Non of the whole animal. TPM made it possible

Non linear processes of light leads to develop different optical
microscopy in last two decades. Arif thesis. Two photon microscopy TPM  is one of the microscopies developed on the
basis of non linear interaction of light. Arif et al reported that TPM has
already achieved its effectiveness in ” biological imaging, especially in thick
tissue and live animal studies”. In the past when linear effet of light was used for imaging of tissue it
was not possible to obtain clear image from a appreciable depth of tissue due
to strong scattering of light.  Non-linear microscopy is less sensitive to
scattering which facilitates it to use in biological tissue imaging in high
resolution 1. In present times TPM is prominent tool for imaging in many
scientific field because of its capability of reducing  photo damage, imaging of live tissue with high
resolution from a appreciable depth.
{important paper

Two photon excitation is a 2nd order
process where a molecule excited to the electronic state from its ground state
by absorbing two photons. The sum of the energies of the two photon is
comparable to the energy differences of the states. The rate of absorption is
dependent on the square of the light intensity which makes it different from
linear process.

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TPM made it
possible to investigate the dynamic of cellular process of living organism in an
increased depth of tissues but with less phototoxicity. With the introduction
of TPM it become possible to address the exact biological and clinical concern
of the whole animal. TPM made it possible to visualize clearly and with more
details even the nearly transparent tissues. So far, its reported that, Impact
of T-cell in brain 3, lipid structure and cell domain 4, kidney dynamics 5
have been visualized by TPM method. This method facilitates to improve the
medical science to address the malfunctioning of organism more accurately.