What was the state of the technology before the development reported in this paper? What problems does the new development solve? Intro Questions for Technique.

What was the state of the technology before the development reported in this paper?

What problems does the new development solve?

Intro Questions for Technique Paper:

2014 Nobel Prize in Chemistry Eric Betzig, Stefan Hell, William Moerner, Eric Betzigfor “surpassing the diffraction limit of light microscopy”

Article #2 outgrowth of Nobel prize winning work (published after prize awarded)

Had developed light sheet microscopy and SIM reconstruction methods to obtain nanoscaleresolution in living specimens

too slow to obtain high resolution data of rapidly dynamic processes in living specimens

Wide Field FluorescenceMicroscopy

Resolution reduced by lightdiffracted from out of focus plane

-hard to get high resolution 3D image (even harder at high mag)

History of Field…..

Development of Confocal Microscopy (~1990s)

Standard Widefield Confocal Microscopy

Provides optical section ofmembrane proteinUnresolved Blur

Fig. 18.9

First Improvements in Light Microscopy Resolution

Confocal Microscopy-Laser light source focused onto specific plane of specimen (but through all planes above that plane)-only diffracted light that is confocal with a pinhole aperture between specimen and photomultiplier allowed to reach photomultiplier (eliminating out-of-focus diffracted light from the image) -3D images reconstructed fromseries of sectional images Problems: -planes above specimen plane also receive excitatory light that is toxic to cells (phototoxicity)-data collected from single point at a time (merge sections for 3D) slow-phototoxicity extended

Improvement:Spinning Disc Laser Microscopy

-spread laser beam projected through spinning disc with array of microlenses focused on specimen

-diffracted light collected throughpinhole array of second spinning disc before reaches photomultiplier

-excitation energy spread over many foci instead of single focus at each scan plane, reducing phototoxicity to specimen

-can also obtain multiple data points in single scan-speeds up3D reconstruction process

2005

Light Sheet Microscopy-

-Light sheet through single plane of specimen that is perpendicular to detection lens

-increased resolution as it eliminates light diffraction from out of focus planes in widefield microscopy (as confocal microscopy does)

-ALSO eliminates photoxicity to planes above and below plane of specimen (don’t receive excitatory light)

Betzig: further improvements:used swept “nondiffracting” Bessel sheet to reduce phototoxicity and SIM (Structured-Illumination Modulation)to increase resolution

2004

2012

Resolution extension through the moiré effect.

Gustafsson M G L PNAS 2005;102:13081-13086

©2005 by National Academy of Sciences

New pattern emerges fromsuperimposition of two patterns:used to derive cell diffractionpattern at high resolution

Basis of SIM (Structures-Illumination Microscopy)

cell diffraction pattern structured light pattern

2008

Lattice Light Sheet Microscopy- Latest Development

-2D opticle lattice light sheet directed through single plane perpendicular to detection lens

-interference pattern of opticle lattice eliminates unwanted diffraction within illuminated plane to further reduce diffraction: increased resolution and reduced phototoxicity/photobleaching

-allows live imaging of dynamic processes at or below the resolution limit of light microscopy (~200 nm)

Article #2

Lattice Light Sheet Microscope-X cylindrical lens stretches beam

-Z cylindrical lens compresses into thin sheet of light

-projected onto SLM (Spatial Light Modulator)-modulateswaveform of light to createoptical lattice

Dithered Mode:fast but lower resolution- light sheet oscillatedin the X axis (not stepped)as scanned along z axis

SIM mode:high resolution but slower -light sheet steppedalong x axis as scannedalong z axis

SLM

y

x

z

s

Dith

ered

Mod

e

Figure 1. Methods of Light Sheet Microscopy

G. beam through Axicon lens

hexa

gona

l latti

ce

squ

are

latti

ce

Bess

el

Gau

ssia

n

intensity at rear pupil intensity at sample swept/dithered intensity overall PSF

point spreads over distance

Square 2DOptical Lattice

Hexagonal 2DOptical Lattice

Excitation Lens

Detection Lens

Specimen

SLM

Gen

erat

ed

Fig. 2. Comparisons of Previous Swept Bessel Beam to New Lattice Light Sheet

Optical Lattice Swept Bessel

dith

ered

(1.

5 s

inte

rval

s )

SIM

(7.5

, 30s

inte

rval

s)

Similar resolution in SIM mode[d=150 nm (x) by 280 nm (z)]BUT cells imaged with sweptBessel beam showed photobleachingand evidence of phototoxicity (retraction)about half way through 100 scans 3Dcell volumes; Lattice sheet did not

Optical Lattice Swept Bessel

In SIM Mode (A vs B):

In Dithered Mode (C vs D):Opticle lattice provided significantly higher resolution during high speed data acquisition [d=230 nm (x) by 370 nm (z)]Also can image indefinitely in opticlelattice dithered mode

To the videos: Live Imaging with Dithered Light Sheets

Fast Dynamic Processes

1) Movie 5GFP-tagged EB1 (found at growing end of microtubules)RFP-tagged histone H2Bthroughout mitosis in HeLa cells (human cervical cancer cells)2) Movie 6mEmerald-ERMitoTracker Dep Red mitochondrial markermApple histone H2Bthroughout mitosis in LLC-PK1 cells (pig kidney epithelial cells)3) Movie 9mCherry labeled Neutrophil cellIn vitro labeled collagen matriximmune system cell migrating through extra cellular matrix material4) Movie 11

Development in Multicellular Systems Nanoscale subcellular processes on time scale of sec. to min. in context of development on time scale of days to hours (increasinglikelihood of phototoxicity)

4) Movie 11GFP-tagged Aurora kinase (AIR-2)Membrane protein and histone H2BDynamics of cell division protein during C. elegans embryogenesis

4) Movie 13Myosin IIDorsal closure during Drosophila embryogenesis, when two epithelial sheets come together overthe “back” of the embryo(See Cadherin in still images)

Remaining Problems?Optical light sheets can only penetrate 20-100 mm, so can only be use at periphery ofsome multicellular organisms. (Drosophila embryo size: 100 x 500 mm)

Goal to use adaptive optics, as used in correction of ground-based astronomy pictures foratmospheric interference Biotechniques