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Arbitrary and Dynamic Patterning in a Programmable Array Microscope

Learn about Arbitrary and Dynamic Patterning in Controlled Light Exposure Microscopy (CLEM) using Programmable Array Microscope (PAM) technology. This innovative method optimizes illumination for reduced photobleaching, offering automated selection of patterns suitable for various samples. Dual-path PAM generates quality confocal images while CLE-PAM efficiently integrates discretized integration time decisions to improve image quality. Discover how CLEM avoids unnecessary photobleaching, addresses border effects, and extends into the time domain for enhanced fluorescence preservation. See remarkable reductions in photobleaching with CLE-PAM illumination compared to traditional methods.

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Arbitrary and Dynamic Patterning in a Programmable Array Microscope

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  1. Arbitrary and Dynamic Patterning in a Programmable Array Microscope (Controlled Light Exposure Programmable Array Microscopy) Wouter Caarls, Anthony H.B. de Vries, Donna J. Arndt-Jovin, Thomas M. Jovin Laboratory of Cellular Dynamics Max Planck Institute for Biophysical Chemistry Goettingen, Germany

  2. What is the PAM? • Optically sectioning microscope using conjugate structured illumination and detection through a spatial light modulator • Reduction of offset by capturing the light rejected by the pinholes (non-conjugate image) • Arbitrary pinhole patterns • Optimized for different samples and objectives • Could be chosen automatically • Patterns can be augmented with masks • Selective photoactivation and photobleaching • Adaptive imaging

  3. Dual-path PAM

  4. Confocal image generation C - 0.5*NC (after registration) Conjugate (duty 1/3) Non-conjugate (duty 2/3) Confocal

  5. Photobleaching • Bleaching occurs above and below the point being illuminated • Also when that point is not part of the sample • And even when the point lies to the side of the sample, due to high NA objectives NA = 1.45 In regular images, much of the experienced photobleaching is unnecessary!

  6. Controlled Light Exposure Microscopy (CLEM*) • Main idea: switch off unneeded illumination to avoid bleaching • Don’t illuminate background • Don’t illuminate bright foreground • Available on (Nikon) laser scanning systems Marsupial/Rho123 Scalebar 10um *Hoebe et al., Nature Biotechnology, 2007

  7. CLE-PAM • Discretized integration time decisions • Per-image decision instead of per-pixel • Allows filtering to avoid noise artifacts Illumination time Black/orange/red/white = Stop after 1/2/3/4 frames

  8. Border effects • Decision to illuminate a pixel affects neighboring pixels, due to illumination PSF • Leads to border effects near decision boundaries Desired illumination Actual illumination Naïve reconstruction

  9. Solving border effects • Dilate illumination, i.e. illuminate more than necessary • Only use that part of the image which is far enough away from the dilated border of illumination Desired illumination Dilated illumination Useful illumination

  10. Background effects • CLE-PAM lowers the signal-to-noise ratio in the background • At very low decision times, this leads to very noisy backgrounds • Especially problematic in maximum intensity projections Single-slice CLEM image Maximum intensity projection

  11. Solving background effects • Basic idea behind CLE-PAM is that background is not important • Trade SNR for spatial resolution by Gaussian filtering • Choose sigma such that resulting SNR is equal to expected SNR with full illumination. Original MIP Background-smoothed MIP The foreground is not affected!

  12. Time-domain extension • Extend CLEM decision into time domain • If a pixel is background now, it will probably be background in the next frame • Additional threshold, below the low threshold • Dilated decision boundary to account for movement • Looks like sample is “pushing” the illumination 0 1 2 3 4 Time

  13. Reduced photobleaching 13m 0m 4m 9m HeLa cells, mitotracker 200nM 20min, normal illumination (67ms) HeLa cells, mitotracker 200nM 20min, CLE-PAM illumination (max 67 ms)

  14. Reduced photobleaching, cont. Using CLE-PAM, the time until half the original fluorescence is bleached is extended by a factor of 2.2 I = mean(img) – mean(bkg) For TD-CLEM, unilluminated pixels are set to mean background Normal t1/2 CLE-PAM t1/2

  15. Conclusions • The Programmable Array Microscope allows dynamic adjustment of illumination • CLEM avoids unnecessary photobleaching by reducing the illumination outside the sample • With the PAM, CLEM can easily be integrated into a full-field system • Border effects are solved by dilating the illumination • Background is smoothed for presentation • Extension to the time domain • Photobleaching is reduced by more than a factor of 2

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