Studies of both normal and disease-state physiology would benefit from the capability to visualize a broad variety of cell types simultaneously, in vivo. Multiphoton excited fluorescence (MPM) microscopy has become the technique of choice for visualization of fluorescently labeled features deep into scattering samples (~1 mm in mouse cortex), at subcellular resolution. However, poor spectral resolution hinders the use of current microscopes for in vivo imaging of more than a few fluorescent markers, making the simultaneous study of multiple cell types difficult. To date, increasing spectral resolution in MPM imaging has largely relied on the use of diffraction gratings and prisms to spectrally disperse emitted florescence, which is only suitable for collimated fluorescence emission emerging from the objective. For deep in vivo imaging, the light emerging from the objective is highly uncollimated due to scattering in the tissue, so that MPM systems relying on spectral dispersion cannot image well at depth and lose spectral resolution. In previous work (Bares et al., 2020) we built a hyperspectral multiphoton microscope (HMM 1.0) to address the challenge of achieving clear separation of multiple fluorescent species while maintaining the deep imaging capability of MPM. To test the capabilities of the HMM 1.0, we imaged a model of inflammation in the mouse ear using a combination of strategies to label multiple tissue structures and cell types, including adoptive transfer and injection of fluorescently labeled antibodies and fluorescent dyes. These in vivo demonstrations establish the capability of HMM 1.0 to image the interactions of multiple cell types in animal models for normal and disease state physiology studies. HMM 1.0 relies on acquiring separate image frames with several different excitation wavelengths and sequential settings of angle-tunable bandpass filters to acquire a temporally multiplexed set of images that are combined to distinguish different fluorescent markers. HMM 1.0, however, offers room for improvement. Changing excitation wavelength frame-to-frame is a primary factor limiting imaging speed. This factor prevents HMM 1.0 from studying fast biological and cellular dynamics such as neural firing or blood flow. In addition, slow imaging speed may cause motion related artifacts to impact accurate fluorescent species identification. This is due to breathing or movement of the live sample between frames that are later combined, leading to misregistered pixels across excitation or emission colors. We have constructed HMM 2.0 that will excite fluorophores in five different spectral bands using three spectrally distinct femtosecond pulses that are cycled through from excitation pulse to excitation pulse, rather than frame to frame. In addition to sending in each individual pulse wavelength (degenerate excitation), simultaneous pairs of different wavelength pulses provide additional excitation bands (nondegenerate excitation). Our collaborators in the Wise lab here at Cornell University have developed a fiber based novel laser source that provides the three wavelengths at 900, 1100, and 1300 nm. HMM 2.0 has a total of eight detection channels that simultaneously sample the visible spectrum range with ~40- nm spectral resolution. This new instrument will increase hyperspectral imaging speed by ~10X. We have performed an imaging experiment of fluorescent beads using a single wavelength at 1300 nm at about 0.654 MHz laser repetition while assigning data to the corresponding excitation pulse. We have also demonstrated the switching mechanism utilizing Pockels’ cells that generates the five excitation conditions needed for HMM 2.0. Our next stage is to integrate the three-color laser source into the HMM 2.0 and perform further imaging experiments to demonstrate the performance of HMM 2.0 at full scale. We envision HMM 2.0 to have several applications, including in studies of the central nervous system and immune response. Both systems involve multiple cell types and tissue components that need to be visualized simultaneously to understand biological mechanisms. In addition, the increased acquisition speed of the HMM2.0 enables studies involving fast cellular dynamics on the scale of milliseconds such as neuronal calcium transients.