Statistics Resources

I recently came across a great website devoted to how statistics is misused in scientific reasoning (and how to use it correctly): Statistics Done Wrong. It’s a great addition to my other favorite statistics resource on the web, this guide to statistics [PDF] from an MIT intersession course.  While statistics isn’t exactly microscopy, it’s still important to know the right tools to use to analyze your data.

How much information does your microscope transmit?

I want to revisit the subject I discussed in the very first post on this blog – how many pixels does your camera need to capture all the information transmitted by the microscope objective?  I’m revisiting this because of a paper published this summer on a clever method for a acquiring high resolution wide field of view images [1]. The method is called Fourier ptychographic microscopy, and essentially amounts to doing image stitching in the Fourier domain to reconstruct a single high resolution image from many low resolution images. This is done by acquiring low resolution transmitted light images from many angles of illumination; the different illumination angles correspond to imaging different regions of frequency space. Reassembling these regions into a single frequency domain image means that much higher resolution is obtained over the full field of view of the microscope.  The net result is they get an image that has the field of view of the 2x lens they use, but has resolution comparable to that of an 0.5 NA lens.

They quantify the combination of resolution and field of view (FOV) by the space-bandwidth product (SBP), which is a fancy way of measuring the number of pixels required to capture the full area at full resolution. Put another way, this is just the FOV divided by the pixel size required to achieve Nyquist sampling at the resolution of the image.  For example, a Nikon 100x/1.4 NA lens has a field of view of about 250 μm in diameter and a resolution of about 220 nm, requiring pixels 110 nm on a side. The area of a 250 μm diameter circle divided by the area of a square 110 nm on a side is about 4.1 million, so we need 4.1 megapixels to capture the full field of view and full resolution (this assumes a circular camera, so we’d need more if our camera was square). This measure is a nice way of quantifying the amount of information transmitted by a microscopy system; for the Fourier ptychographic microscope above, it’s in the gigapixel range. Continue reading


  1. G. Zheng, R. Horstmeyer, and C. Yang, "Wide-field, high-resolution Fourier ptychographic microscopy", Nature Photonics, vol. 7, pp. 739-745, 2013.

Paper Roundup: October 2013

  • A fluorescent polymeric thermometer for measuring temperatures inside of yeast and mammalian cells. [1]
  • A new yellow fluorescent protein, derived from the tetrameric Zoanthus protein, that has a wavelength intermediate between yellow and orange FPs [2]
  • A new photoswitchable non-fluorescent protein, Phanta. It absorbs strongly at 505 nm but this absorption can be mostly switched off by exposure to cyan light, and switched back on by exposure to violet light. It can be used as a switchable non-fluorescent acceptor for FRET. [3]
  • Live-cell super-resolution imaging with vital dyes [4]
  • Statistical tests of colocalization metrics [5]
  • Single particle imaging (single virus imaging) using a cell phone with a custom attachment. [6]
  • Conversion of a flatbed scanner to a gigapixel fluorescent imager. The resolution is only about 10 um, but the field of view is 19 x 28 cm. [7]
  • Hari Shroff’s Instant SIM (all-optical superresolution system) is published. [8]
  • Hari Shroff’s di-SPIM (dual-view plane illumination microscopy) [9]
  • A fully automated FISH technique for counting RNA numbers using branched DNA probes [10]
  • Super-resolution imaging using blinking quantum dots and frame differencing. [11]
  • Counting molecules in yeast using PALM microscopy [12]
  • A review of and and protocols for super-resolution imaging with single-molecule switching (STORM/PALM-type) microscopies. [13]
  • An optically modulatable blue fluorescent protein has been published. Unlike photoactivatible proteins, this does not switch on or off, rather its fluorescence is increased by simultaneous illumination with 514 nm light (the fluorescence is excited with 405 nm light). The utility of this is not immediately obvious to me, but it’s further evidence of the complicated photophysics of fluorescent proteins. [14]
  • A new, very promising looking deconvolution algorithm, ER-Decon, from the Agard lab. [15]
  • A clever trick for making an fluorogenic probe that actives on binding to an RNA aptamer: fuse a dye to a quencher that is displaced on aptamer binding. [16]
  • A review on photophysical processes in dye molecules relevant to single molecule imaging. [17]
  • A new deconvolution algorithm for light-field microscopy capable of producing high-resolution Z-stacks from a single image. [18]
  • A detailed protocol for performing (fluorescent) in situ hybridization and immunofluorescence in Drosophila ovaries. [19]
  • Re-scan confocal microscopy, another super-resolution approach that gets a factor of sqrt(2) improvement in resolution by rescanning a confocal image onto a camera, like Instant SIM. [20]
  • New red calcium indicators [21]
  • A book chapter on FRET with fluorescent proteins [22]
  • A theoretical paper on optimal channel partitioning for separating lifetime and spectral data [23]
  • A review on fluorophore photostability and how to improve it. [24]


