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
Time

13. Reduced photobleaching0m 4m 9m
13m
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