **Replies:** 14\ **Last updated:** [July 28, 2023, 12:23pm UTC](https://forum.microlist.org/t/fourier-transform-of-2d-point-spread-function-using-fiji/2038 "2023-07-28T12:23:59Z")
Hello all, I was trying to use Fiji’s FFT to get an MTF curve out of a 2D image of a point spread function. I google searched the attached 1st image, import it into Fiji, split its RGB into single color, process the gre… --- ## [2D fourier transform of interferometric images](https://forum.microlist.org/t/2d-fourier-transform-of-interferometric-images/2221)
**Author:** [@Ankit\_singh\_rawat](https://forum.microlist.org/u/Ankit_singh_rawat)\ **Replies:** 6\ **Last updated:** [July 27, 2023, 8:29am UTC](https://forum.microlist.org/t/2d-fourier-transform-of-interferometric-images/2221 "2023-07-27T08:29:52Z")
Hello Everyone I have a stack of images from my white light interferometer to measure the surface topography and the first image is the xz cross section of image where z represents the vertical direction. Now when I per… --- ## [Confocal xy resolution](https://forum.microlist.org/t/confocal-xy-resolution/2135)
**Author:** [@szh141](https://forum.microlist.org/u/szh141)\ **Replies:** 11\ **Last updated:** [April 28, 2023, 3:07pm UTC](https://forum.microlist.org/t/confocal-xy-resolution/2135 "2023-04-28T15:07:25Z")
Hello all. I’d really appreciate it if someone can shed some light on my question about the confocal resolution with different pinhole sizes. Below is a plot I frequently came across illustrating the xy resolution change… --- ## [TIRF light path questions](https://forum.microlist.org/t/tirf-light-path-questions/2050)
**Author:** [@szh141](https://forum.microlist.org/u/szh141)\ **Replies:** 3\ **Last updated:** [January 27, 2023, 6:20pm UTC](https://forum.microlist.org/t/tirf-light-path-questions/2050 "2023-01-27T18:20:51Z")
Hello all. I came across a TIRF light path diagram as below. Can anyone shed some light on the function/purpose of the following components? QP, the quarter wave plate. I guess it’s to make the laser circularly polarize… --- ## [What can you say about the structure of this product](https://forum.microlist.org/t/what-can-you-say-about-the-structure-of-this-product/1717)
**Author:** [@JackWhite](https://forum.microlist.org/u/JackWhite)\ **Replies:** 0\ **Last updated:** [February 10, 2022, 5:30pm UTC](https://forum.microlist.org/t/what-can-you-say-about-the-structure-of-this-product/1717 "2022-02-10T17:30:43Z")
Guys, I’m interested in space satellites. Or rather, their satellite camera system. I would be very grateful if you could help me learn more about the very structure of the insides of these products. I’m interested in … --- ## [Resolution formula for Second Harmonic generation microscopy](https://forum.microlist.org/t/resolution-formula-for-second-harmonic-generation-microscopy/1366)
**Author:** [@Bobafotz](https://forum.microlist.org/u/Bobafotz)\ **Replies:** 2\ **Last updated:** [April 26, 2021, 9:16am UTC](https://forum.microlist.org/t/resolution-formula-for-second-harmonic-generation-microscopy/1366 "2021-04-26T09:16:15Z")
Hello everyone, I would like to calculate the optimal pixel-size to meet the Nyquist-Shannon criterion for SHG microscopy but I can’t find the formula anywhere. Do you guys have anything related to it ? We collect the … --- ## [PSF of SPIM systems and deconvolution](https://forum.microlist.org/t/psf-of-spim-systems-and-deconvolution/1346)
**Author:** [@manerotoni](https://forum.microlist.org/u/manerotoni)\ **Replies:** 13\ **Last updated:** [April 26, 2021, 7:58am UTC](https://forum.microlist.org/t/psf-of-spim-systems-and-deconvolution/1346 "2021-04-26T07:58:18Z")
Hello, I was wondering what is your experience for deconvolution of ligh-sheet data. in particular what is the best way to compute/estimate the PSF. For a classic light-sheet with gaussian beam the light-sheet thickne… --- ## [10x objective for two photon microscopy (XLPLN10XSVMP 3mm WD)](https://forum.microlist.org/t/10x-objective-for-two-photon-microscopy-xlpln10xsvmp-3mm-wd/1332)
**Author:** [@nwatson](https://forum.microlist.org/u/nwatson)\ **Replies:** 5\ **Last updated:** [April 12, 2021, 3:29pm UTC](https://forum.microlist.org/t/10x-objective-for-two-photon-microscopy-xlpln10xsvmp-3mm-wd/1332 "2021-04-12T15:29:32Z")
To enable Zemax simulations for a customized two photon microscope (university research application), I am looking for the presciption for the Olympus 10x 0.6 NA objective, model: XLPLN10XSVMP 3mm WD. The presciption can… --- ## [Controls for FRET efficiency measurements](https://forum.microlist.org/t/controls-for-fret-efficiency-measurements/1306)
**Author:** [@Oleg](https://forum.microlist.org/u/Oleg)\ **Replies:** 4\ **Last updated:** [March 5, 2021, 6:47pm UTC](https://forum.microlist.org/t/controls-for-fret-efficiency-measurements/1306 "2021-03-05T18:47:28Z")
In my experiment, I want to evaluate FRET efficiency using emission measurements. I have cell lines expressing FRET-pair, donor-only, and acceptor-only. The problem is that the expression level of the donor is much highe… --- ## [Confocal Microscopy Dichroic mirror](https://forum.microlist.org/t/confocal-microscopy-dichroic-mirror/1315)
**Author:** [@Chanuka](https://forum.microlist.org/u/Chanuka)\ **Replies:** 2\ **Last updated:** [March 5, 2021, 12:10am UTC](https://forum.microlist.org/t/confocal-microscopy-dichroic-mirror/1315 "2021-03-05T00:10:09Z")
Confocal microscopy dichroic mirror passes two rays to opposite directions (from the laser and specimen) Does this two-way traffic cause problems/ interference? According to theory, it works fine. Can anyone explain how… --- ## [Determine the sampling distance for CLSM](https://forum.microlist.org/t/determine-the-sampling-distance-for-clsm/1239)
**Author:** [@ccshih](https://forum.microlist.org/u/ccshih)\ **Replies:** 2\ **Last updated:** [December 19, 2020, 5:30pm UTC](https://forum.microlist.org/t/determine-the-sampling-distance-for-clsm/1239 "2020-12-19T17:30:21Z")
Hi all, I am currently working on measuring the PSF of the confocal microscope, and then I will use that PSF to deconvolve an image. However, I am not certain with the sampling distance I use. My equipment are listed … --- ## [Doubts on Airyscan](https://forum.microlist.org/t/doubts-on-airyscan/1231)
**Author:** [@lsilva.m](https://forum.microlist.org/u/lsilva.m)\ **Replies:** 1\ **Last updated:** [December 10, 2020, 11:46am UTC](https://forum.microlist.org/t/doubts-on-airyscan/1231 "2020-12-10T11:46:34Z")
\[this is a copy of a post originally posted in image.sc\] Hello, I am a PhD student, and recently begun to use the confocal microscope (LSM710) with Airyscan, and I have some doubts/curiosities—hopefully, you can help m… [Next page](https://forum.microlist.org/c/theory/7.md?page=1)