  1. T. Tsuji, S. Yoshida, A. Yoshida, and S. Uchiyama, "Cationic Fluorescent Polymeric Thermometers with the Ability to Enter Yeast and Mammalian Cells for Practical Intracellular Temperature Measurements", Analytical Chemistry, vol. 85, pp. 9815-9823, 2013.
  2. H. Hoi, E. Howe, Y. Ding, W. Zhang, M. Baird, B. Sell, J. Allen, M. Davidson, and R. Campbell, "An Engineered Monomeric Zoanthus sp. Yellow Fluorescent Protein", Chemistry & Biology, vol. 20, pp. 1296-1304, 2013.
  3. C. Don Paul, C. Kiss, D.A.K. Traore, L. Gong, M.C.J. Wilce, R.J. Devenish, A. Bradbury, and M. Prescott, "Phanta: A Non-Fluorescent Photochromic Acceptor for pcFRET", PLoS ONE, vol. 8, pp. e75835, 2013.
  4. L. Carlini, and S. Manley, "Live Intracellular Super-Resolution Imaging Using Site-Specific Stains", ACS Chemical Biology, vol. 8, pp. 2643-2648, 2013.
  5. J.H. MCDONALD, and K.W. DUNN, "Statistical tests for measures of colocalization in biological microscopy", Journal of Microscopy, vol. 252, pp. 295-302, 2013.
  6. Q. Wei, H. Qi, W. Luo, D. Tseng, S.J. Ki, Z. Wan, Z. Göröcs, L.A. Bentolila, T. Wu, R. Sun, and A. Ozcan, "Fluorescent Imaging of Single Nanoparticles and Viruses on a Smart Phone", ACS Nano, vol. 7, pp. 9147-9155, 2013.
  7. Z. Göröcs, Y. Ling, M.D. Yu, D. Karahalios, K. Mogharabi, K. Lu, Q. Wei, and A. Ozcan, "Giga-pixel fluorescent imaging over an ultra-large field-of-view using a flatbed scanner", Lab on a Chip, vol. 13, pp. 4460, 2013.
  8. A.G. York, P. Chandris, D.D. Nogare, J. Head, P. Wawrzusin, R.S. Fischer, A. Chitnis, and H. Shroff, "Instant super-resolution imaging in live cells and embryos via analog image processing", Nature Methods, vol. 10, pp. 1122-1126, 2013.
  9. Y. Wu, P. Wawrzusin, J. Senseney, R.S. Fischer, R. Christensen, A. Santella, A.G. York, P.W. Winter, C.M. Waterman, Z. Bao, D.A. Colón-Ramos, M. McAuliffe, and H. Shroff, "Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy", Nature Biotechnology, vol. 31, pp. 1032-1038, 2013.
  10. N. Battich, T. Stoeger, and L. Pelkmans, "Image-based transcriptomics in thousands of single human cells at single-molecule resolution", Nature Methods, vol. 10, pp. 1127-1133, 2013.
  11. Y. Wang, G. Fruhwirth, E. Cai, T. Ng, and P.R. Selvin, "3D Super-Resolution Imaging with Blinking Quantum Dots", Nano Letters, vol. 13, pp. 5233-5241, 2013.
  12. E.M. Puchner, J.M. Walter, R. Kasper, B. Huang, and W.A. Lim, "Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory", Proceedings of the National Academy of Sciences, vol. 110, pp. 16015-16020, 2013.
  13. J.R. Allen, S.T. Ross, and M.W. Davidson, "Sample preparation for single molecule localization microscopy", Physical Chemistry Chemical Physics, vol. 15, pp. 18771, 2013.
  14. A.E. Jablonski, R.B. Vegh, J. Hsiang, B. Bommarius, Y. Chen, K.M. Solntsev, A.S. Bommarius, L.M. Tolbert, and R.M. Dickson, "Optically Modulatable Blue Fluorescent Proteins", Journal of the American Chemical Society, vol. 135, pp. 16410-16417, 2013.
  15. M. Arigovindan, J.C. Fung, D. Elnatan, V. Mennella, Y.M. Chan, M. Pollard, E. Branlund, J.W. Sedat, and D.A. Agard, "High-resolution restoration of 3D structures from widefield images with extreme low signal-to-noise-ratio", Proceedings of the National Academy of Sciences, vol. 110, pp. 17344-17349, 2013.
  16. M. Sunbul, and A. Jäschke, "Contact-Mediated Quenching for RNA Imaging in Bacteria with a Fluorophore-Binding Aptamer", Angewandte Chemie International Edition, vol. 52, pp. 13401-13404, 2013.
  17. E.M.S. Stennett, M.A. Ciuba, and M. Levitus, "Photophysical processes in single molecule organic fluorescent probes", Chem. Soc. Rev., vol. 43, pp. 1057-1075, 2014.
  18. M. Broxton, L. Grosenick, S. Yang, N. Cohen, A. Andalman, K. Deisseroth, and M. Levoy, "Wave optics theory and 3-D deconvolution for the light field microscope", Optics Express, vol. 21, pp. 25418, 2013.
  19. S.G. Zimmerman, N.C. Peters, A.E. Altaras, and C.A. Berg, "Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries", Nature Protocols, vol. 8, pp. 2158-2179, 2013.
  20. G.M. De Luca, R.M. Breedijk, R.A. Brandt, C.H. Zeelenberg, B.E. de Jong, W. Timmermans, L.N. Azar, R.A. Hoebe, S. Stallinga, and E.M. Manders, "Re-scan confocal microscopy: scanning twice for better resolution", Biomedical Optics Express, vol. 4, pp. 2644, 2013.
  21. H.J. Carlson, and R.E. Campbell, "Circular permutated red fluorescent proteins and calcium ion indicators based on mCherry", Protein Engineering Design and Selection, vol. 26, pp. 763-772, 2013.
  22. H. Hoi, Y. Ding, and R.E. Campbell, "FRET with Fluorescent Proteins", FRET - Förster Resonance Energy Transfer, pp. 431-473, 2013.
  23. A. Esposito, M. Popleteeva, and A.R. Venkitaraman, "Maximizing the Biochemical Resolving Power of Fluorescence Microscopy", PLoS ONE, vol. 8, pp. e77392, 2013.
  24. Q. Zheng, M.F. Juette, S. Jockusch, M.R. Wasserman, Z. Zhou, R.B. Altman, and S.C. Blanchard, "Ultra-stable organic fluorophores for single-molecule research", Chem. Soc. Rev., vol. 43, pp. 1044-1056, 2014.