Our homemade experimental setup for nonlinear laser scanning microscopy (NLSM) have both, reflection and transmission detection arms, and consists of following imaging modalities:
In addition, there is possibility for sample processing: cutting, inscription of arbitrary patterns, selective photo bleaching.
Laser sources used for excitation in NLSM:
Our homemade optical autocorrelator enables measurements of ultrashort pulse durations in the range 10 fs - 100 ps.
More about the autocorrelator
Portable, fiber coupled, USB spectrometer, coupled with NLSM setup, for very weak fluorescence signal measurements and spectral analysis upon the excitation in the arbitrary chosen point on the image.
More about the spectrometer
In near future we plan to develop two more microscopic techniques:
General. TPEF and SHG modalities are fully operational in back reflection arm, while the transmission arm for both THG and SHG is under development through the HEMMAGINERO project. Also, it is possible to perform polarization SHG imaging in backreflection with control of both, polarization of incident laser beam and polarization of SHG signal (analyzer).
Two Photon Excitation Fluroescence - TPEF is the most common effect/signal that is detected in NLSM. It is analogous to the
single photon excitation that is utilized in confocal and epifluroescent microscopy,
but in TPEF the fluorescent molecule is excited by absorbing two photons simultaneously.
Upon the two photon excitation, the fluorescence occurs in the same manner as in the single photon excitation.
|Type||Magnification||NA (immersion)||Working distance||Max field of view|
|Zeiss EC Plan-NEOFLUAR||40||1.3 (oil)||0.21||115.4|
|Zeiss W Plan-Apochromat 2.5||40||1.0 (phys)||2.5||114.5|
* (for 3.75x beam expander)
Sample mounting and positioning. The sample holder is for standard microscope deck glass dimensions on manual XY (lateral) table, whilst Z-axis (axial) is motorized with step size 0.3 um.
Detection and acquisition. For detection in back reflection arm an analogue PMT is used, while in transmission arm a PMT with photon counting module is planned. Maximal recording speed is approximately 1s for 1024x4024 pixels image without averaging, which is limited by the sample rate (1.2 Msamples/s) of the NI-639 acquisition card.
Sample processing. In addition to imaging mode, there is an processing mode available which enables controllable sample cutting and selective photo bleaching with arbitrary patterns (images). It is possible to control the speed of the focal spot across the sample and the dwell time in one point. The galvo scanning mirrors are synchronized with shutter, so the beam does not burn the sample between the slices.
Arbitrary patterns inscribed in the butterfly wing scales by selective photo bleaching
Cooled CCD spectrometer for weak signal measurements