New Imaging Modalities of Optical Microscopy Jung Y. Huang 黃中垚 Department of Photonics, Chiao Tung University Hsinchu, Taiwan http://www.jyhuang.idv.tw September 23, 2006

What are the real issues for optical microscopy? 1. Increased transverse resolution Rayleigh criterion Δx = 0.61 λ / (NA) NA = numerical aperture = n sin θ 2. Increased longitudinal resolution Rayleigh criterion Δz = 2 λ / (NA)2 (longitudinal resolution typically lower than transverse) 3. Ability to image through scattering medium Scattering leads to loss of contrast Scattering gets worse at shorter wavelengths

Current Methods for Increasing Spatial Resolution See the review by M.G.L. Gustafsson, Current Opinion in Structural Biology, 9:627, 1999.

The best resolution that can be obtained by diffraction-limited optical techniques is coarser than the molecular level by two orders of magnitude. Great progress has been made with superresolution methods including near-field scanning optical microscopy (NSOM), stimulated emission depletion (STED) and saturated structured-illumination microscopy (SSIM), which have potential for ultra-structural imaging at molecular-scale resolution.

NLO and Superresolution 1 Saturated Structured-Illumination Microscopy (SSIM) A structured light interacts with fine patterns in the sample and creates a moiré effect. The fine patterns that were previously below the Abbe-Rayleigh limit can now be visualized as a moiré version. Illuminated Object Structured Light Object Mats G. L. Gustafsson, PNAS 102, 13081–13086 (2005)

Things Are Even Better by using Nonlinear Optics (Saturated Absorption)! Response of a saturable absorber to a sine-wave intensity modulation Here is what is happening in k-space

Typical Laboratory Result of SSIM A field of 50-nm fluorescent beads: (a) imaged by conventional microscopy, (b) linear structured illumination, and (c) saturated structured illumination using illumination pulses with 5.3 mJ/cm2 energy density. Mats G. L. Gustafsson, PNAS 102, 13081–13086 (2005)

NLO and Superresolution 2 Stimulated Emission Depletion (STED) Microscopy Axial and transverse resolution better than 50 nm. Hell, Dyba, and Jakobs, Current Opinion in Neurobiology, 14:599, 2004.

Rayleigh Criteria Becomes: STED Principle: an initial excitation pulse is focused on a spot. The spot is narrowed by a second, donut-shaped pulse that prompts all excited fluorophores to STED. This leaves only the hole of the donut in an excited state, and only this narrow hole is detected as an emitted fluorescence. The light doing the turning off is diffraction limited, and so it cannot provide any greater resolution alone. The trick is the saturated depletion, which helps to squeeze the spot down to a very small scale—in principle infinitely.

Typical Laboratory Result of STED Imaging neurofilaments in human neuroblastoma. (left) Sub region of the confocal image after linear deconvolution (LD); (right) the deconvolved STED image to reveal object structures that are below 30 nm.

Photoactivated Localization Microscopy (PALM) Eric Betzig, et al., SCIENCE 313, 1642 (2006) PALM Movie The principle of PALM: a sparse subset of photoactivated fluorescent protein (PA-FP) molecules that are attached to proteins of interest and then fixed within a cell are activated with a brief laser pulse at =0.405 m and then imaged at =0.561 m until most are bleached. This process is repeated many times until the population of inactivated, unbleached molecules is depleted. The location of each molecule is first determined by fitting the expected molecular image given by the PSF of the microscope to the actual molecular image. The molecule can be plotted as a Gaussian that has a standard deviation equal to the uncertainty x,y in the fitted position. Repeating with all molecules across all frames and summing the results yields a superresolution image

Typical Laboratory Result of PALM PALM image of dEosFP-tagged cytochrome-c oxidase localized within the matrix of mitochondria in a COS-7 cell is compared to its corresponding TEM image.

Stochastic Optical Reconstruction Microscopy (STORM) A STORM imaging sequence for an object labeled with red fluorophores Cy5 that can be switched between a fluorescent and a dark state by a RED and GREEN laser, respectively. Red laser light that produces fluorescent emission from Cy5 can also switch the dye to a stable dark state. Exposure to green laser light converts Cy5 back to the fluorescent state, but the recovery rate depends critically on the close proximity of a secondary dye, Cy3. X. Zhuang, et al., Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) Nature Methods http://www.nature.com/naturemethods/

X. Zhuang, et al., Nature Methods http://www.nature.com/naturemethods/ A circular DNA plasmids coated with RecA protein was imaged by using indirect immunofluorescence with switch-labeled secondary antibody. STORM images of the RecA filaments reveal the circular structure with greatly increased resolution.

Probing into the nanoworld with femtosecond resolution Lensed-fiber launched optical waveguide device under SNOM Heterodyne Interferometric SNOM

Probing into the nanoworld with femtosecond resolution Verify the distributions of the amplitude and phase of an optical field at nanometer scale by combining SNOM and heterodyne fiber interferometry Signal intensities Is 110-12 W 1107 photons/sec are below the noise floor of photodiode detectors. By interfering this signal with Iref 110-4 W , however, the signal at the detector is boosted to Is 110-8 W , which is well within the detection limits of photo detectors.

Topography S FFT of the complex field corresponds to a projection in a basis of plane waves The spatial frequencies in the FFT spectrum are related to the propagation constants of the optical guided modes.

Tracking optical-field propagation in nanoworld Triple-Line-Defect Triple line defects 1mm GaAs AlO SiO2 Transmittance (ar. un.) 20% Triple-Line Waveguide (provided by Prof. S. Y. Lin, RPI)

Tracking optical-field propagation in real time Y. Fainman’s group at UCSD

Raman Imaging with Photon Counting Lock-in Detection Lock-in detection functionality was developed with a conventional photon counting hardware for the detection of an extremely weak optical signal.

Raw Data of Lock-in Detected Raman Spectrum Unmodulated Raman Spectrum of SSFLC Raw Data of Lock-in Detected Raman Spectrum Lock-in Detected Raman Spectrum by removing BKG

Amplitude of LI Raman Phase of LI Raman Optic Image The amplitude (filled circles) and phase (open squares) of a phase-resolved Raman peak (a) at 1118 cm-1 and (b) at 1610 cm-1 as a function of modulated frequency Ω.

Raman Imaging with Photon Counting Lock-in Detection The technique not only effectively suppresses the nonresonant background of Raman spectrum, but also yields useful information about the modulation amplitudes and phases of specific Raman peaks. The reduction factor from the phase relaxation time to the amplitude relaxation time is useful for revealing the disordering effect of the FLC alignment from the high-frequency driving. 2D distribution of the electro-optic active species that are related to the application properties of complex optical films can be obtained.

Conclusions Optical microscopy is an useful technique to yield an intuitive image to our mind. New imaging modalities in optical microscopy have been developed to allow researchers probing into nano scale at the molecular level . There are essentially no fundamental limit on how far we can go beyond the Abbe’s diffraction limit.