Fluorescence photoactivation localization microscopy (FPALM)

Sum Frequency Generation Vibrational Spectroscopy (SFS)

FPALM

Above images A-F:
An area containing photoactivatable molecules (here, PA-GFP) is illuminated simultaneously with two frequencies of light, one for readout (here, an Ar⁺ ion laser, its spatial illumination profile shown in A), and a second one for activation (here, a 405-nm diode laser, its profile superimposed in B). Within the region illuminated by the activation beam, inactive PA-GFPs (small dark blue circles) are activated (C) (small green circles) and then localized (D). After some time, the active PA-GFPs (E) photobleach (red Xs) and (F) become irreversibly dark (black circles). Additional molecules are then activated, localized, and bleached until a sufficient number of molecules have been analyzed to construct an image.

Above schematic G:
The experimental geometry shows the 405-nm activation laser (X405), which is reflected by a dichroic mirror (DM1) to make it collinear with the Ar⁺ readout laser.  A lens (L1) in the back port of an inverted fluorescence microscope is used to focus the lasers, which are reflected upward by a second dichroic mirror (DM2), onto the back aperture of the objective lens (OBJ). The sample, supported by a coverslip (CS), emits fluorescence which is collected by the objective, transmitted through DM2, filtered (F), and focused by the tube lens (TL) to form an image on a camera (charge-couple-device, CCD).

SFS

Sum frequency generation vibrational spectroscopy (SFS) is a surface-specific technique that provides vibrational spectra of molecules at interfaces.  SFS relies on the non-linear optical phenomenon of sum frequency generation (SFG).  SFG occurs when two pulsed laser beams, one of fixed visible frequency, ω_VIS, and the other of tunable infrared frequency, ω_IR, achieve spatial and temporal overlap at an interface.  Light is emitted at the sum of the two incident frequencies, i.e., ω_SF = ω_VIS + ω_IR.  The intensity of the light is resonantly enhanced when the frequency of the tunable infrared beam coincides with a vibrational mode of the molecules at the interface.  By detecting the sum frequency light (SF) as a function of infrared frequency, a vibrational spectrum is obtained, which is up-shifted into the visible region of the electromagnetic spectrum.

In the figure above: 
An 800nm pulsed Ti:sapphire laser is pumped by a frequency-doubled 527nm diode-laser-pumped Nd:YLF laser.  Within the regenerative amplifier the Nd:YLF laser acts to amplify (double) the energy level of the Ti:sapphire “seed” laser.  The now amplified output beam hits a beam splitter (70/30 BS): 70% of the power continues to a TOPAS amplifier, and 30% is reflected to a spectral shaper.  Multiple mirrors are used to create paths of equal distance from the beam splitter to the TOPAS and spectral shaper respectively.  The TOPAS outputs a pulsed infra-red laser beam (3.4µm, invisible to the human eye) of tunable frequency ω_IR; the spectral shaper outputs a visible beam (800nm) of fixed frequency ω_VIS.  These two pulsed laser beams achieve spatial and temporal overlap (traveling in the same space at the same time) at a sample interface, which responds by emitting light at the sum of the two incident frequencies, ω_sf=ω_IR+ω_VIS.  This sum frequency (SF) light (invisible due to its low intensity) is reflected into a spectrograph, which separates the SF light to obtain a vibrational spectrum.  The obtained vibrational spectrum is sent to a charge coupled device (CCD) detector, which converts the spectrum into a series of voltages that are ready for computer analysis.

In the figure below: 
A representation of the effect of increased surface disorder on an SF spectrum.  Surface disorder increases from (a) to (d). The depictions of the surface conformations and the simulated spectra illustrate the qualitative trends that occur in an SF spectrum with increasing disorder in the surface species.