Wide-field multiphoton microscopy refers to an optical non-linear imaging technique tailored for ultrafast imaging in which a large area of the object is illuminated and imaged without the need for scanning. High intensities are required to induce non-linear optical processes such as two-photon fluorescence or second harmonic generation. In scanning multiphoton microscopes the high intensities are achieved by tightly focusing the light, and the image is obtained by beam scanning. In wide-field multiphoton microscopy the high intensities are best achieved using an optically amplified pulsed laser source to attain a large field of view (~100 μm). The image in this case is obtained as a single frame with a CCD without the need of scanning, making the technique particularly useful to visualize dynamic processes simultaneously across the object of interest. With wide-field multiphoton microscopy the frame rate can be increased up to a 1000-fold compared to multiphoton scanning microscopy. Wide-field multiphoton microscopes are not yet commercially available, but working prototypes exist in several optics laboratories.
Introduction The main characteristic of the technique is the illumination of a wide area on the sample with a pulsed laser beam. In nonlinear optics the amount of nonlinear photons (N) generated by a pulsed beam per (illuminating) area per second is proportional to
N ∝ E 2 τ A f {\displaystyle N\varpropto {\frac {E^{2}}{\tau A}}f} , where E is the energy of the beam in Joules, τ is the duration of the pulse in seconds, A is the illuminating area in square meters, and f is the repetition rate of the pulsed beam in Hertz. Increasing the illumination area thus reduces the amount of generated nonlinear photons unless the energy is increased. Optical damage depends on the energy density, i.e. peak intensity per area Ip=E/(τA). Therefore, both the area and energy can be easily increased without the risk of optical damage if the peak intensity per area is kept low, and yet a gain in the amount of generated nonlinear photons can be obtained because of the quadratic dependence. For example, increasing both the area and energy 1000 fold, leaves the peak intensity unchanged but increases the generated nonlinear photons by 1000 fold. This 1000 extra photons are indeed generated over a larger area. In imaging this means that the extra 1000 photons are spread over the image, which at first might not seem an advantage over multiphoton scanning microscopy. The advantage however becomes evident when the size of the image and the scanning time are considered. The amount of nonlinear photons per image frame per second generated by a wide-field multiphoton microscope compared to a scanning multiphoton microscope is given by
N w i d e − f i e l d N s c a n n i n g = n f w i d e − f i e l d f s c a n n i n g {\displaystyle {\frac {N_{\mathrm {wide-field} }}{N_{\mathrm {scanning} }}}=n{\frac {f_{\mathrm {wide-field} }}{f_{\mathrm {scanning} }}}} , when assuming that the same peak intensity is used in both systems. Here n is the number of scanning points such that A w i d e − f i e l d = n A s c a n n i n g {\textstyle A_{\mathrm {wide-field} }=nA_{\mathrm {scanning} }} .
Limitations The technique is not suitable for imaging deep in scattering tissue (e.g. brain), as the image quality rapidly degrades with increasing depth The limit to which the energy can be increased depends on laser system. Optical amplifiers such as a regenerative amplifier, can typically yield energies of up to mJ with lower repetition rates compared to oscillator based systems (e.g. Ti:sapphire laser). Possible damage of the optics if the beam is focused somehow somewhere in the optical system to a small area. Different methods exist to achieve the required illumination without risk of damaging the optics (see Methods). Depth cross-sectioning may be missing.
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