We successfully overcome the low photon density in wide-field TF by using a regenerative laser amplifier producing >1 µ
J pulses. Illumination with such high laser intensity allows for efficient two-photon absorption over large areas with commonly used biological fluorophores. In this case, with a maximum incident average power on the SLM of 600 mW, the largest illumination area obtainable with enough signal for 30 ms exposure time is 8100 µ
at 40X magnification with a peak irradiance of 40 GW/cm2
. In , the average laser irradiance at the sample was 0.07 mW/µ
over the 4200 µ
area. Compared to other custom-made scanning video-rate microscopes (1 mW/pixel, 16 pixels/µ
, 80 MHz, 200 fs) [21
], the average irradiance is approximately 200 times lower, while the instantaneous irradiance is comparable (approximately 100 GW/cm2
). Because the two-photon absorption depends on the square of the peak intensity, there is a real gain by diminishing the average irradiance at the sample and increasing the instantaneous intensity. The low repetition rate of the amplified system (250 kHz) also provides more dead time between pulses (1 pulse every 4 µ
s compared to 1 pulse every 12.5 ns for a 80 MHz Ti:Sapph) to let the photochemical system return to its fundamental state before the next pulse, hence reducing the photo- and thermal damage [22
The coupling of an SLM with TF is essential to obtain a truly scanless microscope. Even with amplified pulses, the excitation field is limited to 90×90 µ
for 30 frames-per-second (fps) imaging. Patterned illumination allows efficient random-access to the entire field-of-view defined by the maximum lateral size accessible by the SLM (180×180 µ
with a 40X microscope objective) (equation 5 in [20
]) without any moving parts.
The axial resolution of our setup () is slightly lower but of the same order as previously reported values [15
]. In order to decrease the out-of-focus excitation, one needs to increase the DG groove density or the objective NA. Nevertheless, shows that much of the out-of-focus background present in standard wide-field imaging is absent with TF. Rejecting non-contributing noise is important when imaging changes of fluorescence as a reporter of cellular dynamics. Also, conversely to 3D structural imaging, functional imaging does not require thin optical section. The contributing signal occurs from all planes in cells that often span more than 10 µ
m. Imaging the whole cell body while rejecting the major out-of-focus background leads to better signal ( and ). Nevertheless, TF is essential and gives the flexibility to image large or small ROIs with sectioning.
Similarly, the transverse resolution is decreased by the laser speckles induced by the SLM. The pixelized phase-only modulator cannot perfectly shape the incident beam and random interference zones occur at the sample plane, leading to illumination hotspots. On the other hand, high frame rate transfer with CCD cameras required pixel binning, reducing the lateral resolution. Speckles are thus not a problem for high speed dynamics imaging, considering they are fixed for a given phase mask. If temporal resolution is not imperative, uniform illumination can easily be achieved by averaging speckle variations over time (rotating diffuser [15
], phase mask shift-averaging [23
]). Another technique is the generalized phase contrast method (GPC). TFGPC has recently been implemented for photostimulation patterns generation, where uniform illumination is essential for uniform stimulation [11
The temporal resolution of the microscope is limited by the readout time of the camera. shows that sufficient fluorescence photons are available with 30 ms exposure time. Smaller ROI on the camera (50×50 µm2) can lead to imaging speed as high as 10 ms/frame (100 fps), which is more than enough to monitor fast cellular dynamics. Indeed, transient calcium signals in dendrites and spines are on the order of hundred milliseconds. Because these structures are spatially extended, network spatiotemporal dynamics are challenging to measure properly. By enabling multi-region imaging, our setup makes possible the measurements of the dynamics at multiple places simultaneously, providing contextual network information (, ).